Functional Regulation of N-methyl-D- Aspartate Receptors By

SerineIThreonine Protein Kinases

Ramin K. Raouf

A thesis submitted in conformity with the requirements for the degree of Master of Science

Graduate Department of Physiology University of Toronto

O Copyright by Ramin K. Raouf 1997

r \Y.,"IYI.IVI .Y UI .Y ..-y-.-...-..- -- Bibliographie Services services bibliographiques 395 Wellington Street 395. rue Wellington Ottawa ON K I A ON4 Ottawa ON K I A ON4 Canada Canada

Your fiie Votre réference

Our lile Notre réldrence

The author has granted a non- exclusive licence allowing the National Library of Canada to reproduce, loan, distnbute or sell copies of this thesis in microform, paper or electronic formats.

The author retains ownership of the copyright in this thesis. Neither the thesis nor substantial extracts fiom it may be printed or otherwise reproduced without the author' s permission.

L'auteur a accordé une licence non exclusive permettant a la Bibliothèque nationale du Canada de reproduire, prêter, distribuer ou vendre des copies de cette thèse sous la forme de microfiche/film, de reproduction sur papier ou sur format électronique.

L'auteur conserve la propriété du droit d'auteur qui protège cette thèse. Ni la thèse ni des extraits substantiels de celle-ci ne doivent être imprimés ou autrement reproduits sans son autorisation.

-----A ---

The N-methyl-D-aspartate (NMDA) subtype of glutamate receptor

plays a crucial role in a variety of neuronal processes such as long-term

potentiation (LTP). A major consequence of NMDA receptor activation is a

large influx of calcium into the ce11 which in turn leads to the activation of

protein kinases. Chen and Huang (1992) reported that PKC enhances NMDA

currents by reducing the Mg2+ sensitivity of these channels in trigeminal

neurons. In the hippocampal neurons activation of PKC has been reported to

enhance or reduce the NMDA activated currents (Wang et al., 1994,

Markram and Segal 1992). In these experiments, we examined the effects of

intracellular perfusion of PKCM (a constituitively active form of PKC) on

NMDA-evoked currents in cultured and acutely isolated hippocampal

neurons. PKCM enhanced NMDA-evoked currents in cultured hippocampal

neurons by 22 f 5% (n=ll) as compared to the control (prior to application of

PKCM). Inclusion of PKCM in the patch pipette potentiated NMDA currents

in acutely isolated neurons by 26 k 10 % (1144). Co-application of PKCI (a

specific inhibitor of PKC) and PKCM abolished this enhancement.

Furthermore, PKCM increased the open probability of NMDA single

channels in inside-out patches by 78%. The open times were not significantly

affected, however, the long closed time was significantly decreased by PKCM.

PKCM did not significantly change the Mg2+ sensitivity of NMDA currents.

The Tc50 values for the Mg2+ block prior to and after PKCM application were

- - . . *

cultured neurons. NMDA-evoked currents in acutely isolated CA1 neurons

were more sensitive to Mg2+ blockade, however, this sensitivity was not

affected by application of PKCM (IC50 contr0160 f 8 PM, n=6; PKCM 68 f 12

PM, n=6). Also the effects of Ca2+/calrnodulin dependent protein kinase II

(CaMK II) on NMDA-evoked currents were examined. Intracellular

perfusion of CaMKat (catalytic fragment of CaMK II) enhanced NMDA-

evoked currents by 22 f 5 %.

These results indicate tha t PKC acts via a phosphorylation-dependent

mechanism to enhance NMDA-evoked currents in hippocampal neurons

without altering the Mg2+ sensitivity of the channels.

mxnowieagmenr;s

1 wish to express my deepest gratitude to Dr. John F. MacDonald, my

mentor and supervisor, for his guidance, understanding and patience. 1

benefited greatly Tom my stay in his lab. 1 especially would like to thank my

friend Dr. Zhigang Xiong, for his collaboration and overall support

throughout this project. 1 would like to thank Drs. Bev Orser, Milton

Charlton a n d Lyanne Schlichter for their advice and work on my graduate

cornmittee. 1 a m grateful to Drs. Bev Orser and Wei-Yang Lu for their

participation i n some of the experiments. I appreciate and thank Ms Lidia

Brandes a n d Ms Ella Czerwinska for their technical assistance. And most

importantly, 1 thank my parents, for their love and support, and dedicate this

thesis to them as they inspire me to go further everyday.

Page

Abstract

Acknowledgments

List of Figures

Protein phosphorylation as a regulatory mechanism

Modulation of ligand-gated ion channels by protein phosphorylation

Modulation of glutamate receptor function by protein phosphorylation

NMDA subtype of Glutamate receptors

The NR1 subunit

The NR2 subunit

Recombinant NMDA receptors

Transmembrane topology

Modulation of NDMA receptors by protein phosphorylation.

Regulation of NMDA receptors by phosphatases

NMDA receptor subunits are substrates for protein kinases

Protein kinase C

PKC modulation of NMDA

Calcalmodulin-dependent protein kinase II

Regulation of CaM kinase II

viii

1

3

5

7

9

11

13

16

1 aiole or bonxenm

Modulation of glutamate receptors by CaM kinase II

Objectives and Hypotheses

Methods

Hippocampal Cell Cultures

Preparation of acutely isolated CA1 neurons

Electrophysiological recording

Whole-ce11 recording

Intr aceliular Perfusion

Single channel recording

Materials

Results

NMDA-evoked currents in hippocampal neurons

Interna1 perfusion of PKCM enhanced NMDA-evoked

currents in cultured hippocampal neurons

Enhancement of NMDA-evoked currents in acutely

isolated CA1 neurons

PKCI abolished the PKCM enhancement of

Page

34

Table of Contents

Page

the NMDA-evoked currents 59

Application of PKCM increased the activity of NMDA single channels 62

PKCM modulation of Mg2+ sensitivity of

NMDA-evoked currents in hippocampal neurons

PKCM did not change the apparent

affinity of NMDA receptors for Mg2+

Potentiation of NMDA currents by CaMKat II.

Discussion

PKC enhancement of NMDA-evoked currents

Modulation of NMDA channel activity by PKCM

PKCM modulation of the sensitivity of hippocampal NMDA

receptors to Mg2+ block

Baseline Mg2+ sensitivity of the cultured

and acutely isolated hippocampal neurons

CaMK II modulation of hippocampal NMDA receptors

Future directions

References

vii

-

Page

Figure 1. NR1 protein subunit 18

Figure 2. Whole ce11 recording with the interna1 perfusion technique 43

Figure 3. The catalytic fragment of PKC (PKCM) enhances

NMDA-evoked currents in cultured hippocampal neurons 53

Figure 4. PKCM enhancement of NMDA-evoked currents in acutely

dissociated CA1 neurons 57

Figure 5. PKCI blocked the PKCM enhancement

of NMDA-evoked currents. 60

Figure 6. PKCM enhanced the channel activity in inside-out patches 64

Figure 7. PKCM modulation of open and shut times of NMDA channels 66

Figure 8. MgzC sensitivity of NMDA-evoked currents in cultured

hippocampal neurons 70

Figure 9. PKCM did not affect the Mg2+ sensitivity of

NMDA-evoked currents 73

Figure 10. Intracellular perfusion of CaMKat enhanced NMDA-evoked

currents in cultured hippocampal neurons

viii

Protein phosphorylation is the most common modulatory mechanism

used by organisms to regulate their cellular processes (Edelman et al., 1987;

Cohen, 1989). Reversible phosphorylation is a n enzymatic reaction catalyzed

by protein kinases that involves the transfer of a high energy phosphate

group fkom adenosine triphosphate (ATP) to serine, threonine, or tyrosine

residues of a substrate protein (Edelman e t al., 1987). The reverse reaction,

dephosphorylation, is catalyzed by phosphoprotein phosphatases (Edelman et

al., 1987). Mounting evidence has supported the idea that activation of

protein kinases is the common final pathway in many intracellular second

messenger signaling systems.

The first protein kinase to be implicated in mediation of hormonal

responses was CAMP-dependent protein kinase A (Walsh et al., 1968). CAMP

had been shown to be the second messenger involved in mediating many

cellular responses to extracellular stimuli such as hormones (see Robinson et

al., 1971), however the molecular mechanisms underlying the actions of

CAMP were largely unknown. The major advancement in understanding the

mechanisms of second-messenger signal transduction came when Walsh et

al., (1968) demonstrated that a protein kinase purified from skeletal muscle

(Le., PKA) was activated by CAMP and further that this kinase is involved in

the epinephrine regulation of glycogen metabolism in the liver (Walsh et al.,

1968). Subsequently it was found that PKA is present in a wide variety o f

tissues and especially a t high levels in the brain (Miyamoto et al., 1968)

WIIILLI leu CU b u t : ~ J S C U L ~ ~ L U L I m a L au UL ut: u~ve~s t : eLlel;t,s 01 C;IILVK, in m e

nervous and non-nervous tissue, are mediated by PKA (Kuo & Greengard,

1969). Since then a large body of evidence has supported the involvement of

protein kinase A in the CAMP regulation of different metabolic and

physiological processes (Edelman et al., 1987; Cohen, 1989). Further, many

different protein kinases and phosphatases have been identified and their

substrate specificity determined. The best studied serinehhreonine kinases

are those regulated by intracellular second messengers: P M , cGMP-

dependent protein kinases, Caz+/calmodulin-dependent protein kinases

(CaMK's), and the phospholipid/Ca2+ -dependent protein kinases (PKC)

(Edelman et al., 1987). Also many other second-messenger independent

serinekhreonine kinases as well as protein tyrosine kinases have been

identified and shown to be involved in modulation of cellular processes

(Cohen, 1989; Hunter & Cooper, 1985). Phosphoprotein phosphatases also

play a critical role in modulation of cellular processes, as the signals initiated

by kinases are terminated by phosphatases. PP1, PP2A and B are examples

of phosphatases which dephosphorylate the proteins phosphorylated by

serinehhreonine kinases. Although the phosphorylation state of a protein is

determined by the balance kinases and phosphatases activity at any given

time, however activation of kinases appears to be the most wide spread

means of protein function regulation.

Modulation of ligand-gated ion channels by protein phosphorylation

The majority of the kinases characterized to date are highly

concentrated in the brain, strongly suggesting tha t phosphorylation plays an

important role in modulation of neuronal function. There is now considerable

evidence suggesting that the function of ion channels is modulated by protein

phosphorylation. Many ion channels have been shown to be substrates for

kinases and that protein phosphorylation modulates activity of the ion

channels (Levitan, 1994). The phosphorylation of ligand-gated ion channeIs

has been the focus of many studies in the past 15 years. The superfamily of

ligand-gated ion channels, the members of which include GABA, glycine,

ace tylcholine (ACh), and glutamate receptors, combine a n agonist binding

site with a closely associated ion channel in one multimeric complex. These

ligand-gated channels are involved in the synaptic transmission in the

nervous system. The interaction of the agonist (rnainly neurotransmitters)

with its binding site on the receptor results in a conformational change in the

protein complex and opening of the channel which in turn alters the

membrane potential depending on the ionic permeability of the channel.

Phosphorylation of ligand-gated ion channels can modulate their function in

different ways. Protein phosphorylation can for example alter their single

channel conductance, open probability, the time channel stays open, and also

the rate of and onset of receptor desensitization (Soderling, 1997). Further

the phosphorylation of the receptor or secondary proteins interacting with the

- V - - - - - - -- - -

localization of the receptor a t synaptic loci.

One of the best characterized examples of modulation of a ligand-gated

ion channel by phosphorylation is modulation of the nicotinic ACh (nACh)

receptor by serinekhreonine and tyrosine kinases. Biochemical studies have

shown that P M , PKC, and tyrosine kinases phosphorylate different subunits

of nACh receptors, and the location of the phosphorylation sites on different

subunits have been mapped out (Yee & Huganir, 1987; Schroeder et al., 1990;

Safran e t al., 1987). Electrophysiological studies on recombinant nACh

receptors indicate tha t protein phosphorylation leads to a marked increase in

the rate of desensitization of the receptor. However the phosphorylation by

either PKA or tyrosine kinase does not appear to affect the single-channel

conductance or the mean open time of the channel (for a review see Swope et

al., 1992). Also in addition to modulating the single-channel activity, protein

phosphorylation has been shown to affect the assembly and aggregation of

the receptor subunits a t synaptic loci (Ross et al., 1991; Ross et al., 1987).

Phosphorylation by PKA for instance promotes assembly of mature receptors

(Green e t al., 1991) and tyrosine phosphorylation causes d C h receptors to

aggregate in clusters at neuromuscular junctions (Wallace e t al., 1991).

lvioaulation or glutamate recep to r xunctxon by prote in

phosphorylat ion

Modulation of glutamate receptors by protein phosphorylation has

been the focus of many studies in the past ten years (Soderling, 1997;

Mammen & Huganir, 1997). Glutamate receptors are ubiquitously expressed

throughout the CNS where they mediate the fast component of the excitatory

synaptic transmission. Based on their physiological and pharmacological

properties, glutamate receptors have traditionally been divided into non-

NMDA (AMPA and kainate) and NMDA receptors. There is also a group of

glutamate receptors which are coupled via G-proteins to intracellular second

messenger systems (Nakanishi e t al., 1990; Schoepp & Conn, 1993). These

metabotropic glutamate receptors are believed to play an important role in

mediating the effects of released glutamate (Pin & Duvoisin, 1995; Knopfel et

al., 1995). Modulation of glutamate receptors have been implicated in the

enhancement of excitatory synaptic transmission observed during long term

potentiation (LTP). LTP, which has been used as a working mode1 for

learning and memory in the hippocampus, involves a persistent enhancement

of synaptic transmission, lasting from hours to weeks, and is induced by a

brief repetitive stimulation of monosynaptic excitatory pathways (Kennedy,

1989). The exact mechanisms underlying this enhancement are subject to

debate, however, there is considerable evidence that postsynaptic

mechanisms that modulate the function of glutamate receptors partly

contribute to this potentlation (Collingridge & Singer, 1990; Muller et al.,

- -

and to some extent the maintenance of LTP appear to involve activation of

kinases such as PKC and CaMK II in the postsynaptic neuron suggesting

that protein phosphorylation plays a n important part in regulation of

postsynaptic glutamate receptors (Kennedy, 1989, Soderling, 1996a, Ben-Ari

e t al., 1992). Many lines of evidence now have confirmed the modulation of

glutamate receptors by protein phosphorylation. The AMPA receptor types

(non-NMDA) have been shown to be functionally modulated by PKA, PKC,

and CaMK II (Wang e t al., 1994; Roche e t al., 1996; Kolaj et al., 1994). The

AMPA receptor subunits are phosphorylated in vivo and the they contain

consensus phosphorylation sites for PKA, PKC, and CaMK II (Leonard &

Hell, 1997; Roche e t al., 1996; Leonard & Hell, 1997) which indicates that

they are under regulation by endogenous kinases.

The NMDA class of glutamate receptors have also been shown to

undergo protein phosphorylation. However the precise role of

phosphorylation in the function of the receptor remains unclear. The NMDA

subclass of glutamate receptors posses unique physiological and

pharmacological properties (see below) and are involved in many aspects of

nervous system function such as plasticity and differentiation. The

elucidation of the mechanisms involved in the regulation of the function of

NMDA receptors is necessary for better understanding of the function of

nervous system and design of drugs to battle pathological conditions of the

brain.

NMDA subtype of Glutamate receptors

NMDA receptors are multiheteromeric complexes that form ligand-

gated channels permeable to cations. While the fast component of the

excitatory postsynaptic currents (EPSC's) is mediated by AMPA receptors,

NMDA receptors are responsible for the slow component of the EPSC's

(Collingridge & Singer, 1990; Lester et al., 1990). NMDA receptors have

been implicated in a multitude of processes in the CNS including long term

potentiation and depression (LTP and LTD) (Aroniadou & Teyler, 1991;

Randic e t al., 1993), development and growth (Artola & Singer, 1994),

neuronal differentiation (Komuro & Rakic, 1993). There is also good evidence

implicating NMDA receptors in neurodegeneration associated with a number

of neurological diseased and disorders including epilepsy, ischemia and

Huntington's chorea (Choi, 1992). Also NMDA receptors are targeted by

anesthetic drugs (such as ketamine see MacDonald e t al., 1991) and many

other neuromodulators such as PCP (Domino and Luby 1981).

One of the unique features of NMDA receptors is their high

permeability to Ca2+ : they are 10 times more permeable to Ca2+ than they

are to Nai (MacDermott e t al., 1986; Burnashev et al., 1995; Wollmuth et al.,

1996; Mayer & Westbrook, 1987) but they are also inhibited by intracellular

and extracellular Ca2+ (Legendre et al., 1993). Other divalent cations such as

Cozf, Zn2+, and Mg2+ block NMDA-evoked currents (Nowak et al., 1984;

lvlayer <x; vvesr;iorooa, LYU 1 ) . m e moclcaae 01 lulvlun cnannels by lvlgaf 1s

voltage dependent which gives rise to a non-linear current-voltage

relationship a t negative membrane potentials (Nowak et al., 1984; Mayer e t

al., 1984). These two characteristics of NMDA receptors, i.e., the high

permeability to Ca2+ and the voltage-dependent blockade by M@+, are

important in LTP and other neuronal processes (see Muller e t al., 1991 for

review).

Another interesting feature of NMDA receptors is their requirement

for glycine or D-serine a s a CO-agonist (Johnson & Ascher, 1987). Glycine

highly potentiates the function of NMDA receptors, reduces the apparent

desensitization of the receptor and also interacts with Mg2+ (Paoletti e t al.,

1995; Johnson & Ascher, 1987; Wang & MacDonald, 1995). The physiological

concentration of glycine at the synaptic cleft is not known hence the

physiological significance of this CO-agonism is not clear.

Molecular cloning techniques have revealed that NMDA receptors

consist of two subunits: the mandatory NR1 and the regulatory NR2. NR1

subunits form homomeric channels, whereas NR2 subunits do not, however

NRl receptor function is highly potentiated when expressed with NR2. NRI

channels expressed homomerically or heteromerically with NR2 exhibit al1

the major pharmacological properties of native NMDA receptors (see below).

The recombinant heteromeric NMDA channels have pharmacological

properties similar to those observed in the native NMDA receptor subtypes in

,the CNS (Williams et al., 1993; Buller e t al., 1994). This and other

omervarions suggesr; m a t native lulvlun receptors are neteromers 01

NRllNR2 composition. For example, most brain regions express both NR1

and NR2 subunits and mature cerebellar Purkinje cells which express NRI

but not NR2 do not respond to NMDA (Brose e t al., 1993; Monyer e t al.,

1994). Further, coimmunoprecipitation studies suggest that NR1 and NR2

subunits are CO-localized on the ce11 soma and the dendrites (Sheng e t al.,

1994; McBain & Mayer, 1994).

The discovery and cloning of NR1 and NR2 subunits has lead to

extensive analysis of the properties of NMDA receptors expressed in Xenopus

oocytes and mammalian expression systems and better understanding of the

structural features giving rise to those properties.

The NRI subunit

Although the NR1 subunit is coded by one gene, the NR1 mRNA

undergoes alternative splicing which can generate 8 different splice variants

(Sugihara et al., 1992; Durand e t al., 1992; Anantharam et al., 1992;

Nakanishi et al., 1992; Yamazaki et al., 1992). These splice variants are

generated by inserting a 2 1 amino acid splice cassette (termed NI) in the N-

terminus of the NRloii protein or omission of two independent consecutive

splice cassettes of 37 amino acids (Cl) and 38 amino acids (C2), respectively,

that constitute the last stretch of the C-terminus of NR1 protein (Hollmann

et al., 1993). Each of the NR1 splice variants can form homomeric functional

- - - - - * *

expressed in Xenopus oocytes (Durand et al., 1992; Hollmann et al., 1993).

Al1 the NR1 splice variants form functional homomeric receptors with many

of the properties of the native NMDA receptors, but they differ in their

agonist affinity, non-cornpetitive antagonist affinity, modulation by

polyamines and ZnZ+ , and regulation by protein kinase C (Durand et al.,

1992; Hollmann et al., 1993; Zheng et al., 1994; Traynelis et al., 1995).

Considering that the majority of NMDA receptors in the CNS also contain

one or more of the four NR2 subunits, it is not clear to what extent the

alternative splicing of NR1 subunits contribute to the overall heterogeneity of

the native NMDA receptors.

The NR1 splice variants in the adult brain exhibit differential

distribution patterns that appear to be established a t around time of birth

(Laurie & Seeburg, 1994; Zhang et al., 1994). The NRloxx and NRlXoi (NI-

lacking and CS-containig, respectively) splice variants exhibit a wide and

abundant distribution throughout the brain where as the NRlxli (the splice

variants containing both C l and C2) were mainly expressed in the

hippocampus and more rostral structures (Laurie & Seeburg, 1994). In the

hippocampus itself, a cell-specific and development regulation of alternative

splicing of NR1 subunit is observed (Paupard et al., 1997). In the

hippocampus the N1 lacking splice variants (NRloXx) are expressed

uniformly in the CA1, CA3, and dentate gyrus. In contrast the NI-containing

variants (NRlxx) are expressed at low levels until P8, at which time the

- - - - _ruuiu-i uu -u-iJ U V Y V I Y V V -UV& V y & V L Y I A L Y I A V , V U p Y V I W A I J A,.& V I A U V I I V . L b & A V I A

(Paupard e t al., 1997). By PZ1 the CA1 and dentate gyrus mainly express

the NI-lacking variants whereas the CA3 neurons also express the NI-

containig splice variants of NR1. Splicing of the cassettes Cl and C2 is

regulated independently within the hippocampus. Whereas NRlxii and

NRlxoi are uniformly expressed throughout the hippocampus, NRlxoo are

expressed more prominently in pyramidal neurons of CA3. NRlxio receptor

expression is very low in the hippocampus. Interestingly the cell-specific

expression of NRlxii receptor mRNAs matches that of NRloxx, and the

expression patterns of NRl ixx parallels those of NRlxoo (Cl-, CS-lacking)

receptor mRNAs during development. These observations suggest that some

of the heterogeneity of the endogenous NMDA receptors may be attributed to

the regulation of the splicing of the NMDA receptor NR1 subunit mRNAs

(Paupard et al., 1997).

The NR2 subunit

The NR2 subunit is coded by 4 genes, NRZA, B, C, and D. The NR2

subunits are considerably larger and share little homology (less than 20 %)

with the NR1 subunit (Meguro e t al., 1992; Watanabe et al., 1992; Meguro e t

al., 1992; Monyer et al., 1992; Ishii et al., 1993; Ikeda et al., 1992). These

proteins however are structurally similar and enjoy approximately 50 to 70 %

sequence identity with each other (Ishii et al., 1993). The NR2 subunits have

- - - - - - - - - - a -- ---- - ---J --------- "--" -"- -"-a-- b--"UY-UYY A VVYr " V I V I I V -*-A.,".

The C-terminus region is highly variable between the NR1 and NR2

subfamilies and is the locus of interaction between cytoskeletal elements and

other regulatory proteins and the NMDA receptor complex (Ikeda et al., 1992;

Meguro e t al., 1992; Monyer e t al., 1992; Moriyoshi e t al., 1991; Yamazaki et

al., 1992).

NR2 subunits do not form functional receptors on their own but

strongly potentiate channel function and confer specific pharmacological

properties on the receptor when CO-expressed with NR1 subunits (Monyer et

al., 1992; Ishii et al., 1993). Affinity for the agonist, sensitivity to divalent

cations such as Mg2+, single channel conductance are some of the properties

controlled by the NR2 subunits (Monyer et al., 1992; Meguro e t al., 1992;

Ishii et al., 1993; Stern e t al., 1992).

Although most brain regions express the NR1 subunit, the distribution

of the NR2 subunits exhibits a differential pattern that is both tissue and

cell-type specific (Zhang et al., 1994; Monyer et al., 1992; Meguro et al.,

1992). NR2A is distributed widely in the brain, whereas the distribution of

NR2B is more selective for the forebrain, with high levels of expression in the

hippocampal formation, the septum, the caudate-putamen, and the cerebral

cortex. The NRZC subunit is mainly expressed in the cerebellum (Watanabe

et al., 1994b). Low levels of NR2D subunit are found in the thalamus, the

brainstem and the olfatory bulb (Watanabe et al., 1993).

---- --- r'--'--" -' -"- - .--- Y -Y --a-., A-- YI-- U A ...A-- SV U Y . " * V ~ Y I V * I " U I I

regulated (Akazawa et al., 1994; Monyer et al., 1994; Watanabe et al., 1992;

Watanabe et al., 1994a). At the early embryonic stage, NRSB and NR2D are

the only subunits that are expressed by the developing brain. NRZB is

widely distribute throughout the brain, whereas the expression of NR2D is

localized to the diencephalon and the brainstem. Wide expression of NR2A

subunit mRNA begins to appear during the first two weeks after birth. At

this point NR2C mRNA appears in the cerebellum and the expression of

NR2D subsides considerably. The expression of NR2B subunit mRNA also

becomes restricted in the forebrain. These observations suggests that the

differential and developmental expression of different NR2 subunits reflects

the diversity in the pharmacological properties of NMDA receptors in the

adult and developing brain (Mori & Mishina, 1995).

Recombinant NMDA receptors

Many studies have focused on the properties of the recombinant

heteromeric NMDA receptors and have found that different combination of

the NRlfNR2 subunits are characterized by distinct physiological and

pharmacological properties. The majority of these studies have examined

recombinant heteromeric NMDA receptors containing the NRloil (NRla as

defined by Sugihara et al., 1992) splice variant (which is the most common

form in the brain) and one of the NR2 subunits. The NRl/NR2A complex for

exampre snows a rower arrinity ror agonists sucn as glutamate or ~WUJA

(Kutsuwada et al., 1992; Ikeda et al., 1992; Buller et al., 1994) as compared

to the other heteromers. The single channel properties of heteromeric

channels are also dependent on the NR2 subunit present. In general

NRllNR2A and NRl/NR2B channels have higher conductance than

NR1lNR2C or NRlINR2D channels (Stern et al., 1992; Stern et al., 1994;

Behe et al., 1995; Wyllie et al., 1996). The low conductance type channels,

i.e., NRlINRZC and NRlINR2D also differ from the high conductance type in

their longer mean open time of subconductance and temporal asymmetry

(Wyllie et al., 1996).

The high Ca" permeability of NMDA receptors have been implicated

in the initiation of some forms of LTP in the hippocampus and neurotoxicity

of NMDA and glutamate (Choi, 1992; Tokita et al., 1996). Al1 the

heteromeric NMDA receptors are highly permeable to Ca2+ (Monyer et al.,

1992) which correlates with the observations in the properties of the native

NMDA receptors in the brain (Ascher & Nowak, 1988; MacDermott et al.,

1986). There are however subtle differences between NRlINR2A and

NRllNR2C in some of their Ca2+-dependent characteristics (For review see

Burnashev, 1996). The relative fraction of whole-ce11 current carried by Ca2+

is slightly higher in HEK 293 cells expressing NRlINR2A channels than

those expressing NRlINRSC (Villarroel et al., 1995) which is in agreement

with the results that NRlINR2A expressing HEK 293 cells are more

. ---------- -- ------ "'H" - .A"-. - .A"- V Y--r-YVV---b *..Y** LVY "YI....,

(Cik et al., 1994).

Heteromeric NMDA channels expressed in Xenopus oocytes or HEK

293 cells also differ in their sensitivities to Mg2+ block. NMDA whole-ce11

currents in HEK 293 cells expressing NRlINR2A or NRlINR2B channels are

more sensitive to Mg2+ than the currents in cells expressing NRlINR2C or

NRlINRZD channels (Monyer e t al., 1994; Monyer et al., 1992). Similar

results are obtained for heteromeric channels expressed in Xenopus oocytes

(Kutsuwada e t al., 1992; Ishii e t al., 1993; Wagner & Leonard, 1996; Kuner &

Schoepfer, 1996a). The latter two studies have examined the Mg2+ sensitivity

of all the heteromeric combinations in more detail. Both studies con£irmed

that heteromeric channels containing NR2A or NR2B are more sensitive to

Mg2+ block than NR2C or NR2D containing channels and that NR2D

channels have slightly higher sensitivity than NR2C channels. However

Kuner and Schoepfer (1996) have reported that concentration values of

extracellular Mg2+ at which half-maximal block occurs (IC50) a t a holding

potential of -100 mV to be (in PM) 2.4, 2.1, 14.2, and 10.2 for NRlINRPA,

NRBB, NRPC, and NR2D respectively, whereas the 1C50 values reported by

Wagner and Leonard (1996) are (in PM) 2.8, 10.7, 349, and 147 a t -80 mV

respectively. The apparent discrepancy in the IC5o values may be due to the

different holding potentials in different studies (Kuner & Schoepfer, 1996a).

p hosp horylation (see below).

Transmembrane topology

Although the NMDA receptor channel subunits have four hydrophobic

segments (Ml - M4) in the middle of the molecule (similar to nicotinic

acetylcholine receptors) the initial four transmembrane segment model

suggested for NMDA receptor channel subunits (Moriyoshi et al., 1991) is

highly disputed by several lines of evidence. Phosphopeptide map analysis

suggest that the C-terminus of the NR1 subunit is phosphorylated in vivo

(Tingley e t al., 1993). Further the C-terminus of the NRllNR2b channel is

responsible for the potentiation by TPA (Mori e t al., 1993) suggesting that

the carboxy terminus of the NR1 and NR2B subunits is probably

intracellular. I n contrast the four transmembrane model places the C-

terminus on the extracellular side of the membrane. Also mutational

analysis of the glycine binding and redox modulation sites of the N R l subunit

suggest a n extracellular localization of the segments between regions M3 and

M4 (Kuryatov e t al., 1994; Sullivan et al., 1994). Further, analysis of the N-

glycosylation sites on the GluRl and GluR6 (Roche et al., 1994) subunit

suggests a three transmembrane model in which the putative channel lining

segment M2 does not span the membrane but loops into the membrane

without traversing it (Hollmann et al., 1994; Sutcliffe e t al., 1996; Wo &

NMDA receptor topology is obtained from a recent study by Kuner et al.,

1996. In this study the extracellular and cytoplasmic faces of cystein-

substituted NRl/NRSC channels with sulfhydryl-specific agents were

investigated. The pattern of accessibility of the residues along M2 suggest

that this segment forms a channel lining loop originating and ending in the

cytoplasmic side of the membrane, placing the functionally critical

asparagine (N-site) at the tip of the loop (Kuner e t al., 1996b). Also mutation

studies on the structural determinants of the Mg2+ block suggest a re-entrant

topology for M2 segment (Kupper et al., 1996). The currently accepted

topology of NMDA receptor subunits is depicted in Figure 1.

Similar to ACh receptors, the segment M2 is the channel lining region

of the NMDA receptor subunits. Ali subunits of the NMDA receptor channel

have a n asparagine in segment M2, corresponding to the QIR site that

determines the Ca2+ permeability of AMPAkainate channels (Hume et al.,

1991; Verdoorn et al., 1991). Mutation of this asparagine to glutamine on the

NR2B and NR1 subunits strongly affects the sensitivity to Mg2+ blockade of

the heteromeric NMDA receptor channels (Burnashev et al., 1992; Mori et

al., 1992; Sakurada et al., 1993). Electrophysiological studies on the kinetics

of the blockade by Mg2+ suggest that Mg2+ binds to a site located deep within

the pore (Ascher & Nowak, 1988). A re-entrant topology for M2 segment is

also supported by the fact that there probably are two different binding sites

for the external and interna1 Mg2+ block (Kupper et al., 1996), and that the

A schematic diagram of the proposed topology of the NR1 subunit (see text

for details). The phosphorylation sites for PKC (shaded circles) and PKA

(open circle) are located in the Cl cassette.

residues five to seven positions downstream form QIRIN site (Kupper et al.,

1996). However the overall Mg2+ sensitivity of the receptor appears to be

determined by multiple structural elements including Ml , M2-M3 linker, and

M4 segments (Kuner & Schoepfer, 1996a).

Modulation of NDMA receptors by protein phosphorylation

The involvement of serinekhreonine kinases in the functional

modulation of NMDA receptors is supported by a large body of evidence,

however, the exact role played by these kinases in this functional regulation

is not clear. Al1 NMDA receptor subunits contain numerous consensus sites

for phosphorylation by PKC and CaM-kinase II (Moriyoshi et al., 1991;

Kutsuwada e t al., 1992; Nakanishi, 1992). Also direct phosphorylation of

NMDA receptors in neuronal preparations has been shown (Hall & Soderling,

1997; Lau & Huganir, 1995).

Investigation of the mechanisms underlying the run-down of NMDA

receptors provided the first indication that protein phosphorylation may be

involved in the functional regulation of these receptor channels. Run-down

or wash-out of the NMDA-evoked currents is a phenornena that is observed

during whole-ce11 recording of NMDA-evoked currents in neurons. The

responses to NMDA gradually decrease and a t values of about 50% of the

initial values (Mody e t al., 1988; MacDonald et al., 1989). This run-down of

the current can be partially blocked by inclusion of Mg-ATP in the pipette.

- -

recovered by perfusion of the support solution into the pipette following

establishment of run-down (MacDonald et al., 1989).

Functional regulation of MMDA receptors by protein phosphorylation

may be important in such processes as LTP where the entry of Ca2+ through

the receptor plays an important role in initiating the events leading to the

changes in the synaptic efficacy. I t has been reported, for example, that the

NMDA-mediated component of EPSC's is also enhanced following induction

of LTP in hippocampal slices (Bashir et al., 1991; Xie et al., 1992). Also the

activation of the metabotropic class of NMDA receptors which leads to

induction of several phosphorylation cascades resulting in the enhancement

of NMDA-rnediated currents in hippocampal slices (Kelso e t al., 1992; Kinney

& Slater, 1993). PKC and CaMK II are two serinelthreonine kinases that are

involved in the induction of LTP in the hippocampus (Suzuki et al., 1992;

Ben-Ari e t al., 1992; Muller et al., 1991; Barria et al., 1997; Fukunaga et al.,

1996; Wang & Kelly, 1995). Both of these enzymes are regulated by Ca2+ and

pharmacological inhibitors and genetic knock-out of these kinases block the

induction of LTP (Wang & Feng, 1992; Silva et al., 1992). These data suggest

that PKC and CaMK II can regulate the function of NMDA receptors in vivo.

- - - -

The phosphorylation state of a given protein at any given time is the

net balance of phosphorylation and dephosphorylation in the cell. Hence

equally important to the modulation of the cellular functions is the activity of

phosphatases. Recently regdation of NMDA receptors by phosphatases have

been demonstrated. Wang et al. (1994) have shown that in perforated patch

recordings of the cultured hippocampa.1 neurons, application of calyculin A,

an inhibitor of serinekhreonine protein phosphatases PP1 and PPBA,

enhances NMDA-evoked cwrrents. This enhancement is reflected in an

increase in both the frequency and the duration of channel opening in cell-

attached patches following the application of calyculin A. Also direct

application of PP1 and PP2A to inside-out patches decreases the probability

of channel opening (Wang et al., 1994). Similarly, inhibition of calcineurin, a

Ca2+/calmodulin dependent serinelthreonine phosphatase, has been shown to

potentiate the activity of NMDA channels (Lieberman & Mody, 1994). In the

seudy by Lieberman and Mody (1994) application of okadaic acid and FK506,

two inhibitors of calcineurin, prolong NMDA channel openings in dentate

gyrus granule cells. Also, these inhibitors of calcineurin block the run-down

of NMDA receptors in outside-out patches and glycine-insensitive

desensitization a t synapses between cultured rat hippocampal neurons (Tong

& Jahr, 1994; Tong et al., 1995).

i ! A v l u n L t;~;t;pbuI 3UUUlllC13 il1 t: 3UU3bl-ilbt53 IU1- pL-Ubt5111 K l l l a S e S

Al1 of the cloned NMDA receptor subunits contain consensus sites for

phosphorylation by CaM-kinase II, PKA, and PKC (Moriyoshi et al., 1991;

Kutsuwada et al., 1992; Nakanishi, 1992). Also it has been shown recently

that the NMDA receptor subunits are phosphorylated in vivo, strengthening

the hypothesis that direct phosphorylation of these receptor subunit may

underlie the modulation of NMDA receptors by phosphorylation.

The NR1 subunit contain several sites for phosphorylation by serine

threonine kinases. Tingley e t al. (1993) demonstrated that the NR1 subunit

is phosphorylated in the primary cultures of cortical neurons, and that the

phosphorylation increases following phorbol ester treatment. Further

analysis has revealed that PKC-induced phosphorylation occurs mainly a t

four serine residues located on cassette II (Cl) (Tingley et al., 1997). W o of

these residues, serines 890 and 896, are phosphorylated by PKC and serine

897 is phosphorylated by PKA. Interestingly, cassette II (Cl) has been found

to decrease the potentiation by phorbol esters of homomeric NR1 subunits

containing that insert (Durand et al., 1993), suggesting that direct

phosphorylation of the NR1 subunit a t the carboxy-terminal may not be

important for the potentiation of the function of the homomeric receptors

(Yamakura e t al., 1993). Phosphorylation of serine 890 by PKC results in the

reversible dispersion of surface-associated clusters of the NR1 subunit

expressed in fibroblasts (Tingley et al., 1997; Ehlers et al., 1995). This

suggests a role for phosphorylation of these residues in trafficking of the

- * * * "

the NR1 subunits of NMDA receptors in vivo determines the clustering of

these receptors at the synaptic sites hence modulating the contribution of

these receptors to synaptic events.

Activation of PKC increases phosphorylation of both NR2A and NR2B

when expressed in HEK 293 cells (Fung et a1.,1994). In a recent study in

cultured rat hippocampal neurons, Hall and Soderling (1997) reported that

NR2A and NRZB subunits are highly phosphorylated under basal conditions,

whereas the basal phosphorylation of NR1 subunit is very low. Also

stimulation of PKC by phorbol ester treatment enhanced the phosphorylation

of NR1 by 3-5 fold, whereas that of NR2 was enhanced by less than 2 fold

(Hall & Soderling, 1997). It appears the majority of the residues

phosphorylated on NR2 are serines and not threonine or tyrosine (Hall &

Soderling, 1997).

Protein kinase C

Protein kinase C (PKC) is one of the most extensively studies signaling

kinases in the nervous system. PKC activity has been implicated in a variety

of neuronal processes such as neurotransmitter release (Malenka et al., 1986;

Parfitt & Madison, 1993). regulation of growth and differentiation (Burgess

et al., 1986; Spinelli & Ishii, 1983; Ponzoni et al., 1993), modulation of ion

channels and neurotransmitter receptors (Levitan, 1988; Levitan, 1994;

- . -

(Routtenberg et al., 1986; Ben-Ari et al., 1992). A large body of evidence

exists implicating PKC as a crucial player in the events leading to the

induction of LTP. Many of PKC inhibitors block induction of the NMDA-

dependent form of LTP in the hippocampal slices. Furthermore, activators of

PKC such as phorbol esters facilitate the induction of LTP. Also intracellular

injection of PKC into the CA1 neurons generates LTP, which provides

compelling evidence that PKC activation is required for the induction of some

forms of LTP in the hippocampus. It has been suggested that PKC may

mediate the induction of LTP by modulating the activity of NMDA receptors,

that is potentiating the receptor's response by releasing the Mgz+ block hence

ampli%ing the Ca2+ signal which would ultimately initiate the cascades

leading to maintained enhancement of synaptic activity (i.e. LTP). Th' 1s was

mainly based on the evidence from the work of Chen and Huang (1992) in the

trigeminal neurons. However no such report has looked a t the effects of PKC

activity on the responses of native NMDA receptors i n hippocampal neurons.

PKC family of serinelthreonine phospholipid dependent kinases are

subdivided in to three categories based on their CO-factor requirements: the

conventional PKC's (a, P, y) that require Ca2+ and diacylglycerol (DAG) or

phorbol ester in addition to phosphatidylserine, conventional PKC's (6, E, q,

and o) that do not require Ca2+ as a cofactor, and the atypical PKC's (6 and li)

that do not need either of Ca2+ or DAG for maximal activity (Jaken, 1996).

enriched in the neuronal tissue. Further this distribution exhibits a

differential pattern suggesting that different isozymes play different roles in

the regulation of function of the nervous system. Upon activation of the

enzyme translocated to the cytoplasmic membrane where it interacts with

phospholipids and DAG and the active site is uncovered leading to the

phosphorylation of the substrate.

PKC modulation of NMDA receptors

There is considerable evidence supporting a role for PKC in functional

modulation of NMDA receptors. However the exact role played by PKC is not

clear. Studies done using neuronal preparations suggests that PKC plays a

role in the modulation of NMDA receptor function. In these experiments

topical application of phorbol esters, potent activators of PKC, significantly

enhanced or depressed the NMDA-evoked currents depending on the

preparation.

In the presence of TTX, phorbol esters selectively enhanced, in a

reversible manner, the depolarizing responses of dorsal horn neurons to

NMDA and the potentiation of the NMDA response was blocked by APV, a

specifïc NMDA receptor antagonist (Gerber et al., 1989). H-7 reversibly

reduced this potentiation. In the CA1 neurons of the hippocampus activation

of metabotropic glutamate receptors potentiates NMDA-evoked currents

\L A r a r n u u u G j A A G U UA., I U U Y J . A LLLJ G L A I A Q L ~ L G J . U C ; I ~ ~ W aù UrULht5U Uy 1 1 1 I d ë l G ~ l ~ U ~ ~ ~

perfusion of PKCI 19-31 or the PKC antagonist sphingosine. Application of

phorbol esters also enhanced NMDA-evoked currents in 3 of the 5 cells tested

which suggest that activation of metabotropic receptors enhances NMDA-

evoked currents via a PKC dependent mechanism. In two neurons however,

phorbol ester treatment actually reduced the response of NMDA receptors.

In a study done by Markram and Segal (1992), it was found that topical

application of phorbol esters to hippocampal slices reduced the amplitude of

NMDA-evoked currents in the CA1 neurons. The application of H-7 blocked

this potentiation. Similar results have been obtained fkom cerebellar granule

cells. Application of phorbol ester reduces the NMDA-evoked cytoplasmic

[Ca2+] in cultured cerebellar granule cells (Courtney & Nicholls, 1992). These

results suggest that phorbol esters in fact down-regulate NMDA receptors in

these preparations.

The reason behind the inconsistencies observed in the effects of

phorbol esters on NMDA-evoked currents in different preparation is not

known. Non-specific actions of phorbol esters a t higher concentrations or

differential sensitivity of NDMA receptors of different subunit compositions

to PKC activation have been suggested as the underlying cause for the

observed discrepancies (Mamrnen & Huganir, 1997). These results

emphasizes the importance of PKC in modulation of NMDA receptors

although the exact role played by PKC in the modulation of function remains

uncle ar.

A , - -- - - - a

MePhe4-Gly-015-enkephalin (DAGO), increased NMDA-evoked currents in

spinal trigeminal neurons in thin medullary slices of rats. Intracellularly

applied protein kinase C (PKC) mimics the effect of DAGO, and a specific

PKC inhibitor interrupted this enhancement (Chen & Huang, 1991).

Application of a specific PKC inhibitor, PKC 19-31, which acts as a

pseudosubstrate and binds to the catalytic site of the enzyme, blocked the

enhancement of current by PKC and DAGO. Chen and Huang (1992)

proposed a mechanism for the enhancement of NMDA receptor responses by

PKC. They reported that PKC activity increases the probability of channel

openings and reduces the sensitivity of the channel to the voltage-dependent

Mg2+ block. Hence PKC activity leads to potentiation of the NMDA

component of EPSP's by increasing the probability of channel openings and

reducing the blockade by Mgzc.

The modulation of the recombinant NMDA receptors by protein

phosphorylation has been extensively studies ever since the first NMDA

receptor subunit was cloned. Initial experiments with oocytes injected with

the ra t whole brain mRNA demonstrated the potentiation of the NDMA-

induced currents by external application of phorbol esters (Kelso et al., 1992;

Urushihara et al., 1992). With the cloning of different splice variants of the

NR1 subunit the effects of PKC activation on the homomeric NMDA

receptors was extensively studied. Homomeric receptors composed of

different splice variants of the NRl subunit exhibit differential sensitivities

-

Durand et al., 1993; Yamazaki et al., 1992; Yamakura et al., 1993; Mori et

al., 1993). The splice variants that contain the N-cassette insert in the

putative extracellular N-terminus show the highest sensitivity to treatment

with TPA. (Durand e t al., 1992). Presence the cassettes II and III (Cl and

C2) on the putative intracellular C-terminus reduce the extent of potentiation

by TPA of homomeric NR1 channels (Durand e t al., 1993). Interestingly the

majority of the sites known for PKC potentiation are located within cassette

II (Tingley e t al., 1993). This finding however is consistent with the study by

Yamakura e t al. 1993, where they report that mutation of al1 the c-terminal

serines and threonines to alanine does not alter the potentiation by TPA

(Yamakura e t al., 1993). Also the heteromeric NRllNR2A receptors in which

the NR1 subunit contains the N-cassette insert are more sensitive to

treatment by TPA than receptors that do not have cassette one (N-cassette).

Analysis of the heteromeric recombinant NMDA receptors has also

revealed tha t the potentiation of these receptor complexes by PKC activators

depends on the NR2 subunit present. In a series of experiments mouse NR1

subunit was coexpressed with mouse NRZA, NRBB, NRBC, and NR2D

subunits and it was found that NRllNR2A or B receptors are highly

potentiated by TPA treatment whereas NRIINRPC and D combinations were

not (Kutsuwada et al., 1992; Wagner & Leonard, 1996). Mori e t al. (1993)

have shown that replacing the carboxy-terminus of NR2C subunits with that

of NR2B renders the resulting chimera sensitive to TPA treatment; when

carboxy-terminus are no longer sensitive to TPA treatment (Mori et al.,

1993). It is not clear how NR2C blocks the effects of TPA on NR1 subunit.

The mechanism of the potentiation of the heteromeric recombinant

NMDA receptors by phorbol esters has not been completely elucidated. The

enhancement of the receptor function may be due to direct phosphorylation of

the receptor subunits (Mammen & Huganir, 1997). On the other hand

increasing evidence suggests that regulatory proteins associated with NMDA

receptor complex play an important role in functional modulation of these

receptor channels. Several lines of evidence suggest that NMDA receptors

are closely associate and functionally modulated by cytoskeletal proteins.

For example Ca2+ - induced actin depolymerization reduces NMDA channel

activity (Rosenmund & Westbrook, 1993b). Also it has been reported that

NMDA receptors are mechanosensitive, so that the amplitude of the response

is modulated by changes in osmotic and hydrostatic pressure (Paoletti &

Ascher, 1994). Furthermore Zhang et al. (1996) have reported that stretch-

induced injury reduced the Mg2+ blockade of NMDA-evoked currents in

cultured cortical neurons by a PKC dependent mechanism. Both NR1 and

NR2 subunits interact with PSD95, a postsynaptic density protein, through

their carboxy-termini Kornau et al., 1996(Niethammer et al., 1996). Recently

it has been shown that recombinant proteins containing the carboxy-terminal

tail of NMDA receptor subunit NR2B interact with SAP102 from rat brain

homogenates (Muller et al., 1996). Subcellular distribution of NMDA

- " 1 A Y - - - - - -

dependent manner (Ehlers et al., 1995).

Homorneric NMDA receptors assembled £rom NRl splice variants

differ in their regulation by PKC (Durand et al., 1992; Tingley et al., 1993;

Ehlers et al., 1995). NRlloo display a sevenfold greater potentiation by PKC

than do NRloil receptors (Durand et al., 1993; Nakanishi et al., 1992). It

appears that the NI and C2 inserts are essential for the regulation of the

sensitivity to PKC activators (Durand et al., 1993). In fact splicing in the Cl

insert significantly reduces the potentiation by TPA (Durand et al., 1993),

however the majority of the phosphorylation sites for PKC are located within

the Cl insert. Also it is not clear why insertion of N I in the N-terminus,

which is located extracellularly, might regulate the sensitivity to potentiation

by PKC activating agents such as TPA.. Splicing out the N1 or C2 cassettes

has no noticeable effect on the phosphorylation pattern of NR1 protein

(Tingley et al., 1993). I t is possible that phosphorylation at C l sites reduces

the effect of phosphorylation a t other sites. Yet another possibility is that

potentiation of the homomeric NR1 receptor response by TPA is due to an

increased expression or translocation of the receptors at the ce11 surface. The

NRI splice variants XI0 and XOO (also the c-terminal of NR2 subunits)

contains a specific sequence (tSXV motif, where S is a serine, X is any amino

acid, and V is valine), that can specifically interact with the PDZ domains of

PSD95, a postsynaptic density protein (Kornau et al., 1995; Ehlers et al.,

1995). The association of NMDA receptors with PSD95 leads to aggregation

- - - - - -. - - * I - - - -, -

Hence insertion of C l cassette may result in maximal translocation of MMDA

receptors at the ce11 surface and hence reduced potentiation by TPA. Further

experiments are needed to clari& the exact nature of the potentiation by TPA

in oocytes expressing homomeric NR1 receptors.

Ca2+/calmodulin-dependent protein kinase II

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is

another serinekhreonine protein kinase that due to the evidence for its

regulatory role i n LTP has enjoyed considerable attention in the past few

years. CaM kinase II belongs to a family of calmodulin-dependent

serinelthreonine kinases consisting of substrate specific phosphorylase

kinase, myosine light chain kinase, and multifunctional CaM kinases 1, III,

IV and CaM kinase (Hanson & Schulman, 1992; Nairn & Picciotto, 1994).

The multifunctional CaM kinases have a wide substrate specificity and are

believed to be involved in signal transduction and other neuronal processes

such as neurotransmission, gene expression, axonal transport, neurite

outgrowth and plasticity (for review see Hanson & Schulman, 1992; Nairn &

Picciotto, 1994; Braun & Schulman, 1995; Williams et al., 1995). The

involvement of CaM kinase II in synaptic plasticity - LTP in particular - has

extensively been studied. Its wide distribution in the nervous system and

localization at the post-synaptic density, a putative locus for post-synaptic

suggest a n important role for CaM kinase II in LTP.

Regula t ion of CaM kinase II

Studies on the regulation of CaMK II have revealed that the kinase is

chronically inhibited by an autoinhibitory domain in the C-terminus (Colbran

e t al., 1988). The autoinhibitory domain interacts with the catalytic domain

at the N-terminus blocking the ATP binding and hence the catalytic activity

of the enzyme. Binding of Ca2+/calmodulin to a site adjacent to and

overlapping to the autoinhibitory domain weakens this interaction resulting

in dissociation of the inhibitory domain fkom the catalytic site and activation

of the enzyme (Colbran et al., 1988, 1989). The activated CaM kinase II has

a low affinity for calmodulin a t this state and the catalytic activity is

dependent on the presence of Ca2+/CaM, however, subsequent to the binding

of CaWCaM cornplex, CaMK II undergoes a rapid autophosphorylation on

Thr286 (a subunit, Thr287 on B subunit) which increases the affinity of the

enzyme for calmodulin by approximately 1000 fold (Meyer T. et al., 1992;

Schulman et al., 1992). CaMK II activity is now independent of the Ca2+

levels in the cell. This trapping of calmodulin has been suggested as a

mechanism by which the Ca2+ transients in the ce11 are coded (Hanson et al.,

1994; Schulman et al., 1992). After the dissociation of calmodulin the

- - - - - - - - - . - - -. - - - - -- - - - - - - Y

- ---- - - - ------ --------- --- --

phosphorylate the substrate in a Ca2+-independent fashion. This conversion

of the enzyme to a Ca2+-independent form has been suggested as a

mechanism for detection of Ca2+ signals in the ce11 (Meyer et al., 1992). The

enzyme then undergoes autophosphorylation a t two inhibitory sites, Thr305

and Thr 306, which prevent the activation of the enzyme by calmodulin (the

phosphorylation of the two inhibitory threonines prevents the binding of

calmodulin) locking the enzyme in the autonomously partially active state

until dephosphorylation (Lou & Schulman, 1989; Hanson & Schulman, 1992).

This constituitively active form also has a higher affinity for CaM-dependent

protein phosphatases 1, 2A, and 2C which in turn dephosphorylate the

enzyme back into the Ca2+/calmodulin-dependent form (Soderling, 199613).

Its wide distribution in the nervous system an localization to the PSD

strongly suggests that this enzyme plays an important role in the regulation

of the neurotransmitter receptors, hence neuronal function.

Modulation of glutamate receptors by CaM kinase II

The involvement of CaMK II in LTP has been extensively studied and

there is now considerable evidence suggesting an important role for this

enzyme in the generation and maintenance of elevated synaptic activity (see

Soderling TR., 1996 for review). However the exact role of CaMK II in the

enhancement of the excitatory neurotransmission (LTP) is not understood.

Glutamate receptors are likely targets for modulation by CaMK II of

- - a a

be highest a t known excitatory synapses in the CNS (Benson et al., 1992).

The a-subunit of CaMK II and glutamate receptors CO-localize in the PSD

(Kelly e t al., 1984; Kennedy e t al., 1983). Glutamate receptors are substrates

for CaMK II. AMPA-type GluR's purified form the PSD's of ra t forebrain are

phosphorylated by endogenous CaMK II (McGlade-McCulloh e t al., 1993).

Recombinant GluRl receptors are phosphorylated in vitro by CaMK II

(McGlade-McCulloh e t al., 1993). Further the phosphorylation sites for

CaMK II on GluRl has recently been identified to be Ser627 (Yakel et al.,

1995) and also that the site is conserved in GluR2-6 (Yakel et al., 1995;

McGlade-McCulloh et al., 1993).

There is also evidence tha t CaMK II functionally modulates glutamate

receptors. Introduction of the autophosphorylated form of CaMK II, which is

constituitively active, into cultured hippocampal neurons resulted in a

threefold increase in whole-ce11 kainate-activated currents (McGlade-

McCulloh et al., 1993). The responses of recombinant GluRl and GluR6 is

enhanced by the introduction of activated CaMK II through the patch pipette

(Yakel e t al., 1995) Similarly in the acutely isolated dorsal root ganglion

neurons, AMPA- and kainate-evoked currents are potentiated by application

of autophosphorylated CaMK II (Kolaj et al., 1994).

The modulation of NMDA receptors by CaMK II however is largely

unknown. Both the a subunit of CaMK II and NR2 subunit are major PSD

increased 32P labeling of CaMK II (autophosphorylation and activation) and

GluR's in cultured hippocampal neurons suggesting that the Ca2+ entering

through NMDA receptors can activate endogenous CaMK II (Tan e t al.,

1994). Also CaMK II may play a role in the expression of NMDA receptors as

inhibition of CaMK IIIIV by KN-62 depressed NMDA receptor expression in

cultured cerebellar granule cells (Resink et al., 1996b; Resink et al., 1996)

which in turn can regulate the contribution of NMDA receptors to synaptic

transmission. More direct evidence for functional regulation of NMDA

receptors by CaMK II is provided in two reports. Okumar et al. (1996) have

reported that the NR2B and NR2A subunits are substrate for CaMK II both

in vitro and in vivo. NR2B and possibly a cognate site in NR2A are

phosphorylated by CaMK II on Ser1303 in the carboxy-terminus of the

protein (Omkumar e t al., 1996). The functional modulation of NMDA

receptors by CaMK II i n DRG neurons was investigated by Kolaj et al.

(1994). The whole-ce11 NMDA-activated currents in acutely isolated DRG

neurons is enhanced by the introduction of autophosphorylated CaMK II into

the patch pipette (Kolaj e t al., 1994). Whether or not the NMDA receptors

are modulated by CaMK II in other regions of the brain where a different

complement of NMDA receptor subunits may be expressed is not yet clear.

- -

Phosphorylation by protein kinases such as PKC or CaMK II

represents the most widespread mechanism of modulating the function of ion

channels through second messenger signaling systems. Both PKC and CaMK

II have been shown to play an important role LTP. Functional modulation of

NMDA receptors could potentially play an important role in LTP and other

neurophysiological processes. Despite compelling evidence for modulation of

NMDA receptor function by PKC, the nature of this modulation remains

unclear. For example both PKC enhancement and depression of NMDA

receptor function have been reported in hippocampal neurons (Lozovaya &

Klee, 1995; Markram & Segal, 1994). The effects of CaMK II on NMDA

receptors in hippocampal neurons have not been examined up until

preparation of this manuscript. The general objective of this study was to

examine the effects of phosphorylation by PKC and CaMKII on hippocampal

NMDA receptors. We used patch-clamp recording technique and fast

application of agonist (NMDA) in cultured and acutely isolated hippocampal

neurons to examine the effects of PKC and CaMK II on NMDA-evoked

currents. The following questions were asked: Does PKC activity enhance

the magnitude of NMDA-evoked currents in hippocampal neurons? If PKC

enhances NMDA-evoked currents how is the enhancement reflected in the

single-channel activity of NMDA receptors? Chen and Hunag (1992) have

been reported that PKC decreased the affinity of NMDA channels for Mg2+

block in acutely isolated trigeminal neurons so we also wished to examine the

G L l C L b D U 1 i. .LIU U11 i~llt: 1VLÈ;- ù~llDI~lVlbJ' V I 1 Y 1 V I U n I'SL'ZYLU13 111 1LlppUL;LLILL~iTIl

neurons. Next we considered the modulation of NMDA receptors by CaMK

II. There is evidence suggesting that CaMK II enhances NMDA receptor

activity in spinal chord neurons (Kolaj e t al., 1994), however no reports to

date have examined the functional modulation of NMDA receptors in

hippocampal neurons. Thus we asked the question does CaMK II affect the

function of NMDA receptors in hippocampal neurons?

Given the important role played by NMDA receptors in LTP and other

neuronal processes, elucidating the mechanisms underlying the modulation

of this receptor by such serinelthreonine kinases is of paramount importance

to the understanding of the physiological function of the receptor.

-.-- ---- --

Hippocampal Cell Cultures

Cultures of fetal hippocampal neurons were prepared according to the

previously described techniques (MacDonald et al., 1989). Briefly, time-

pregnant mice were sacrificed by cervical dislocation. Fetuses were removed

and hippocampi were microdissected and placed in cold Hanks' solution. The

hippocampi were then mechanically dissociated by trituration and plated in

35 mm collagen-coated culture dishes a t densities below 1 x 106 ml-'. The

cells were grown in dissociated tissue culture using standard techniques

(MacDonald e t al., 1987). The cultures were used 12 to 17 days after plating

for electrophysiological recordings. The culture media was replaced with

extracellular solution prior to recording.

Preparation of acutely isolated CA1 neurons

Acutely isolated hippocampal neurons were obtained a s described

previously (Mody et al., 1989). Rats (approximately 2 weeks old) were

sacrificed by decapitation using a guillotine. The whole brain was removed

and placed in cold extracellular solution (see below for composition).

Hippocampi were then microdissected and cut into 400-500 pm thick slices by

hand using a razor blade. The slices were incubated in extracellular solution

containing 0.3 - 0.5 mglml papain (from papaya latex, Sigma) at room

temperature for 30 minutes. Al1 solutions were bubbled with a carbogen

\--A V I V , V A V U I V / a . & A A r O i V U & b . I. V & L V V l A A A b V A A U & A A U U U U U A V A A 1 V A V A A W A A L b A A U J III- V L L b

slices were washed and kept in enzyme free extracellular solution until used.

To obtain dissociated neurons from hippocampal slices one slice was

transferred to a 35 mm culture dish containing 2 ml of extracellular solution.

For isolation of CA1 neurons, one or two slices were transferred into a 35 mm

culture dish containing 2 ml of extracellular solution and the dish was placed

on the stage of a n inverted phase-contrast microscope (Nikon or Olympus).

Using two fire polished (round-tipped) glass pipettes, the CA1 region of the

hippocampal slices were cut out an mechanically abraded to obtain single

cells. Electrophysiological recording of the isolated neurons began

approximately 15 minutes after the mechanical dissociation.

Electrophysiological recording

Whole-ce11 recording

Whole-ce11 voltage clamp recordings were performed using the patch

clamp technique (Neher & Sakmann, 1992) on cultured and acutely isolated

hippocampal neurons. Whole-ce11 recording, the most common configuration

of patch clamp recording, allows on to record fiom ce11 while modiSring their

interna1 environment by the recording pipette's solution. Hence, for instance,

to study the modulation of ligand-gated ion channels by protein

phosphorylation, substances such as protein kinases or their modulators can

recording ligand-activated currents in the cell.

Patch electrodes were constructed fkom thin-walled borosilicate glass

(1.5 mm diameter, WPI) on a two stage puller (PPS3, Narishige). The tips of

the e1ectrod.e~ were heat polished on a Narishige microforge (Scientific

Instruments Laboratory, Tokyo, Japan, Mode1 MF-83) to a final diameter of 1

- 2 Fm. The patch electrodes had resistance between 3 to 5 MW. The whole-

ce11 currents were recorded using Axopatch 1-D or Axopatch 200A (Axon

Instruments) amplifiers in the voltage-clamp mode. Data were filtered a t 2

KHz and digitized on-line using either the TL-1 or Digidata 1200 DAC units

(Axon Instruments). The on-line acquisition was done using pClamp

software (ver. 6.03, Axon Instruments) running on a P66 based PC.

In order to secure a rapid and robust exchange of solution around the

cells, multi-barre1 fast perfusion systems were employed (manufacturer of the

second system). In majority of experiments a house-made fast perfusion

system was employed (Johnson and Ascher 1987, Wang & MacDonald 1995).

Three square capillary tubes (400 x 400 pm) were glued together and

mounted on a modified Leitz manipulator (see references for details). The

ce11 being recorded from was continuously perfused with regular extracellular

solution by aligning one of the barrels (containing control solution) with the

ce11 (placed within 100-200 pm of the cell). The barre1 assembly then could be

moved rapidly in the lateral plane using a computer-driven stepper motor, in

- -

containing the desired test solution aligned with the cell. In the experiments

done to determine the baseline ~ ~ 2 + sensitivity of the cultured hippocampal

neurons a similar perfusion system was used (Manufacturer). The

movements of the barrels in the lateral plane were controlled by the pClamp

software. The pClamp software was programmed to output a waveform

which in turn was used to control the movements of the barre1 assembly.

In al1 experiments (unless otherwise stated) the neurons were voltage-

clamped at -60 mV. During some experiments a voltage pulse of amplitude -

10 or -20 mV was applied prior to each application of the agonist to monitor

the ce11 capacitance and the access resistance. The recordings in which the

access resistance or the capacitance changed by more than 10 percent were

not included in data analysis.

Intracellular Perfusion

The intracellular perfusion was carried out as described previously

(MacDonald et al., 1994). Conventional patch-electrodes were constructed

(see above) from 0.5 mm 0.d. thin walled glass tubing (TW 150F-4, WPI) and

modified by creating an expansion a t the shank near the tip of the electrodes

(MacDonald et al., 1994, see Figure 2). The "bubble" in the taper was created

within 100 Pm of the tip of the electrode. This expansion allowed for the

An schematic drawing of the intracellular perfusion apparatus. The

Drawing is not to scale. The tip of the internal electrode is placed within

100 p m of the tip of the recording pipette. A fast perfusion system

consisting of 3 square glass capillary tubes glued together and placed

aligned with the ce11 being recorded. A stepper motor moves the barrels in

the lateral plane, placing the next barre1 aligned with the cell. The

internal perfusion is achieved by perfusing the test solution through the

internal pipette into the recording pipette. Vc sets the command potential

in the voltage clamp circuitry.

" - * * * a

the patch-electrode, also it reduced the possible built-up of pressure in the

patch-electrode during the perfusion by allowing better drainage of excess

fluid Fom the tapered area of the patch-electrode.

The internal pipette was constructed from 0.75 mm 0.d. thick-walled

glass tubing. The pipettes were pulled on a one-stage electrode puller (David

Kopf Instruments, Mode1 700C) to a relatively long taper (1-1.5 cm). The tip

of the internal pipette was broken down to a diameter of 10 -15 Pm. A

modified electrode holder (MacDonald et al., 1994) was used to hold the

internal pipette inside the patch-electrode. The position of the internal

pipette was adjusted under a microscope to place the tip of the internal

pipette within the "bubble" near the tip of the patch-electrode.

The internal pipette was then connected to the perfusion assembly.

The perfusion assembly consisted of a piece of polyethylene tubing that was

connected to a lcc syringe (pressure trap). The syringe was connected to a

picospritzer II (General Valve Corp., Hanover NJ). The polyethylene tubing

was filled with the desired test solution and connected to the internal pipette.

The solution could be forced out of the tubing by applying pressure through

the 1 cc syringe (via the picospritzer). Both the internal pipette and the

patch-electrode were first filled with regular intracellular solution . Once the

holder was mounted on the headstage the perfusion tubing was connected to

the internal pipette and clamped off. After whole-ce11 recording had begun,

- - - - w

applying pressure through the syringe (via the picospritzer). The pressure

was adjusted (to a maximum of 15 psi) to give a steady rate of perfusion of 1 -

3 pL per minute. The excess solution drained through the suction port.

Single channel recording

Single channel recordings were done in the inside-out configuration of

patch clamp recording (Harnill et al. 1981) fiom cultured hippocampal

neurons. Patch electrodes with resistance between 4 - 8 M a were

manufactured as described above. The pipette shanks were then coated with

Sylgard (Sigma, pre-mixed and kept at -20 O C ) using a small glass pipette, to

within 100 p m of the tip. The Sylgard was subsequently cured by holding the

pipette shank in a stream of hot air fiom a heat gun (Master-Mite, USA) for

10 to 15 seconds. The pipettes were then placed on a microforge and the tips

were heat polished to a final diameter of 0.7 to 1.0 Pm.

Single-channel events were either captured on-line or were first

recorded on video tape using a digital data recorder (VR-10, Instrutech Corp.,

Mineola, NY) and later acquired using pClamp6 software (Axon

Instruments). The single-channel currents were filtered at 2 kHz and

sampled at 5 kHz. The records were analysed using the pClamp software.

The records were analyzed using pClamp 6.0 (Axon Instruments).

Materials

For whole-ce11 recordings the extracellular solution contained (in mM):

140 NaCl, 5.4 KCl, 25 HEPES, 33 glucose, 1.3 Ca2+, 0.003 glycine, and 0.001

tetrodotoxin (pH = 7.3-7.4, 320 - 35 mOsm). The solution used for acute

isolation of hippocampal neurons contained 4.0 mM Mg2+ to reduce the

excitotoxic effects of glutamate released during dissection of tissue. The

recording solution contained (in mM): 140 KC1, 10 HEPES, 5 EGTA, 2

tetraethylammonium chloride (TEA), 1 CaCl2 , and 4 K+-ATP (pH=7.5).

Inside-out patch recordings were carried out in regular recording solution

The patch pipette was fïlled with regular extracellular fluid containing 10 -

15 pM NMDA and 3 p M glycine. MgCl2 and NMDA were added as stated in

the text fiom stock solutions.

PKCM (the catalytic fragment of PKC) was prepared by E. Dudek and

M. Browningas as described (Wang et al., 1994). Protein kinase C was

purified Tom rat brain as described (Roth et al., 1989) using sequential

chromatography on diethyl aminoethyl (DEAE)-cellulose, phenyl sepharose

and protamine agarose. The catalytically active fragment of PKC was

prepared by digestion with trypsin as described by Huang & Huang (1986).

After trypsinization the 45 kDa catalytic fragment (PKCM) was purified by

chromatography on DEAE-52 or by soy bean trypsin inhibitor affinity

chromatography. The PKCM fractions which exhibited phosphorylating

concentrated and dialysed against the recording buffer. The samples were

frozen in small aliquots and thawed just before use. The specific activity of

PKCM was 1-2 pmolh idmg using Histone III-S a s substrate. The PKCM

solution for recording was prepared by a 2:l dilution of the stock solution

(PKCM dialysed against regular intracellular solution) with regular

intracellular solution containing 8 mM K+-ATP to a final concentration of 2

p M PKCM and 4 mM K+-ATP. To prevent hydrolysis of ATP al1 intracellular

solutions containing ATP were kept on ice before filling the electrodes.

The PKC inhibitory peptide, PKCI (PKC (19-36)), was purchased from

CalBiochem (and was used a t 10 p M concentration dissolved in regular

recording solution.

CaMKat (the catalytic fragment of CaMK II) was prepared by E.

Dudek and M.D. Browning).

--------

NMDA-evoked currents in hippocampal neurons

The major advances in understanding the pharmacology of NMDA

receptors came about because of the availability of specific agonist and

antagonists for this receptor. The NMDA receptor, named after its selective

agonist NMDA, is present on the soma and dendrites in both cultured and

native hippocampal neurons (Mattson et al., 1991). Inward NMDA-evoked

currents could routinely be elicited by using a fast application system in both

cultured and acutely dissociated hippocampal neurons. Sample currents are

shown in Figure 3 (top). At -60 mV the amplitude of the NMDA-activated

currents exhibited considerable variability form one neuron to another.

NMDA is a specific agonist for the NMDA receptors and does not interact

with any of the other excitatory amino acid (glutamate) receptors so that

application of NMDA activated NMDA receptors only (Patneau & Mayer,

1990). The NMDA-evoked currents observed were most likely not

contaminated by voltage-activated currents since the neurons were voltage

clamped a t -60 mV, also agents such as TTX (0.3 pM in ECF) and TEA

(tetraethylammonium chloride 2 mM in ICF) were used to block these

currents.

-

cu l tu red h ippocampal neurons.

The majority of the studies on the effects of PRC on NMDA receptors

have used agents such as phorbol esters to activate endogenous PKC's.

However the effects of phorbol esters on NMDA-evoked currents remain

unclear as both enhancement and depression of these currents have been

reported (Markram & Segal, 1992; Aniksztejn e t al., 1992; Courtney &

Nicholls, 1992). For example Markram and Segal (1992) have observed a

depression of NMDA-evoked currents in the CA1 neurons by topical

application of phorbol ester on hippocampal slices whereas Aniksztejn et al.

(1992) have reported a n up-regulation of NMDA currents in these neurons.

Several possible explanations may exist for this apparent discrepancy. For

example phorbol esters may have influenced the channel directly a t higher

concentrations (Hockerberger et al., 1989). Differences among various

phorbol esters in their specificity of action on PKC's have also been described

(Liu et al., 1993). Also differences in basal levels of endogenous PKC or the

particular isoforms expressed by different ce11 types may be responsible for

the inconsistencies observed. Considering that the effects of indirect

activation of endogenous PKC are uncertain we used PKCM, a constitutively

active form of PKC, to study the effects of direct intracellular application of

PKC on whole ce11 NMDA-evoked currents. PKCM is the catalytic fragment

of PKC which, unlike the whole enzyme, does not require Ca2+ or

phospholipids for its activation. PKCM has previously been used to study the

V - - - -. -- - - -- - - - - . - . - - - -

glutamate receptors (Hall et al., 1995; Wang et al., 1994).

In order to study the modulation of NMDA receptors by PKC in

cultured hippocampal neurons, we used an intracellular perfusion technique

(see methods) to introduce PKCM directly inside the ce11 through the

recording pipette. This method is advantageous since the NMDA responses

can be monitored before and afier application of PKCM, and as a result each

ceIl can be used as its own control reducing the effects of variability of the

responses observed in different cells.

Patch clamp recordings in whole-ce11 configuration, modified for the

internal perfusion technique (see methods), were performed in 14 - 21 days'

old cultured hippocampal neurons. Using the intracellular perfusion

technique PKCM was introduced inside the recording pipette while

continuously recording whole-ce11 current responses to rapid applications of

NMDA. NMDA solution was applied a t 1 minute interval following the patch

rupture. The patch pipette contained Mg2+ (6 mM) and ATP (4 mM) as they

are both required for the phosphorylating activity of PKCM. NMDA-evoked

currents recorded immediately prior to the application of PKCM were used as

controls to normalize the responses recorded following the perfusion of

PKCM. NMDA-evoked currents exhibited some degree of wash-out after the

start of the recording but stabilized in 10 - 15 minutes. The internal

perfusion was started only after NMDA currents had stabilized. The

amplitudes of these currents were on average 1600 * 200 pA (n=14), prior to

ï,llC iIppllGtZiAUl1 Ul 1 ~1UlV.i.. A11 L'lgUL-C 13 Llle lilt5ZlllS L C Y U l V L V l LHk! JJe?ï& U l LIlk!

NMDA-evoked currents normalized to the current amplitude prior to the

application of PKCM are plotted against time, for 11 neurons treated with

PKCM and 6 neurons treated with the vehicle (control). Representative

current records before and after application of PKCM are shown in Figure 3

(bottom). The internal perfusion of PKCM (2 PM) resulted in the

enhancement of NMDA-evoked currents to 122 f 5 % of the response prior to

perfusion of PKCM in 11 out of 15 cells tested. (Figure 3). This enhancement

of the NMDA currents began approximately 3 minutes after the start of the

perfusion and reached a peak in 10 to 15 min. In the other four cells tested,

NMDA currents did not increase after the perfusion of PKCM but slightly ran

down (data not shown). With the inclusion of the 4 cells in the analysis the

average enhancement of NMDA-evoked currents was 18 k 5 % which was

significantly different than the control (p < 0.05, two-way ANOVA).

It is not clear why the perfusion of PKCM had no apparent effect in

these cells. Although utmost care was taken during the assembly of the

intracellular perfusion system (see methods) to ensure that the tip of the

internal pipette was not broken or blocked and that it was placed near the tip

of the patch pipette it is not inconceivable that perfusion of PKCM was

somehow hindered in those recordings. Yet the lack of PKCM effect in these

cells may also have been due to higher basal PKC phosphorylation levels in

these cells. For example NMDA receptors (or associated proteins) may have

A .--a. v V. A A A V VLIYLIIJ Y A W L A U b A A A V L A U V A -C A L V \I 1 X V A l L j G 1 1 1 1 U A A b G 3

NMDA-evoked currents in cultured hippocampal neurons.

Top) Whole-ce11 recordings of the peak NMDA (100 FM)-evoked currents

before and during the interna1 perfusion of PKCM (2 pM ) or control

(dialysis solution for PKCM, see methods). The responses are normalized

to the response prior to the s tar t of internal perfusion and the means f

SEM for 11 neurons are depicted. NMDA-evoked currents were

significantly potentiated by application of PKCM (122 k 5 % of the

response prior to application of PKCM, n = 11, pc0.05 two-factor ANOVA).

Interna1 perfusion of control did not enhance the currents (n = 6) .

Bottom) Representative current traces elicited by a fast application of

NMDA prior to and after internal perfusion of PKCM. The holding

potential for this and subsequent figures is -60 mV, unless otherwise

stated. The horizontal bar in A depicts the onset, the duration and the

offset of the internal perfusion.

1 0 PKCM (n=l l )

PKCM I I 1 1 '7

-4 -2 O 2 4 6 8 10 12 14 16 18 20

Time (min)

NMDA

been highly phosphorylated by endogenous PKC so that application of PKCM

did not have any further effects on the function of these receptors.

Next we conducted control experiments in which the solution used to

dialyze the kinase (see methods) was intracellularly perfused during the

whole ce11 recordings. The control solution did not enhance NMDA-evoked

currents (Figure 3) and the effects of PKCM were significantly different fi-om

that of the control (control) (~~0.05, two factor ANOVA with treatment and

time as factors). This result indicates that the effects observed were due to

perfusion of PKCM itself and not the recording conditions used.

Enhancement of NMDA-evoked currents in acutely isolated CA1

neurons

Subsequently we examined the effects of PKCM on NMDA-evoked

currents in acutely dissociated CA1 neurons. This preparation has routinely

been used in our laboratory and the NMDA-activated currents have been

shown to have similar characteristics as those in cultured hippocampal

neurons (Mody et al., 1989; Wang & MacDonald, 1995). Cultured neurons

however are embryonic neurons hence they are not fully differentiated when

they are placed in dissociated culture. The acute dissociation technique

allows isolation of adult cells (presumably fully differentiated) from small

defined brain regions. Considering that the expression of the NMDA receptor

subunits is developmentally regulated we also looked a t the modulation of

NMDA receptors in acutely dissociated neurons. Also acutely isolated

- - reducing the space clamp problems (Kay and Wong 1986). In these

experiments the neurons were isolated from the CA1 region of hippocampus

as this area has been the focus of many studies on LTP and the pharmacology

of the neurons (pyramidal) in this area has been well characterized.

Whole-ce11 recordings were performed in cells obtained nom the CA1

area of ra t hippocampus. In these experiments PRCM was included in the

intracellular solution and comparisons were made to the control recordings in

which the only the dialysis solution for PKCM (with ATP and Mg2+ added)

was included in the patch pipette. The current amplitudes were normalizes

to the first response recorded at the start of the recording. Recordings made

with regular ICF (control) in the pipette exhibited a rnoderate 'run-down'

with the current amplitude gradually decreasing throughout the recording

(88.5k4.8 % of the first response tested after the breakthrough, 12 minutes

following the start of the recording). Inclusion of PKCM (2 PM) in the

recording pipette however consistently resulted in a "run-up" of NMDA-

induced currents in acutely isolated CA1 neurons. NMDA-evoked currents

were enhanced to 126 * 9.8 % of the current responses recorded 1 minute

following the breakthrough (n = 15, 12 minutes following the start of the

recording) (Fig 4). This enhancement was gradual, reaching a plateau 7 to 8

minutes after the start of the recording (Fig 4).

-

acutely dissociated CA1 neurons.

Top: Representative current traces from a CA1 ce11 with PKCM in the

pipette 1 min and 15 min after the start of the recording (patch rupture).

Bottom: Time course of whole-ce11 peak NMDA-evoked currents in acutely

dissociated CA1 neurons with the intracellular solution containing 0.4 pM

PKCM (n = 15) or control (n = 14; 4 mM K+ ATP and 6 mM Mg2+). The

current amplitudes are normalized to the amplitude of the first current

recorded after the rupture of the patch and the data are presented as

means f SEM. PKCM enhanced NMDA-evoked currents by 126 k 10 %

whereas in control recordings the currents decreased by 88.5 +_ 4.8 % 12

minutes after the start of recording (p<0.05, Student's t-test).

PKCM 1 min 15 min

2 sec

PKCM Control

Time (min)

PKCI abolished t h e PKCM enhancement of the NMDA-evoked

cu r r en t s

In these experiments PKCI (PKC (19-36)) 10 PM, a potent inhibitor of

PKCM, was used to determine if the effects of PKCM were due to its

phosphorylating activity. PKCI is the peptide fragment corresponding to the

autoinhibitory (pseudo-substrate) domain of PKC which interacts with the

catalytic domain including the active site of the enzyme hence inhibiting its

phosphorylating action (House & Kemp 1987). PKCI has been shown to be

highly specific for PKC a t low micromolar concentrations (IC50 = 0.3 PM).

PKCI is an extremely poor inhibitor of PKA (IC50 > 60 PM) and can

moderately inhibit CaMK II at higher concentrations (I& = 30 PM) (Smith

et al., 1991).

Co-application of PKCI with PKCM completely prevented the PKCM

enhancement of NMDA-evoked currents in both cultured and acutely isolated

hippocampal neurons. Interna1 perfusion of PKCI (10 PM) and PKCM (2 PM)

(in regular ICF) in cultured neurons failed to potentiate NMDA-evoked

currents but the currents exhibited a moderate run-down following the

perfusion of the mixture (Figure 5). This depression of the currents however

was not significantly different from that observed in the control (p0.05, two-

factor ANOVA). Similarly, in acutely isolated CA1 neurons, inclusion of

PKCI (10 FM) along with PKCM (2 PM) in the intracellular solution

abolished the PKCM-mediated potentiation (n = 6, two-factor ANOVA).

Left: The peak whole-ce11 NMDA-evoked currents before and during the

internal perfusion of a mixture PKCM (2 PM) and PKCI (10 PM) in

cultured hippocampal neurons are plotted. Co-application of PKCI totally

blocked the PKCM enhancement of the currents. Application of PKCI (10

PM) by itself resulted in a significant depression of control currents (84 If: 5

%, p< 0.05 two-factor ANOVA).

Right: The peak responses of acutely dissociated neurons to NMDA in the

presence of PKCI (10 FM) and PKCM (2 PM) mixture or PKCI (10 PM)

alone in the pipette. PKCI blocked the PKCM enhancement (n = 6).

Application of PKCI alone caused a moderate inhibition of the currents (n

= 5). The horizontal bar in A represents the onset, the duration and the

offset internal perfusion.

V Y ' - - - - - -

I - d ----- - \ - - r - . - i ---.- ----

added) lead to a significant inhibition of NMDA-evoked currents in cultured

and acutely dissociated hippocampal neurons. In culture hippocampal

neurons NMDA-evoked currents were depressed by 13 f 5.0 % (n = 4, 15

minutes after start of perfusion) (Figure 5). Also in acutely dissociated CA1

neurons, inclusion of PKCI (10 PM) by itself reduced NMDA-evoked currents

by 24.2 + 6.9 % compared to the first response recorded following the

breakthrough (15 minutes after start of the recording, n = 5, p< 0.05, t-test)

(Fig 5). The reduction associated with PKCI is most likely due to inhibition

of endogenous PKC.

Application of PKCM increased the activity of NMDA single channels

To further examine the mechanism of PKCM enhancement of NMDA-

evoked currents, NMDA single-channel recordings were carried out in inside-

out patches excised from cultured hippocampal neurons. In the excised

inside-out patches the modulation of channels by substances applied to the

cytoplasrnic side of the patch can be readily studied. PKCM, PKCI or both

were dissolved in ICF containing 4 mM ATP and 6 mM Mg2+ (see methods)

and were applied to the cytoplasmic side of the patch. The patch pipette

contained low concentrations of NMDA (10 FM) and saturating

concentrations of glycine (3 PM) with no added Mgzc in order to minimize

desensitization of NMDA channels and ensure distinct channel activation

\UAIJU w VVI~UIIUUII, ~ r l d ~ , u l u u CYi ~ U ~ ~ u l l U U l l , AQCJA, V V i X 1 1 g C;.b #l., i; l;7Lt). lile

cytoplasmic side of the patch was continuously perfused with ICF containing

4 mM ATP and 6 mM Mg? Under these conditions, assuming a n NMDA

reversa1 potential of -OmV, NMDA-activated channels exhibited a main

conductance level of approximately 38 pS at -80 mV patch holding potential

(+80 pipette potential). Transitions to subconductance levels were

occasionally observed however they were not analyzed due to their poor

resolution. Application of PKCM (0.8 PM) to the cytoplasmic side of the patch

increased the single-channel open probability by 71% (Popen = 0.021 f 0.008

before and 0.057 + 0.02 after application PKCM, p < 0.05 Wilcoxon signed

rank test, n = 7, Figure 6), without affecting the single-channel conductance

(37.8 f 2.5 pS before and 38.6 f 3.0 after application of PKCM, n = 6,

p>0.005). The open times (G and zi) were not significantly affected by PKCM

(see Figure 7) however the average duration of channel closings were

significantly reduced. The long closed times (ri) was reduced by 55 % after

application of PKCM, reflecting the increase in the open probability (n = 4, p

< 0.05, paired t-test, Figure 7). Co-application of PKCI and PKCM did not

have any discernible effects on the channel activity in two patches tested (not

shown).

We applied PKCI alone to two patches (data not shown) and the effects

on the single channel conductance, open probability or average open time of

L. - - - - - - - - -

Y - - - - - - -

patches.

Top: Single-channel currents before (control, on the left) and during (on

the right) application of 0.3 pM PKCM. The channel conductance was not

affected by PKCM (40 pS)

Bottom: A continuous record of NMDA channel open probability (Popen)

before (left) and during application of PKCM. The mean open probability

of the channe1 was 0.043 before and 0.100 during application of PKCM.

The holding potential for the inside-out patch was -80 mV (+80 mV pipette

potential). The bin width for Popen calculations was 0.4 ms.

Representative dwell-time histograms of open and shut times before (left)

and during application of PKCM (right) are depicted. The distribution of

open times and closed times were fitted with two and three Gaussian

components. The solid line represent the sum of the components and N is

the number of events. The time constants (parameters) for the fits are

given in the diagram. Application of PKCM did not signifïcantly affect the

distribution of open times (Before zl= 0.38 f 0.07 and ~2 = 2.19 f 0.47 ms;

After TI= 0.41 f 0.14 and 22 = 2.06 10.64, p > 0.05, n=4, paired t-test).

The long closed times however were significantly reduced by application of

PKCM (before rl= 0.74 + 0.19, .c2= 10.94 f 3.62 and 2% 215.2 + 32.61;

after 21= 0.62 1 0.2, 22= 7.73I 2.61 and r3= 118.13 k 17 ms, ~3 was

significantly reduced p < 0.05, n= 4, paired t-test).

Gr"

A "

by 40%. This patch was estimated to contain 3 channels and hence was not

analyzed further. In the other patch PKCI had no discernible effect on

channel activity. This lack of effect of PKCI on the single-channel activity

may be due to the absence of endogenous PKC in the patch.

PKCM modulation of Mg2+ sensitivity of NMDA-evoked currents in

hippocampal neurons

Baseline sensitivity of NMDA-evoked currents to Mg2+ in cultured

hippocampal neurons

In our laboratory we had routinely observed that NMDA-activated

currents in cultured hippocampal neurons exhibit a much lower sensitivity to

Mg2+ block than the currents evoked in acutely dissociated neurons. In these

experiments we looked to quanti@ the Mg2+ sensitivity of NMDA-evoked

currents in Our cultured hippocarnpal neurons. Whole-ce11 voltage clamp

recordings were performed and the neurons were clamped at -60 mV. NMDA

(50 or 100 PM) was applied using a fast perfusion system a t 1 minute

intervals. The data were collected only after the currents had stabilized (the

cells exhibited some degree of run-down despite presence of ATP in the

recording pipette). The dose-response data were obtained by varying the

concentration of Mgz+ (50 - 1000 PM) in the NMDA solution or alternatively

- - - * * . - 8 ). The experiments were conducted in two different agonist concentrations

(50, 100 pM NMDA) however the results were combined as there was no

significant difference between the data obtained using either concentration

with any of the two protocols. The dose-response data obtained using either

protocol with 50 or 100 pM NMDA were not significantly different hence the

data were combined and analysed.

In one set of experiments the recording pipette was filled with ICF

containing 11 mM EGTA and 1 mM Ca2+. Whole-cell NMDA (100 or 50 p M

NMDA and 10 FM glycine) currents recorded in cultured neurons exhibited

sensitivities to Mg2+ blockade ranging fkom (Ic50 in PM) 48.2 to 884 with a

mean IC50 of 275 f 67 pM and Hill coefficient of 1.2 f 0.1 (n = 6) (Figure 8).

This value is considerably higher than that observed in acutely dissociated

hippocampal (see below) or trigeminal neurons (Chen & Huang 1992). In

order to determine if the rise in the intracellular Ca2+ and the consequent

inactivation of NMDA channels affects the Mg2+ sensitivity of these neurons,

experiments were also conducted in which the pipette solution contained 10

mM BAPTA, a high capacity fast Ca2+ chelator, and O added Ca2+ (BAPTA

ICF). Whole-ce11 NMDA currents recorded using BAPTA ICF exhibited Mg2+

sensitivities similar to those obtained using EGTA ICF (Figure 8 ). The IC50

hippocampal neurons.

A. Representative current traces r o m the dose-response data. Dose-

response data were obtained by application of Mg2+ of various

concentrations superimposed on a long NMDA application (top) or,

alternatively, by simultaneous application of Mg2+ and NMDA (bottom).

B. The effect of intracellular Ca2+ buffering on the Mg2+ sensitivity of

NMDA-evoked currents. The graph shows the dose-response relationship

for the Mgz+blockade of NMDA-evoked currents in cultured hippocampal

neurons with either EGTA (left) or BAPTA (right) as the main Ca2+ buffer

in the recording pipette. The dose-response curves were fitted using the

logistic equation in the for 1 = 1- (1 - 11(1+ I C ~ O / W ~ ~ + ] ) ~ ~ ) , where 1- is

the NMDA-evoked current at O mM external Mg2+ (no blockade), ICS0 is

the concentration of Mg2+ that blocks 50% of I w and nH is the Hill

coefficient. The extent of Ca2+ did not significantly affect the Mg2+

sensitivity of the NMDA evoked currents (ICMJ = 304.2 + 99 with EGTA

and 244.8 + 63 with BAPTA, p> 0.05 Student's t-test).

PKCM did not change the apparent affinity of NMDA receptors for

Mg2+

In this series of experiments the modulation of the Mgz+ block of

NMDA-evoked currents by PKCM was investigated. In cultured hippocampal

neurons the dose-inhibition data, before and during the internal perfusion of

PKC, were collected by varying the concentration of Mg2+ in the agonist

solution (NMDA 100 PM) between O to 1000 mM. The dose-inhibition data

were collected 10 to 15 minutes following the start of the internal perfusion of

PKCM when the enhancement was at its peak. These recordings were made

a t a holding potential of -60mV. The IC50 for the Mg2+ block of NMDA-evoked

currents prior to application of PKCM was 489 + 180 PM, n = 7. Intracellular

application of PKCM did not alter the apparent sensitivity of NMDA-evoked

currents to Mg2+ block (IC50 = 519 + 183 pM after application of PKCM, n = 7,

Figure 9). There was no significant change in the Hill coefficient for the dose-

inhibition curve before and after the perfusion of PKCM (0.87 + 0.04 before

compared to 0.92 t- 0.07 after intracellular perfusion of PKCM, p > 0.05

Wilcoxon Signed Rank test).

The NMDA-evoked currents in acutely dissociated CA1 neurons

exhibited a much higher and more consistent sensitivity to Mg2+ block than

evoked currents

Right: The dose-response relationship for the Mg2+ block of NMDA-evoked

currents in cultured and dissociated hippocampal neurons are shown

(left). The dose-response curves were fitted as in Figure 8. Intracellular

application of PKCM did not significantly affect the Mg2+ sensitivity of the

NMDA-evoked currents (n=7, p>0.05 t-test). The Ic50 value was 489 1:

180 PM before (control) and 519 + 183 pM during application of PKCM.

The Hill coefficients were 0.87 + 0.04 before and 0.92 I 0.07 after PKCM.

The Mg2+ sensitivity of NMDA-evoked currents in acutely dissociated

neurons was also not affected by inclusion of PKCM in the pipette (ICSo=

68.5 ?I 11.9 pM with control and 60.0 k 7.9 pM with PKCM, n=6, pz0.05 t-

test).

Left: The IC5o values for Mg2+ block of NMDA-evoked currents were

significantly lower in dissociated CA1 neurons than the values in the

cultured hippocampal neurons (pC0.05, t-test) in both control and PKCM

treated neurons.

Inhibition (%)

ru A 6, O

00 O O O

V

evoked currents a t -60 mV ranged from 40 to 118 pM with a mean of 69 k 12

pM (n=6). Inclusion of PKCM in the recording pipette did not affect this

apparent affinity OC50 = 60 f 8.0, n = 6, p>0.05 t-test, Figure 9).

Potentiation of NMDA currents by CaMKat.

mole-ce11 recordings were performed in cultured hippocampal

neurons to determine the effects on NMDA-evoked currents the of

intracellular perfusion of CaMKat II. NMDA currents were monitored before

and after the application of CaMKat. Intracellular perfusion of CaMKatII

caused a gradua1 enhancement of currents reaching 122 f 5 % (n = 8) of the

control (before application of CaMKat) in cultured hippocampal neurons

(Figure 10). Intracellular application of the control (see methods) solution

resulted in a small potentiation of NMDA current (3.7 f 6 %, n = 6) (Figure

10). However i t was found later that the control solution had some residual

kinase activity (see methods), nevertheless CaMKatII enhancement was

significantly different fkom that of the mock @ < 0.05, t-test).

- -

evoked c u r r e n t s in c u l t u r e d hippocampal neurons.

Top) Whole-ce11 recordings of the peak NMDA (100 PM)-evoked currents

before and during the internal perfusion of CaMKat (2 p M ) or control (see

methods). The responses are normalized to the response prior to the start

of internal perfusion and the means f SEM for 6 neurons are depicted.

NMDA-evoked currents were significantly potentiated by application of

CaMKat (123 + 5.5% of the response prior to application of CamKatII, n =

6, p<0.05 two-factor ANOVA). Interna1 perfusion of control enhanced the

currents by 103.7 f 6.1 %, (n-= 6).

Bottom, Representative current traces elicited by a fast application of

NMDA prior to, 6 minutes and 10 minutes after the s tar t of internal

perfusion of CamKat. The horizontal bar depicts the onset, t he duration

and the offset of the internal perfusion.

Control

CaMKat @r

Interna1 perfusion

-6 -4 -2 O 2 4 6 8 I O 12 14 16

Time (min)

CaMKat 6 min CaMKat 10 min

2 sec

Discussion

PKC enhancement of NMDA-evoked currents

The results presented here demonstrate that PKCM enhances NMDA-

evoked currents in cultured and acutely dissociated hippocampal neurons. In

cultured hippocampal neurons intracellular perfusion of PKCM potentiated

the NMDA-evoked currents by 22 %. Co-perfusion of PKCI and PKCM did

not enhance the currents. Similarly, inclusion of PKCM in the recording

pipette resulted in a 26% enhancement of NMDA-evoked currents in acutely

isolated CA1 neurons, and this enhancement was not observed when PKCI

was also included in the pipette solution. The application of PKCI alone

resulted in a depression of the NMDA-evoked currents in both dissociated

and cultured hippocampal neuron (24% and 13% respectively). These results

are consistent with the previous reports on the enhancement of NMDA-

evoked currents in hippocampal neurons by PKC (Wang e t al., 1994; Ben-Ari

e t al., 1992). In trigeminal neurons intracellular perfusion of PKC (whole

enzyme) potentiated the NMDA receptor function by 30% in the absence of

extracellular Mg2+ (Chen & Huang, 1992). I n that study however, in order to

boost the activity of the PKC whole enzyme, diolein and was included in the

perfusate solution and intracellular Ca2+ was buffered a t a high level (Chen

& Huang 1992). Although these conditions boost the activity of the PKC

whole enzyme in the perfusate, they presumably could also activate the

eriuugenuus rhbs. m s o ir; nas Deen snown mar; inn-aceuular calcium can

influence the state of actin polymerization which may affect the activity of

NMDA channels (Rosenmund & Westbrook, 1993a; Whatley & Harris, 1996;

Paoletti & Ascher, 1994).

The extent of potentiation of NMDA receptor function in hippocampal

neurons by PKC activity is much less than that observed for recombinant di-

heteromeric receptors. Native and cultured hippocampal neurons express

mainly the NR1, NR2A and NR2B subunits. NMDA-evoked responses in

Xenopus oocytes expressing the NRl/NR2A or NRlINRZB receptor

combinations are consistently enhanced by application of phorbol esters by

up to 200 and 400 % of the control, respectively (Wagner & Leonard, 1996).

This potentiation is independent of extracellular Ca2+ or Mg2+ concentrations

(Wang & Leonard 1996). It is not clear why phorbol esters potentiate the

NMDA receptors function in this preparation to such a degree. One possible

explanation for these apparent discrepancy may be that native NMDA

receptors are composed of more than one type of NR2 subunits (Sheng e t al.,

1994) and hence are modulated differently by protein phosphorylation than

the di-heteromeric recombinant receptors, although recent evidence suggest

that the native NMDA receptors may contain only one type of NR2 subunit

(Blahos & Wenthold, 1996). PKC may regulate NMDA receptors by

phosphorylating the NR1, NR2 subunits or several of the regulatory proteins

associated with the receptorlchannel cornplex. The trafficking (Le., targeting,

assembly, and localization) of the NMDA receptor subunits i n different

a a - - - - - - A- - - - -- - - -. . - - - -- - - --- - - -- - ---- - " --O-- --

different mechanism in each preparation. Although recombinant receptors

exhibit characteristics similar to those of the native receptors, subtle

differences exist (for review see (Sucher et al., 1996). For example NR1

subunits do not form functional channels when expressed in mammalian ce11

lines such HEK 293 cells (Grimwood et al., 1995; Chazot e t al., 1992). The

formation of functional channels in mammalian cells requires the CO-

expression of both NR1 and NR2 subunits (Ishii et al., 1993; Meguro et al.,

1992). It is possible that an as of yet unidentified endogenous subunit

present in Xenopus oocytes promotes the formation of functional channels by

NR1 subunit (Sucher et al., 1996). The possible presence of this subunit in

the channel complex may be responsible for the high degree of up-regulation

of NMDA receptors expressed in oocyte by PKC. Also it has been reported

that localization of NR1 receptors to the plasma membrane in mammalian

cells is dependent upon the presence of NR2 subunit (McIlhinney et al., 1996;

Boeckman & Aizenman, 1994) suggesting that the processes involved in the

trafficking of these receptors in Xenopus oocytes may be different than those

taking place in mammalian cells. For example the basal phosphorylation

level of NMDA receptor subunits may be different in the two preparations.

In cultured hippocampal neurons it has recently been shown that NR2

subunits are highly phosphorylated under basal conditions, whereas basal

phosphorylation of NR1 subunits is very low (Hall & Soderling, 1997).

Further following stimulation of the cultures with glutamatelglycine or

piluruul esLers, l v n l priuspliurylabluri w a s luuriu b u ut: eLl1laIlC;t:U Uy 3-3-lUlU,

whereas phosphorylation of NR2 subunits was enhanced by less than 2-fold

(Hall & Soderling, 1997). This is consistent with our observation that PKCI

reduced NMDA-evoked currents, presumably by inhibiting the endogenous

PKC's. These results suggest that the basal level of phosphorylation of

NMDA receptor subunits in cultured hippocampal is relatively high which

may have resulted in the lower level of PKCM enhancement observed.

Interestingly Markram and Segal (1992) have reported that topical

application of phorbol ester to hippocarnpal slices reduces the amplitude of

NMDA currents in the CA1 pyramidal neurons. In their study topical

application of PMA (phorbol 12-myristate 13 acetate) depressed the

amplitude of NMDA-evoked currents in majority of the cells tested although

occasionally it potentiated the responses to NMDA (Markram and Segal

1992). The depression of NMDA receptor responses by phorbol esters has

also been reported in cerebelar purkinje cells (Courtney & Nicholls, 1992).

In cultured hippocampal neurons application of phorbol esters leads slight

depression of NMDA evoked currents, however if the neurons are treated

with a calpain inhibitor a potentiation of NMDA response is observed

(Bartlett e t al., 1989). In this case it is plausible that Ca2+ entry through

NMDA receptors leads to activation of calpain, a proteolytic enzyme that

cleaves activated PKC to terminate its actions. Hence stimulatory effects of

PKC activation by phorbol esters are antagonized by the proteolytic cleavage

of the enzyme by activated calpain. Also it is possible that as of yet

UIILII~L QLWZI ILCU DCbuuuaL y t;utxm UA ~ I I V I UUI GD IXL 3 u a y ut: I t:ùyullb~ult: IUL but:

depression of NMDA currents. For example phorbol esters may activate

other serinelthreonine kinases. Recently it has been shown that phorbol

esters can activate protein kinase D (PKD) both in vitro and in vivo

Rozengurt E, 1997). PKD is a serinelthreonine protein kinase that binds

phorbol esters and diacylglycerol and contains membrane localization signals

and a cysteine-rich repeat sequence homologous to that seen in the

regulatory domain of PKC (Valverde et al., 1994). The substrate specificity of

this kinase has not yet been fully characterized but it is suggested that PKD

may be an unusual component in the transduction of phorbol ester signals

(Valverde et al., 1994). Activation of different isozymes of PKC may also

underlie the reduction of NMDA response by phorbol esters.

Modulation of NMDA channel activity by PKCM

Application of PKCM to the cytoplasmic side of the inside-out patches

increased Popen by 170 %. This increase in Popen was reflected in the

decreased closed times although the open times did not significantly change.

Application of PKCI and PKCM together failed to potentiate the singe

channel activity. Chen and Huang (1992) have reported that PKC

potentiates NMDA single-channel activity by a factor of 1.44 in outside-out

patches excised from isolated trigeminal neurons (Popen = 0.09 in control and

0.13 with PKC in the patch pipette). These data suggest that PKC activity

leads to an increased probability of the channel opening contributes to the

enhancement of NMDA-evoked currents by PKC observed in whole-ce11

recordings. According to the formula, the whole-ce11 current, 1, is equal to N

x i x P,,,,, where N is the number of channels, i is the single-channel current,

and P o p * is the probability of channel opening. Hence an increase in the

Papen should have translated into a comparable enhancement of the whole-ce11

currents, however several factors may have contributed to the much smaller

degree of potentiation observed a t the whole-ce11 level: 1) The increase in the

open probability will not be the same for every channel. 2) In this study we

used inside-out patches to asses the effect of PKCM on the activity of the

channel. One of the difficulties with inside-out patches is the loss of cytosolic

factors that may modulate the behavior of the channel (Sackmann & Neher,

1995). For example it is possible that the kinases that normally

phosphorylate the channel in the ce11 are not present in the patch so that the

basal level of phosphorylation of the channels is much lower than it is in the

cell. Hence PKCM has a bigger effect on the patch than on the whole-ce11

currents. 3) Since the number of channels in the each patch are not known it

si not clear how each the channels is affected by PKC. For example if one

channel in the patch is silent and becomes active after PKM a big change in

the open probability in the patch is detected so that the average Popen will be

overestimated.

Although the enhancement of the single-channel activity by PKCM

suggests that direct phosphorylation of the channel is responsible for the

possibility of other mechanisms being involved in the process. For example

PKCM may potentiate NMDA receptor function by increasing the number of

receptors expressed on the ce11 surface.

PKCM modulation of the sensitivity of hippocampal NMDA receptors

to Mg2* block

NMDA channels are blocked by Mg2+ ions in a voltage-dependent

manner (Ascher & Nowak, 1988; Mayer & Westbrook, 1987; Mayer et al.,

1984). A reduction in the affinity of NMDA channels for Mg2+ has been

reported as a mechanism for the potentiation by PKC in acutely isolated

trigeminal neurons (Chen & Huang, 1992). Hence one of the main objectives

of this project was to determine if PKCM affects the Mgz* blockade of NMDA

channels in hippocampal neurons. The dose-inhibition curves constructed

prior to and during PKCM application indicated that the affinity of

hippocampal NMDA receptors for Mg2+ at -60 mV was not significantly

affected by the kinase activity. The discrepancy between these results and

the results obtained in acutely dissociated trigeminal neurons is not clear.

The acutely isolated hippocampal neurons exhibit a baseline Mg2+ sensitivity

similar to that of the isolated trigeminal neurons (IC5o = 27.3 PM). However

intracellular application of PKC in trigeminal neurons resulted in a 4-fold

increase in Ic50 (Kd) for Mg2+ block at -60 mV (Chen & Huang, 1992) which

function in vivo. The work from recombinant receptors however does not

support this hypothesis as application of phorbol esters to Xenopus oocytes

expressing NRlINR2A or NRllNR2B subunit combinations enhances the

currents by 200% and 440 % respectively, whereas the affinity for MgZC is

decreased by only a factor of two. Also treatment with phorbol esters did not

affect the voltage-dependence of the Mg2+ block of any of the subunit

combinations, NRlINRBA, B, C, and D (Wagner & Leonard, 1996). Hence the

PKC enhancement of the NR2A and NRZB containing di-heteromeric

receptors cannot entirely be explained by a change in the affinity for Mg2+

(Wagner & Leonard, 1996). One explanation for the disagreement between

the data obtained from trigeminal neurons and those from hippocampal

neurons is that the two population of neurons express different receptor-types

that are modulated differently by PKC. This idea is supported by the fact

that the Mg2+ sensitivity of the NMDA receptors in trigeminal neurons is

different from that of the acutely isolated hippocampal neurons (27 and 68.5

pM respectively). For example trigeminal neurons may express different

splice variants of the NR1 subunit, which are differentially modulated by

PKC (Koltchine et al., 1996; Durand et al., 1993), and also different

combination of NR2A and NR2B subunits which could potentially contribute

to the differences observed.

- - - - - - - - - - - - - a - ------- . - - -- ---- - - - - - V V ' - " Y I

hippocampal neurons

Although the affinity for Mg2+ block of NMDA single-channel currents

have been extensively studied, this is the first time tha t the apparent

sensitivity of whole-ce11 NMDA-evoked currents to Mg2+ block in both

cultured and acutely isolated hippocampal neurons has been reported. In

this study cultured hippocampal neurons expressed NMDA channels that

exhibited a considerably lower affinity for Mg2+ than acutely isolated CA1

neurons. In cultured hippocampal neurons voltage-clamped a t -60rnV, the

apparent affinity for Mg2+ of NMDA channels was 489 k 180 p M compared to

69 f 12 pM for dissociated neurons. It is difficult to explain this apparent

discrepancy on the basis of different subunit expression alone since

hippocampal neurons express almost exclusively the NR2A and NR2B

subunits which confer the highest sensitivity to the recombinant receptors

containing them (Hall & Soderling 1997; Wagner & Leonard 1996). Studies

on recombinant NMDA receptors indicate that di-heteromeric receptors

containing NR2A or NR2B are most sensitive to Mg2+ (IC5o = 2.8 k 0.5 pM

and 10.7 i 3.1 pM respectively) (Ishii e t al., 1993; Kutsuwada e t al., 1992;

Monyer et al., 1994; Mori et al., 1992; Mori e t al., 1993; Wagner & Leonard,

1996). Cultured hippocampal neurons have also been reported to express the

NR2A and NR2B subunits (Lau & Huganir, 1995) although it is not

inconceivable that they may also express other NR2 subunits due to

conditions of the culture.

In a recent study Zhang et al. (1996) have reported that stretch-

induced injury in cultured cortical neurons reduces the Mg2+ blockade of

NMDA-evoked currents and the reduction is partially relieved by calphostin

C, a PKC inhibitor . In that study the ICm for Mg2+ blockade prior to the

stretch-induced injury was 78 p M which is considerably lower than the value

we observed in Our cultured hippocampal neurons (IC50 = 489 +- 180 FM)..

Zhang et al (1996) also reported that the reduction in Mg2+ block is partially

relieved in cells treated with calphostin C prior to mechanical stretching

which suggests that PKC mediates the process. This suggest that the basal

PKC phosphorylation of the our hippocampal cultured neurons may be higher

resulting in the decreased sensitivity to Mg2+ blockade of NMDA-evoked

currents. In cultured hippocampal neurons the NR2 subunits are highly

phosphorylated on serine residues (Lau & Huganir, 1995) which fits with this

hypothesis. Also a higher basal phosphorylation level in cultured

hippocampal neurons may explain the relatively small effects of PKCM on

NMDA currents in these neurons (see above).

CaMK II modulation of hippocampal NMDA receptors

Our preliminary results suggest that NMDA receptor function is

potentiated by CaMK II. Interna1 perfusion of CaMKat enhanced NMDA-

. . - . . - - - A- - - - -- -- - -- - -- - - iI - - - - - - , -- - , - - --

control experiments, the application of the 'mock' enhanced the NMDA

currents by 3.7 k 6 %, n = 6. It was later found that the mock had some

residual kinase activity which may explain the potentiation observed with its

application. Although preliminary, these results are in agreement with the

reported potentiation of NMDA-evoked currents by the a subunit of CaMK II

in acutely isolated spinal cord neurons (Kolaj et al., 1994). In that study

Kolaj et al. (1994) reported that intracellular application of

autothiophosphorylated form CaMK II (70% Ca2+ -independent activity)

enhanced NMDA currents by 36 f 14 %.

CaMK II activity has been implicated in the induction of LTP (Silva e t

la., 1992, Malenka et la., 1989, Malinow e t al., 1989). CaMK II is highly

abundant in the nervous system and is specially concentrated in the

postsynaptic density (Benson et al., 1992, Basbaum and Kennedy 1989,

Kennedy et al., 1983; Kelly et al., 1984) at glutametergic synapses. There is

evidence that both AMPA and NMDA receptors are directly phosphorylated

by CaMK II (McGlade-McCulloh et al., 1993; Tan et al., 1994, Kitamura e t

al., 1993). Intracellular application of CaMK II in hippocampal neurons has

been shown to potentiate kainate-activated currents (McGlade-McCulloh et

al., 1993) which may be mediated in part by direct phosphorylation of the

AMPA receptor subunits GluR1 - GluR4 which contain consensus

phosphorylation sites for CaMK II (Barria e t al., 1997). NMDA receptor

-

by CaMK II (Hollmann and Heinemann 1994). The NR2B subunit is

phosphorylated in vitro by CaMK II on serines (Hall & Soderling, 1997).

Phosphorylation a t these sites may have contributed to the potentiation of

NMDA-evoked currents by CaMK II. However given the multifunctional

nature of CaMK II the effects of CaMK II may have been mediated through

phosphorylation of other proteins such a s microtubule-associated proteins or

calcineurin (Hanson and Schulman, 1992).

- - - - - - - - - - -

Despite considerable efforts in delineating the role of PKC in

modulating the function of NMDA receptors the exact mechanisms involved

have not yet been resolved. Our results suggest that PKC activity enhances

the function of NMDA receptors without significantly affecting the Mg2+

sensitivity of these channels. However the mechanisms underlying this

enhancement are not clear. A recent report suggests that phosphorylation by

PKC reduces the binding of Caz+/Calmodulin to its high affinity binding site

in the C l cassette of NR1 subunits(Hisatsune et al., 1997). Ca2+/Calmodulin

has previously been shown to decrease the open probability of NMDA

channels hence it's plausible that the potentiation is due to a decrease in the

CaWCalmodulin inactivation. Alternatively phosphorylation of the serine

residues in the Cl cassette may directly mediate the enhancement.

Experiments are planned in which the effects of systematic mutations of the

residues phosphorylated by PKC on the enhancement of the receptor function

are investigated. These experiment should further clariSi the mechanisms

underlying the PKC enhancement.

Alternatively phosphorylation by PKC may affect the interaction of

NMDA receptor with the cytoskeleton. The NMDA receptor subunits have

been shown to interact with PSD95, SAP 107, and a-actinin which is an actin

binding protein (Wyszynski et al., 1997; Lau et al., 1996; Kornau et al.,

1995). Also it has been shown that the activity of many ion channels depends

on the polymerization state of actin filaments (Whatley & Harris, 1996).

This provide strong evidence that the function of NMDA channel may depend

on its interaction with the cytoskeleton which may also explain the stretch

sensitivity of the receptor (Paoletti & Ascher, 1994). This interaction in turn

may be modulated by protein phosphorylation. Questions that still need to

be answered are : Does the enhancement of NMDA receptor function by

serinekhreonine kinases require an intact cytoskeleton? In another words

does phosphorylation alter the specific interaction of the receptor complex

with cytoskeletal elements?

Our preliminary data suggest that CaMK II potentiates NMDA

receptor function. CaMK II is a multifunctional enzyme which modulates a

variety of other kinases and phosphatases. Hence activation of CaMK II can

lead to activation of other phosphorylation cascades. It is not known if the

enhancement of NMDA receptor observed is due to phosphorylation of the

receptor or elements associated with it or due to activation of secondary

kinases or phosphatases. Experiments in which the effects of specific

inhibitors of other kinases or phosphatases are examined could provide some

clues as to the mechanism of potentiation. Subunit dependence of the CaMK

II enhancement and the role of the CaMK II phosphorylation sites on NR2

subunit are yet to be determined.

AKAZAWA, C., OHISHI, H., NAKAJIMA, Y., OKAMOTO, N., SHIGEMOTO,

R., NAKANISHI, S. & MIZUNO, N. (1994). Expression of mRNAs of L-

AP4-sensitive metabotropic glutamate receptors (mGluR4, mGluR6,

mGluR7) in the rat retina. Neurosci.Lett. 171, 52-54.

ANANTHARAM, V., PANCHAL, R.G., WILSON, A., KOLCHINE, V.V.,

TREISTMAN, S.N. & BAYLFX, H. (1992). Combinatorial RNA splicing

alters the surface charge on the NMDA receptor. FEBS Lett. 305, 27-

30.

ANIKSZTEJN, L., OTANI, S. & BEN-ARI, Y. (1992). Quisqualate

metabotropic receptors modulate NMDA currents and facilitate

induction of long term potentiation through protein kinase C. Eur. J.

Neurosci. 4, 500-505.

ARONIADOU, V.A. & TEYLER, T.J. (1991). The role of NMDA receptors in

long-term potentiation (LTP) and depression (LTD) in rat visual

cortex. Brain Res. 562, 136-143.

ARTOLA, A. & SINGER, W. (1994). NMDA receptors and developmental

plasticity in the visual cortex. In The NMDA receptor, 2nd eds.

ASCHER, P. & NOWAK, L. (1988). The role of divalent cations in the N-

methyl-D-aspartate responses of mouse central neurones in culture. J.

Physiol. (Lond) 399, 247-266.

BARRTA, A., MULLER, D., DERKACH, V., GRIFFITH, L.C. & SODERLING,

T.R. (199713). Regulatory phosphorylation of AMPA-type glutamate

receptors by C d - K I 1 during long-term potentiation. Science 276,

2042-2045.

BASHIR, Z.I., ALFORD, S., DAVIES, S.N., RANDALL, A.D. &

COLLINGRIDGE, G.L. (1991). Long-term potentiation of NMDA

receptor-mediated synaptic transmission in the hippocampus. Nature

349, 156-158.

BASHIR, Z.I., BORTOLOTTO, Z.A., DAVIES, C.H., BERRETTA, N.,

IRVING, A.J., SEAL, A.J., HENLEY, J.M., JANE, D.E., WATKINS,

J.C. & COLLINGRIDGE, G.L. (1993). Induction of LTP in the

hippocampus needs synaptic activation of glutamate metabotropic

receptors. Nature 363, 347-353.

, , , - - -- -- - - - - - - - 2 --

COLQUHOUN, D. (1995). Determination of NMDA NRl subunit copy

number in recombinant NMDA receptors. Proc. R. Soc. Lond. B. Biol.

Sci. 262, 205-213.

BEN-ARI, Y., ANIKSZTEJN, L. & BREGESTOVSKT, P. (1992). Protein

kinase C modulation of NMDA currents: an important link for LTP

induction. Trends. Neurosci. 15, 333-339.

BLAHOS, 5.2. & WENTHOLD, R.J. (1996). Relationship between N-methyl-

D-aspartate receptor NR1 splice variants and NR2 subunits. J. Biol.

Chem. 271, 15669-15674.

BOECKMAN, F.A. & AIZENMAN, E. (1994). Stable transfection of the NR1

subunit in Chinese hamster ovary cells fails to produce a functional N-

methyl-D-aspartate receptor. Neurosci. Lett. 173, 189-192.

BRAUN, A.P. & SCHULMAN, H. (1995). The multifunctional

calcium/calmodulin-dependent protein kinase: From form to function.

Annu. Rev. Physiol. 57, 417-445.

BROSE, N., GASIC, G.P., VETTER, D.E., SULLIVAN, J.M. &

HEINEMANN, S.F. (1993). Protein chernical characterization and

NMDAR1. J. Biol. Chem. 268, 22663-22671.

BULLER, A.L., LARSON, H.C., SCHNEIDER, B.E., BEATON, J.A.,

MORRISETT, R.A. & MONAGHAN, D.T. (1994). The molecular basis

of NMDA receptor subtypes: Native receptor diversity is predicted by

subunit composition. J. Neurosci. 14, 5471-5484.

BURGESS, S.K., SAHYOUN, N., BLANCHARD, S.G., LEVINE, H., CHANG,

K.J. & CUATRECASAS, P. (1986). Phorbol ester receptors and protein

kinase C in primary neuronal cultures: development and stimulation of

endogenous phosphorylation. J. Ce11 Biol. 102, 3 12-3 19.

BURNASHEV, N., MONYER, H., SEEBURG, P.H. & SAKMANN, B. (1992).

Divalent ion permeability of AMPA receptor channels is dominated by

the edited form of a single subunit. Neuron 8, 189- 198.

BURNASHEV, N., ZHOU, Z., NEHER, E. & SAKMANN, B. (1995).

Fractional calcium currents through recombinant GluR channels of the

NMDA, AMPA and kainate receptor subtypes. J. Physiol. (Lond.) 485,

403-418.

fi u rcluna~r; v , lu. ( I Y ~ ) . Gamurn permeammy or glutamate-gateà ChannelS

in the central nervous system. Curr. Opin. Neurobiol. 6, 311-317.

CHAZOT, P.L., CIK, M. & STEPHENSON, F.A. (1992). Immunological

detection of the NMDARl glutamate receptor subunit expressed in

human embryonic kidney 293 cells and in ra t brain. J. Neurochem. 59,

1176-1178.

CHEN, L. & HUANG, L.Y. (1991). Sustained potentiation of NMDA receptor-

mediated glutamate responses through activation of protein kinase C

by a mu opioid. Neuron 7, 319-326.

CHEN, L. & HUANG, L.Y. (1992). Protein kinase C reduces Mg2+ block of

NMDA-receptor channels as a mechanism of modulation. Nature 356,

521-523.

CHOI, D.W. (1992). Excitotoxic ce11 death. J. Neurobiol. 23, 1261-1276.

CIK, M., CHAZOT, P.L. & STEPHENSON, F.A. (1994). Expression of

NMDAR1-la (N598Q)INMDAR2A receptors results in decreased ce11

mortality. Eur. 5. Pharmacol. Mol. Pharmacol. 266, R1-R3

COHEN, P. (1989). The structure and regulation of protein phosphatases.

Annu. Rev. Biochern. 58, 453-508.

COURTNEY, M.J. & NICHOLLS, D.G. (1992). Interactions between

phospholipase C-coupled and N-methyl- D-aspartate receptors in

cultured cerebellar granule cells: Protein kinase C mediated

inhibition of N-methyl-D-aspartate responses. J. Neurochem. 59, 983-

992.

DURAND, G.M., GREGOR, P., ZHENG, X., BENNETT, M.V., UHL, G.R. &

ZUKIN, R.S. (1992). Cloning of a n apparent splice variant of the rat N-

methyl-D- aspartate receptor NMDAR1 with altered sensitivity to

polyamines and activators of protein kinase C. Proc. Natl. Acad. Sci.

U.S.A. 89, 9359-9363.

DURAND, G.M., BENNETT, M.V.L. & ZUKIN, R.S. (1993). Splice variants of

the N-methyl-D-aspartate receptor NR1 identiSl domains involved in

regulation by polyamines and protein kinase C. Proc. Natl. Acad. Sci.

U.S.A 90, 6731-6735.

EDELMAN, A.M., BLUMENTHAL, D.K. & KREBS, E.G. (1987). Protein

serinelthreonine kinases. Annu. Rev. Biochem. 56, 567-613.

- - - - - - - - - - - - - I \ - - - - I - --- a ------ - -

subcellular distribution of the NR1 subunit of the NMDA receptor.

Science 269, 1734-1737.

FUKUNAGA, K., MULLER, D. & MNAMOTO, E. (1996). CaM kinase II in

long-terrn potentiation. Neurochem. Int. 28, 343-358.

GERBER, G., KANGRGA, I., RYU, P.D., LAREW, J.S.A. & RANDIC, M.

(1989). Multiple effects of phorbol esters in the rat spinal cord. J.

Neurosci. 9, 3606-3614.

GIBB, A.J. & COLQUHOUN, D. (1991). Glutamate activation of a single

NMDA receptor-channel produces a cluster of channel openings. Proc.

R. Soc. Lond. B. Biol. Sci. 243, 39-45.

GIBB, A.J. & COLQUHOUN, D. (1992). Activation of N-methyl-D-aspartate

receptors by L-glutamate in cells dissociated from adult rat

hippocampus. J. Physiol. (Lond) 456, 143-179.

GREEN, W.N., ROSS, A.F. & CLAUDIO, T. (1991). Acetylcholine receptor

assembly is stimulated by phosphorylation of its gamma subunit.

Neuron 7, 659-666.

GRIMWOOD, S., LE BOURDELLÈS, B. & WHITING, P.J. (1995).

Recombinant human NMDA homomeric NR1 receptors expressed in

mammalian cells form a high-affinity glycine antagonist binding site.

J. Neurochem. 64, 525-530.

HALL, K.E., BROWNING, M.D., DUDEK, E.M. & MACDONALD, R.L.

(1995). Enhancement of high threshold calcium currents in rat primary

afferent neurons by constitutively active protein kinase C. J. Neurosci.

15, 6069-6076.

HALL, R.A. & SODERLING, T.R. (1997). Differential surface expression and

phosphorylation of the N-methyl-D- aspartate receptor subunits NR1

and NR2 in cultured hippocampal neurons. J. Biol. Chem. 272, 4135-

4140.

HANSON, P.I., MEYER, T., STRYER, L. & SCHULMAN, H. (1994). Dual

role of calmodulin in autophosphorylation of multifunctional CaM

kinase may underlie decoding of calcium signals. Neuron 12, 943-956.

HANSON, P.I. & SCHULMAN, H. (1992). Neuronal Ca2+/calmodulin-

dependent protein kinases. Annu. Rev. Biochem. 61, 559-601.

J l U L l _ r l V ~ l V I V , M., DUUL 1 bit, J ., IVMKUlV, C;. , BLAiSLi3 1 , L., 3 U L L l VALU, J .,

PECHT, G. & HEINEMANN, S. (1993). Zinc potentiates agonist-

induced currents a t certain splice variants of the NMDA receptor.

Neuron 10, 943-954.

HOLLMANN, M., BOULTER, J., MARON, C. & HEINEMANN, S. (1994).

Molecular biology of glutamate receptors. Potentiation of N- methyl-D-

aspartate receptor splice variants by zinc. Ren.Physiol.Biochem. 17,

182-183.

HOUSE, C . & KEMP, B.E. (1987). Protein kinase C contains a

pseudosubstrate prototope in its regulatory domain. Science

238(4834), 1726-1728

HUANG, K.P. & HUANG, F.L. (1986). Conversion of protein kinase C from a

Ca" -dependent to an independent form of phorbol ester-binding

protein by digestion with trypsin. Biochem. Biophys. Res. Commun.

139, 320-326

HUME, R.I., DINGLEDINE, R. & HEINEMANN, S.F. (1991). Identification

of a site in glutamate receptor subunits that controls calcium

permeability. Science 253, 1028-1031.

Biochem. 54:897-930, 897-930.

IKEDA, K., NAGASAWA, M., MORI, H., ARAKI, K., SAKIMURA, K.,

WATANABE, M., INOUE, Y. & MISHINA, M. (1992). Cloning and

expression of the e4 subunit of the NMDA receptor channel. FEBS

Lett. 313, 34-38.

ISHII, T., MORIYOSHI, K., SUGIHARA, H., SAKZJRADA, K., KADOTANI,

W., YOKOI, M., AKAZAWA, C., SHIGEMOTO, R., MIZUNO, N. &

MASU, M. (1993). Molecular characterization of the family of the N-

methyl-D- aspartate receptor subunits. J. Biol. Chem. 268, 2836-2843.

JAKEN, S. (1996). Protein kinase C isozymes and substrates. Current Opin.

in Cell Biol. 8, 168-173.

JOHNSON, J.W. & ASCHER, P. (1987). Glycine potentiates the NMDA

response in cultured mouse brain neurones. Nature 325, 529-53 1.

KELLY, P.T., MCGUINNESS, T.L. & GREENGARD, P. (1984). Evidence

that the major postsynaptic density protein is a component of a

Ca2+/calmodulin-dependent protein kinase. Proc. Natl. Acad. Sci.

U.S.A. 81, 945-949.

WLDU, a . ~ , I Y ~ L D V I Y , 1.m. (x; hnul\Anu, d . r . (IYYL). rrocein Kinase G-

mediated enhancement of NMDA currents by rnetabotropic glutamate

receptors in Xenopus oocytes. J. Physiol. (Lond) 449, 705-718.

KENNEDY, M.B. (1989). Regulation of synaptic transmission in the central

nervous system: long- term potentiation. Ce11 59, 777-787.

KENNEDY, M.B., BENNETT, M.K. & ERONDU, N.E. (1983). Biochemical

. and imrnunochemical evidence that the "major postsynaptic density

protein" is a subunit of a calmodulin-dependent protein kinase. Proc.

Natl. Acad. Sci. U.S.A. 80, 7357-7361.

KINNEY, G.A. & SLATER, N.T. (1993). Potentiation of NMDA receptor-

mediated transmission in turtle cerebellar granule cells by activation

of metabotropic glutamate receptors. J. Neurophysiol. 69, 585-594.

KNOPFEL, T., KUHN, R. & ALLGEIER, H. (1995). Metabotropic receptors:

novel targets for drug development. J. Med. Chem. 38, 1417-1425.

KOLAJ, M., CERNE, R., CHENG, G., BRICKEY, D.A. & RANDIC, M. (1994).

Alpha subunit of calcium/calmodulin-dependent protein kinase

enhances excitatory amino acid and synaptic responses of rat spinal

dorsal horn neurons. J. Neurophysiol. 72, 2525-2531.

nub J. Ullli'ifi, v . v ., M L Y f i L W 1 L U U l a l V l , v ., RA ILFA 1, n. OG 1 nbm 1 l V H l \ , D.Pl.

(1996). Alternative splicing of the NMDARI subunit affects modulation

by calcium. Mol. Brain Res. 39, 99-108.

KOMURO, H. & RAKIC, P. (1993). Modulation of neuronal migration by

NMDA receptors. Science 260, 95-97.

KORNAU, H.C., SCHENmR, L.T., KENNEDY, M.B. & SEEBURG, P.H.

(1995). Domain interaction between NMDA receptor subunits and the

postsynaptic density protein PSD-95. Science 269, 1737-1740.

KUNER, T. & SCHOEPFER, R. (1996a). Multiple structural elements

determine subunit specificity of Mg2+ block in NMDA receptor

channels. J. Neurosci. 16, 3549-3558.

KUNER, T., WOLLMUTH, L.P., KARLIN, A., SEEBURG, P.H. &

SAKMANN, B. (199613). Structure of the NMDA receptor channel M2

segment inferred from the accessibility of substituted cysteines.

Neuron 17, 343-352.

KUO, J.F. & GREENGARD, P. (1969). Cyclic nucleotide-dependent protein

kinases. IV. Widespread occurrence of adenosine 3',5'-monophosphate-

UC~CLIUCIIC> ~ L U V Z I I I Aluaac LAI v alluua A AD VU CD auu puy r a ur urtz aululai

kingdom. Proc. Natl. Acad. Sci. U.S.A. 64, 1349-1355.

KUPPER, J., ASCHER, P. & NEYTON, J. (1996). Probing the pore region of

recombinant N-methyl-D-aspartate channels using external and

interna1 rnagnesium block. Proc. Natl. Acad. Sci. U.S.A. 93, 8648-

8653.

KURYATOV, A., LAUBE, B., BETZ, H. & KUHSE, J. (1994). Mutational

analysis of the glycine-binding site of the NMDA receptor: Structural

similarity with bacterial amino acid- binding proteins. Neuron 12,

1291-1300.

KUTSUWADA, T., KASHIWABUCHI, N., MORI, H., SAKIMURA, K.,

KUSHIYA, E., ARAKI, K., MEGURO, H., MASAKI, H., KUMANISHI,

T., ARAKAWA, M. & MISHINA, M. (1992). Molecular diversity of the

NMDA receptor channel. Nature 358, 36-41.

LAU, L.F. & HUGANIR, R.L. (1995). Differential tyrosine phosphorylation of

N-methyl-D-aspartate receptor subunits. J. Biol. Chem. 270, 20036-

20041.

. .

GARNER, C.C. & HUGANIR, R.L. (1996). Interaction of the N-methyl-

D-aspartate receptor complex with a novel synapse-associated protein,

SAP102. J. Biol. Chem. 271, 21622-21628.

LAURIE, D.J. & SEEBURG, P.H. (1994). Regional and developmental

heterogeneity in splicing of the rat brain NMDARl mRNA. J.

Neurosci. 14, 3180-3194.

LAWRENCE, J.H., TOMASELLI, G.F. & MARBAN, E. (1993). Ion channels:

structure and function. Heart Dis. Stroke 2, 75-80.

LEGENDRE, P., ROSENMUND, C. & WESTBROOK, G.L. (1993).

Inactivation of NMDA channels in cultured hippocampal neurons by

intracellular calcium. J. Neurosci. 13, 674-684.

LEONARD, A.S. & HELL, J.W. (1997). Cyclic AMP-dependent Protein

Kinase and Protein Kinase C Phosphorylate N-Methyl-D-aspartate

Receptors a t Different Sites. J. Biol. Chem. 272, 12107-12115.

LESTER, R.A., CLEMENTS, J.D., WESTBROOK, G.L. & JAHR, C.E. (1990).

Channel kinetics determine the time course of NMDA receptor-

mediated synaptic currents. Nature 346, 565-567.

L ~ V I INLU, I.D. \IYUU). ~v~oaulaT;ion 01 in cnanners in neurons ana otner ceïis.

Annu. Rev. Neurosci. 11, 119-136.

LEVITAN, I.B. (1994). Modulation of ion channels by protein

phosphorylation and dephosphorylation. Annu. Rev. Physiol. 56, 193-

LIEBERMAN, D.N. & MODY, 1. (1994). Regulation of NMDA channel

function by endogenous Ca2+- dependent phosphatase. Nature 369,

235-239.

LOU, L.L. & SCHULMAN, H. (1989). Distinct autophosphorylation sites

sequentially produce autonomy and inhibition of the multifunctional

Ca2+/calrnodulin-dependent protein kinase. J. Neurosci. 9, 2020-2032.

LOZOVAYA, N A . & KLEE, M.R. (1995). Phorbol diacetate differentially

regulates the N-methyl-D-aspartate (NMDA) and non-NMDA receptor-

mediated components of the rat hippocampal excitatory postsynaptic

currents. Neurosci. Lett. 189,101- 104.

MACDERMOTT, A.B., MAYER, M L , WESTBROOK, G.L., SMITH, S.J. &

BARKER, J .L. (1986). NMDA-receptor activation increases

cytoplasmic calcium concentration in cultured spinal cord neurones.

Nature 321, 519-522.

MACDONALD, J.F., BARTLETT, M.C., MODY, I., PAHAPILL, P.,

REYNOLDS, J.N., SALTER, M.W., SCHNEIDERMAN, J.H. &

PENNEFATHER, P.S. (1991). Actions of ketamine, phencyclidine and

MK-801 on NMDA receptor currents in cultured mouse hippocampal

neurones. J. Physiol. (Lond) 432, 483-508.

MACDONALD, J.F., MILJKOVIC, Z. & PENNEFATHER, P. (1987). Use-

dependent block of excitatory amino acid currents in cultured neurons

by ketamine. J. Neurophysiol. 58, 251-266.

MACDONALD, J.F., MODY, 1. & SALTER, M.W. (1989). Regulation of N-

methyl-D-aspartate receptors revealed by intracellular dialysis of

murine neurones in culture. J. Physiol. (Lond) 414, 17-34.

MALENKA, R.C., MADISON, D.V. & NICOLL, R.A. (1986). Potentiation of

synaptic transmission in the hippocampus by phorbol esters. Nature

321, 175-177.

MAMMEN, A.L. & HUGANIR, R.L. (1997). Regulaiton of NMDA receptors by

protein phosphorylation. In The ionotropic glutamate receptors, eds.

Jersey: Humana Press.

MARKRAM, H. & SEGAL, M. (1992). Activation of protein kinase C

suppresses responses to NMDA in rat CA1 hippocampal neurones. J.

Physiol. (Lond) 457, 491-501.

MATTSON, M.P., WANG, H. & M I C W L I S , E.K. (1991). Developmental

expression, compartmentalization, and possible role in excitotoxicity of

a putative NMDA receptor protein in cultured hippocampal neurons.

Brain Res. 565, 94-108.

MAYER, M.L. & WESTBROOK, G.L. (1987). Permeation and block of N-

methyl-D-aspartic acid receptor channels by divalent cations in mouse

cultured central neurones. J. Physiol. (Lond) 394, 501-527.

MAYER, M.L., WESTBROOK, G.L. & GUTHRIE, P.B. (1984). Voltage-

dependent block by Mg2+ of NMDA responses in spinal cord neurones.

Nature 309, 261-263.

MCBAIN, C.J. & MAYER, M.L. (1994). N-methyl-D-aspartic acid receptor

structure and function. Physiol. Rev. 74, 723-723.

- - - -- - - - - - - - - - - - I --, - - i ---, ---- , - - - - I 7 -

& SODERLING, T.R. (1993). Phosphorylation and regulation of

glutamate receptors by calcium/calmodulin-dependent protein kinase

II. Nature 362, 640-642.

MCILHINNEY, R.A.J., M O L N ~ , E., ATACK, J.R. & WHITING, P.J. (1996).

Ce11 surface expression of the human N-methyl-D-aspartate receptor

subunit l a requires the CO-expression of the NRZA subunit in

transfected cells. Neuroscience 70, 989-997.

MEGURO, H., MORI, H., ARAKI, K., KUSHIYA, E., KUTSUWADA, T.,

YAMAZAKI, M., KUMANISHI, T., ARAKAWA, M., SAKIMURA, K. &

MISHINA, M. (1992). Functional characterization of a heteromeric

NMDA receptor channel expressed from cloned cDNAs. Nature 357,

70-74.

MIYAMOTO, E., KUO, J.F. & GREENGARD, P. (1968). Adenosine 3',5'-

monophosphate-dependent protein kinase £rom brain. Science 165, 63-

65.

MODY, I., SALTER, M.W. & MACDONALD, J.F. (1988). Requirement of

NMDA receptorlchannels for intracellular high- energy phosphates and

U - - - - --- - - - .

hippocampal neurons. Neurosci. Lett. 93, 73-78.

MODY, I., SALTER, M.W. & MACDONALD, J.F. (1989a). Whole-ce11 voltage-

clamp recordings in granule cells acutely isolated from hippocampal

slices of adult or aged rats. Neurosci. Lett. 96, 70-75.

MONYER, H., BURNASHEV, N., LAURIE, D.J., SAKMANN, B. &

SEEBURG, P.H. (1994). Developmental and regional expression in the

rat brain and functional properties of four NMDA receptors. Neuron

12, 529-540.

MONYER, H., SPRENGEL, R., SCHOEPFER, R., HERB, A., HIGUCHI, M.,

LOMELI, H., BURNASHEV, N., SAKMANN, B. & SEEBURG, P.H.

(1992). Heteromeric NMDA receptors: Molecular and functional

distinction of subtypes. Science 256, 121% 1221.

MORI, H., MASAKI, H., YAMAKURA, T. & MISHINA, M. (1992).

Identification by mutagenesis of a Mg2+-block site of the NMDA

receptor channel. Nature 358, 673-675.

MORI, H. & MISHINA, M. (1995). Structure and function of the NMDA

receptor channel. Neuropharmacology 34, 1219-1237.

Involvement of the carboxyl-terminal region in modulation by TPA of

the NMDA receptor channel. Neuroreport. 4, 519-522.

MORIYOSHI, K., MASU, M., ISHII, T., SHIGEMOTO, R., MIZUNO, N. &

NAKANISHI, S. (1991). Molecular cloning and characterization of the

rat NMDA receptor. Nature 354, 31-37.

MULLER, B.M., KISTNER, U., KINDLER, S., CHUNG, W.J.,

KUHLENDAHL, S., FENSTER, S.D., LAU, L.F., VEH, R.W.,

HUGANIR, R.L., GUNDELFINGER, E.D. & GARNER, C.C. (1996).

SAP102, a novel postsynaptic protein that interacts with NMDA

receptor complexes in vivo. Neuron 17 , 255-265.

MULLER, D., BUCHS, P.A., STOPPINI, L. & BODDEKE, H. (1991). Long-

term potentiation, protein kinase C, and glutamate receptors. Mol.

Neurobiol. 5, 277-288.

NAIRN, A.C. & PICCIOTTO, M.R. (1994). Calcium/calmodulin-dependent

protein kinases. Semin. Cancer Biol. 5, 295-303.

I Y ~ ~ ~ I Y lr3n1, IY., IIADJJ, n. OL, o n l v n l u n n , IY .fi. \ I J J C S ) . L - U L ~ T I ~ ~ U V ~ spc111g

generates functionally distinct N- methyl-D-aspartate receptors. Proc.

Natl. Acad. Sci. USA 89, 8552-8556.

NAKAMSHI, N., SHNEIDER, N.A. & AXEL, R. (1990). A family of

glutamate receptor genes: evidence for the formation of

heteromultimeric receptors with distinct channel properties. Neuron

5, 569-581.

NAKANISHI, S. (1992). Molecular diversity of glutamate receptors and

implications for brain function. Science 258, 597-603.

NEHER, E. & SAKMANN, B. (1992). The patch clamp technique. Sci. Am.

266, 44-51.

NIETHAMMER, M., KIM, E. & SHENG, M. (1996). Interaction between the

C terminus of NMDA receptor subunits and multiple members of the

PSD-95 family of membrane-associated guanylate kinases. J.

Neurosci. 16, 2157-2163.

NOWAK, L., BREGESTOVSKI, P., ASCHER, P., HERBET, A. &

PROCHIANTZ, A. (1984). Magnesium gates glutamate-activated

channels in mouse central neurones. Nature 307, 462-465.

v i v u x u l v m , D.v. , N U , IVLCI., ~ V ~ ~ J C I I Y ~ ~ I I I ~ I I Y , n.ci., i v u ~ u , A . oz

KENNEDY, M.B. (1996). Identification of a phosphorylation site for

calcium/calmodulindependent protein kinase II in the NR2B subunit of

the N-methyl-D-aspartate receptor. J. Biol. Chem. 271, 31670-31678.

PAOLETTI, P. & ASCHER, P. (1994). Mechanosensitivity of NMDA receptors

in cultured mouse central neurons. Neuron 13, 645-655.

PAOLETTI, P., NEYTON, J. & ASCHER, P. (1995). Glycine-independent and

subunit-specific potentiation of NMDA responses by extracellular

Mg2+. Neuron 15, 1109-1 120.

PARFITT, K.D. & MADISON, D.V. (1993). Phorbol esters enhance synaptic

transmission by a presynaptic, calcium- dependent mechanism in rat

hippocampus. J. Physiol. (Lond) 471:245-68, 245-268.

PATNEAU, D.K. & MAYER, M.L. (1990). Structure-activity relationships for

amino acid transmitter candidates acting a t N-methyl-D-aspartate and

quisqualate receptors. J. Neurosci. 10, 2385-2399.

PAUPARD, M.C., FRIEDMAN, L.K. & ZUKIN, R.S. (1997). Developmental

regulation and cell-specific expression of N-methyl-D- aspartate

PIN, J.-P. & DUVOISIN, R. (1995). The metabotropic glutamate receptors:

Structure and functions. Neuropharmacology 34, 1-26.

PONZONI, M., LUCARELLI, E., CORRIAS, M.V. & CORNAGLIA-

FERRARIS, P. (1993). Protein kinase C isoenzymes in human

neuroblasts. Involvement of PKC epsilon i n ce11 differentiation. FEBS

Lett. 322, 120-124.

RANDIC, M., JIANG, M.C. & CERNE, R. (1993). Long-term potentiation and

long-term depression of primary afferent neurotransmission in the rat

spinal cord. J. Neurosci. 13, 5228-5241.

RESINK, A., VILLA, M., BENKE, D., HIDAKA, H., MOHLER, H. &

BALAZS, R. (1996). Characterization of agonist-induced down-

regulation of NMDA receptors in cerebellar granule ce11 cultures. J.

Neurochem. 66, 369-377.

ROCHE, K.W., O'BRIEN, R.J., MAMMEN, A.L., BERNHARDT, J. &

HUGANIR, R.L. (1996). Characterization of multiple phosphorylation

sites on the AMPA receptor GluRl subunit. Neuron 16, 1179-1188.

(1994). Transmembrane topology of the glutamate receptor subunit

GluR6. J. Biol. Chem. 269, 11679-11682.

ROSENMUND, C. & WESTBROOK, G.L. (1993a). Calcium-induced actin

depolymerization reduces NMDA channel activity . Neuron 10, 805-

814.

ROSENMUND, C. & WESTBROOK, G.L. (1993b). Rundown of N-methyl-D-

aspartate channels during whole- ce11 recording in rat hippocampal

neurons: Role of Ca2+ and ATP. J. Physiol. 470, 705-729.

ROSS, A.F., GREEN, W.N., HARTMAN, D.S. & CLAUDIO, T. (1991).

Efficiency of acetylcholine receptor subunit assembly and its regulation

by CAMP. J. Ce11 Biol. 113, 623-636.

ROSS, A.F., RAPUANO, M., SCHMIDT, J.H. & PRIVES, J.M. (1987).

Phosphorylation and assembly of nicotinic acetylcholine receptor

subunits in cultured chick muscle cells. J. Biol. Chem. 262, 14640-

14647.

ROTH, B.L., MEHEGAN, J.P., JACOBOWITZ, D.M., ROBEY, F. &

IADAROLA, M.J. (1989). Rat brain protein kinase C: purification,

antmoay proauction, ana quantrncation In arscrete regions 01

hippocampus. J. Neurochem. 52(1), 215-221.

ROUTTENBERG, A., COLLEY, P., LINDEN, D., LOVINGER, D.,

MURAKAMI, K. & SHEU, F.S. (1986). Phorbol ester promotes growth

of synaptic plasticity. Brain Res. 378, 374-378.

SAFRAN, A., SAGI-EISENBERG, R., NEUMANN, D. & FUCHS, S. (1987).

Phosphorylation of the acetylcholine receptor by protein kinase C and

identification of the phosphorylation site within the receptor delta

subunit. J. Biol. Chem. 262, 10506-10510.

SAKURADA, K., MASU, M. & NAKANISHI, S. (1993). Alteration of Ca2+

permeability and sensitivity to Mg2+ and channel blockers by a single

amino acid substitution in the N- methyl-D- aspartate receptor. J.

Biol. Chem. 268, 410-415.

SCHOEPP, D.D. & CONN, P.J. (1993). Metabotropic glutamate receptors in

brain function and pathology. Trends Pharmacol. Sci. 14, 13-20.

SCHROEDER, W., COVEY, T. & HUCHO, F. (1990). Identification of

phosphopeptides by mass spectrometry. FEBS Lett. 273, 31-35.

signals by multifunctional CaM kinase. Ce11 Calcium 13, 401-41 1.

SHENG, M., CUMMINGS, J., ROLDAN, L.A., JAN, Y.N. & JAN, L.Y. (1994).

Changing subunit composition of heteromeric NMDA receptors during

development of rat cortex. Nature 368, 144-147.

SILVA, A.J., STEVENS, C.F., TONEGAWA, S. & WANG, Y. (1992). Deficient

hippocampal long-term potentiation i n alpha-calcium- calmodulin

kinase II mutant mice. Science 257, 201-206.

SMITH, M.K., COLBRAN, R.J. & SODERLING, T.R (1990). Specificities of

autoinhibitory domain peptides for four protein kinases. Implications

for intact ce11 studies of protein kinase function. J. Biol. Chem. 265(4),

1837-1840.

SODERLING, T.R. (1996a). Modulation of glutamate receptors by

calcium/calmodulin-dependent protein kinase II. Neurochem. Int. 28,

359-361.

SODERLING, T.R. (1996b). Structure and regulation of calcium/calmodulin-

dependent protein kinases II and IV. Biochim. Biophys. Acta 1297,

131-138.

- - - - - - - - - . -. i - - - - - - - ---- - - - - -- a-"' - "'^Y" S"'-V-SI*UVV

receptor ion channels. In The ionotropic glutamate receptors, eds.

MONAGHAN, D.T. & WENTHOLD, R.J., pp. 121-134. Totowa, New

Jersey: Humana Press.

SPINELLI, W. & LSHII, D.N. (1983). Tumor promoter receptors regulating

neurite formation in cultured human neuroblastoma cells. Cancer Res.

43, 4119-4125.

STERN, P., BEHE, P., SCHOEPFER, R. & COLQUHOUN, D. (1992). Single-

channel conductances of NMDA receptors expressed from cloned

cDNAs: cornparison with native receptors. Proc. R. Soc. Lond. B. Biol.

Sci. 250, 271-277.

STERN, P., CIK, M., COLQUHOUN, D. & STEPHENSON, F.A. (1994).

Single channel properties of cloned NMDA receptors in a human ce11

line: Comparison with results from Xenopus oocytes. J. Physiol.

(Lond.) 476, 391-397.

SUCHER, N.J., AWOBULUYI, M., CHOI, Y.B. & LIPTON, S.A. (1996).

NMDA receptors: from genes to channels. Trends. Pharmacol. Sci. 17,

348-355.

, , , , , - -. - - - . - - - -- . - -- - - - 3 --

(1992). Structures and properties of seven isoforms of the NMDA

receptor generated by alternative splicing. Biochem. Biophys. Res.

Commun. 185, 826-832.

SULLIVAN, J.M., TRAYNELIS, S.F., CHEN, H.-S.V., ESCOBAR, W.,

HEINEMANN, S.F. & LIPTON, S.A. (1994). Identification of two

cysteine residues that are required for redox modulation of the NMDA

subtype of glutamate receptor. Neuron 13, 929-936.

SUTCLIFFE, M.J., WO, Z.G. & OSWALD, R.E. (1996). Three-dimensional

models of non-NMDA glutamate receptors. Biophysical J. 70, 157 5-

1589.

SUZUKI, T., OKUMURA-NOJI, K., OGURA, A., TANAKA, R., NAKAMURA,

K. & KUDO, Y. (1992). Calpain may produce a Ca(2+)-independent

form of kinase C in long-term potentiation. Biochern. Biophys. Res.

Commun. 189, 1515-1520.

TAN, S.E., WENTHOLD, R. J. & SODERLING, T.R. (1994). Phosphorylation

of AMPA-type glutamate receptors by calcium/calmodulin- dependent

protein kinase II and protein kinase C in cultured hippocampal

neurons. J. Neurosci. 14, 1123-1 129.

J.B., RILEY, C.T. & HUGANIR, R.L. (1997). Characterization of

protein kinase A and protein kinase C phosphorylation of the N-

methyl-D-aspartate receptor NR1 subunit using phosphorylation site-

specific antibodies. J. Biol. Chem. 272, 5157-5166.

TINGLEY, W.G., ROCHE, K.W., THOMPSON, A.K. & HUGANIR, R.L.

(1993). Regulation of NMDA receptor phosphorylation by alternative

splicing of the C-terminal domain. Nature 364, 70-73.

TOKITA, Y., BESSHO, Y., MASU, M., NAKAMURA, K., NAKAO, K.,

KATSUKI, M. & NAKANISHI, S. (1996). Characterization of

excitatory amino acid neurotoxicity in N- methyl-D- aspartate

receptor-deficient mouse cortical neuronal cells. Suppl. Eur. J.

Neurosci. 8, 69-78.

TONG, G. & JAHR, C.E. (1994). Regulation of glycine-insensitive

desensitization of the NMDA receptor in outside-out patches. J.

Neurophysiol. 72, 754-76 1.

TONG, G., SHEPHERD, D. & JAHR, C.E. (1995). Synaptic desensitization of

NMDA receptors by calcineurin. Science 267, 1510-1512.

'1'fCHY NELlS, S.F., HAKl'Lk!i Y , IVI. CU; HiSlNElVIANN, B.F.. ( lYY5) . Gontrol OX

proton sensitivity of the NMDA receptor by RNA splicing and

polyamines. Science 268, 873-876.

URUSHIHARA, H., TOHDA, M. & NOMURA, Y. (1992). Selective

potentiation of N-methyl-D-aspartate-induced current by protein

kinase C in Xenopus oocytes injected with rat brain RNA. J. Biol.

Chem. 267,11697-11700.

VALVERDE, A.M., SINNETT-SMITH, J., VAN LINT J. & ROZENGURT, E.

(1994). Molecular cloning and characterization of protein kinase D: a

target for diacylglycerol and phorbol esters with a distinctive catalytic

domain. Proc. Natl. Acad. Sci. U S A 91(18), 8572-8576

VERDOORN, T.A., BURNASHEV, N., MONYER, H., SEEBURG, P.H. &

SAKMANN, B. (1991). Structural determinants of ion fiow through

recombinant glutamate receptor channels. Science 252, 1715-1718.

VILMROEL, A., BURWSHEV, N. & SAKMANN, B. (1995). Dimensions of

the narrow portion of a recombinant NMDA receptor channel.

Biophysical J. 68, 866-875.

activation on the Mg2+-sensitivity of cloned NMDA receptors.

Neuropharmacology 35, 29-36.

WALLACE, B.G., QU, Z. & HUGANIR, R.L. (1991). Agrin induces

p hosp horylation of the nicotinic acetylcholine recep tor . Neuron 6, 869-

878.

WALSH, D.A., PERKINS, J.P. & KREBS, E.G. (1968). An adenosine 3',5'-

monophosphate-dependant protein kinase from rabbit skeletal muscle.

J. Biol. Chem. 243, 3763-3765.

WANG, J. & FENG, D.-P. (1992). Postsynaptic protein kinase C essential to

induction and maintenance of long-term potentiation in the

hippocampal CA1 region. Proc. Natl. Acad. Sci. USA 89, 2576-2580.

WANG, J.H. & KELLY, P.T. (1995). Postsynaptic injection of Ca2+/CaM

induces synaptic potentiation requiring CaMKII and PKC activity.

Neuron 15, 443-452.

WANG, L.Y., DUDEK, E.M., BROWNING, M.D. & MACDONALD, J.F.

(1994). Modulation of AMPAkainate recep tors in cultured murine

WANG, L.Y. & MACDONALD, J.F. (1995). Modulation by magnesium of the

affinity of NMDA receptors for glycine in murine hippocampal

neurones. J. Physiol. (Lond) 486, 83-95.

WANG, L.Y., ORSER, B.A., BMUTIGAN, D.L. & MACDONALD, J.F.

(1994). Regulation of NMDA receptors in cultured hippocampal

neurons by protein phosphatases 1 and 2A. Nature 369, 230-232.

WATANAElE, M., INOUE, Y., SAKIMURA, K. & MISHINA, M. (1992).

Developmental changes in distribution of NMDA receptor channel

subunit mRNAs. Neuroreport. 3, 1138-1 140.

WATANABE, M., INOUE, Y., SAKIMURA, K. & MISHINA, M. (1993).

Distinct spatio-temporal distributions of the NMDA receptor channel

subunit mRNAs in the brain. Ann. N'Y Acad. Sci. 707, 463-466.

WATANABE, M., MISHINA, M. & INOUE, Y. (1994a). Differential

distributions of the NMDA receptor channel subunit mRNAs in the

mouse retina. Brain Res. 634, 328-332.

-- - -- - -, - -- - - - > -

\ - - - - - , - - - - - - - - - Q----

expression of the N-methyl-D-aspartate receptor channel subunit in

peripheral neurons of the mouse sensory ganglia and adrenal gland.

Neurosci. Lett. 165, 183-186.

WHATLEY, V.J. & HARRIS, R.A. (1996). The cytoskeleton and

neurotransmitter receptors. Int. Rev. Neurobiol. 39, 113-143.

WILLIAMS, E.J., MITTAL, B., WALSH, F.S. & DOHERTY, P. (1995). A

Ca2+/calmodulin kinase inhibitor, KN-62, inhibits neurite outgrowth

stimulated by CAMs and FGF. Mol. Cell Neurosci. 6, 69-79.

WILLIAMS, K., RUSSELL, S.L., SHEN, Y.M. & MOLINOFF, P.B. (1993).

Developmental switch in the expression of NMDA receptors occurs in

vivo and in vitro. Neuron 10, 267-278.

WO, Z.G. & OSWALD, R.E. (1994). Transmembrane topology of two kainate

receptor subunits revealed by N- glycosylation. Proc. Natl. Acad. Sci.

U.S.A. 91, 7154-7158.

WO, Z.G. & OSWALD, R.E. (1995). Unraveling the modular design of

glutamate-gated ion channels. Trends Neurosci. 18, 161- 168.

, ,

Differential contribution of the NR1- and NR2A-subunits to the

selectivity filter of recombinant NMDA receptor channels. J. Physiol.

(Lond) 491, 779-797.

WYLLIE, D.J., BEHE, P., NASSAR, M., SCHOEPFER, R. & COLQUHOUN,

D. (1996). Single-channel currents from recombinant NMDA

NRlalNR2D receptors expressed i n Xenopus oocytes. Proc. R. Soc.

Lond. B. Biol. Sci. 263, 1079-1086.

WYSZYNSKI, M., LIN, J., RAO, A., NIGH, E., BEGGS, A.H., CRAIG, A.M. &

SHENG, M. (1997). Cornpetitive binding of alpha-actinin and

calmodulin to the NMDA receptor. Nature 385, 439-442.

XIE, X., BERGER, T.W. & BARRIONUEVO, G. (1992). Isolated NMDA

receptor-mediated synaptic responses express both LTP and LTD. J.

Neurophysiol. 67, 1009-1013.

YAKEL, J.L., VISSAVAJJHALA, P., DERKACH, V.A., BRICKEY, D.A. &

SODERLING, T.R. (1995). Identification of a Ca2+/calrnodulin-

dependent protein kinase II regulatory phosphorylation site in non- N-

methyl- D-aspartate glutamate receptors. Proc. Natl. Acad. Sci. USA

92, 1376-1380.

X A i V L N M . U U , 1 , lVlUK1, il., 3HllVlUJ1, K. & lVllSHlNA, NI. (1393).

Phosphorylation of the carboxyl-terminal domain of the zeta 1 subunit

is not responsible for potentiation by TPA of the NMDA receptor

channel. Biochem. Biophys. Res. Commun. 196, 1537-1544.

YAMAZAKI, M., MORI, H., ARAE(II, K., MORI, K.J. & MISHINA, M. (1992).

Cloning, expression and modulation of a mouse NMDA receptor

subunit. FEBS Lett. 300, 39-45.

YEE, G.H. & HUGANIR, R.L. (1987). Determination of the sites of CAMP-

dependent p hosphorylation on the nicotinic acetylcholine recep tor. J.

Biol. Chem. 262, 16748-16753.

ZHANG, L., ZHENG, X., PAUPARD, M.-C., WANG, A.P., SANTCHI, L.,

FRIEDMAN, K . ZUKIN, R.S. & BENNETT, M.V.L. (1994).

Spermine potentiation of recombinant N-methyl-D-aspartate receptors

is affected by subunit composition. Proc. Natl. Acad. Sci. USA 91,

10883-10887.

ZHENG, X., ZHANG, L., DURAND, G.M., BENNETT, M.V.L. & ZUKIN, R.S.

(1994). Mutagenesis rescues spermine and Zn2+ potentiation of

recombinant NMDA receptors. Neuron 12, 81 1-818.

APPLlED A I M G E , lnc - - - 1653 East Main Stree: - -. - Rochester. NY 14609 USA -- -- - - Phone: i l 61482-0300 -- -- - - Fax: 7161288-5969

Q 1993, Applied Image, Inc., All Rights Reserved