Proc. Nati. Acad. Sci. USAVol. 88, pp. 652-656, January 1991Neurobiology

Intracellular Ca2' buffers disrupt muscarinic suppression of Ca2+current and M current in rat sympathetic neurons

[whole-cell voltage clamp/fura-2/bis(2-aminopbenoxy)ethane-NN,N',N'.tetraactate (BAPTA)]

DAVID J. BEECHt, LAURENT BERNHEIM, ALISTAIR MATHIE, AND BERTIL HILLEtDepartment of Physiology and Biophysics, School of Medicine SJ-40, University of Washington, Seattle, WA 98195

Contributed by Bertil Hille, October 24, 1990

ABSTRACT The role of intracellular Ca2+ concentration([Ca2+]i) in the muscarinic suppression of Ca2' current and M-type K+ current has been investigated in isolated rat sympa-thetic neurons using the whole-cell patch-clamp technique andfura-2 fluorescence measurements. Muscarinic stimulationsuppressed currents without raising [Ca2+J1. Nonetheless, in-tracellular bis(2-aminophenoxy)ethane-N,N,N',N'-tetraace-tate (BAPTA) (11-12 mM), a Ca2+ chelator, reduced Ca2+-current suppression from 84 to 46% and M-type K+ currentsuppression from 82 to 15%. For the latter, we explain theBAPTA action by a requirement for a certain minimum (Ca2+J1for continued operation of the pathway coupling muscarinicreceptors to M-type K+ channels. The pathway couplingmuscarinic receptors to Ca channels also showed some depen-dence on [Ca2+],, but there may also be a blocking action ofBAPTA that is independent of Ca2+ chelation.

Many neurotransmitters affect the excitability of neuronsthrough G protein-coupled receptors and intracellular mes-sengers. In rat sympathetic neurons, Ca2l currents (ICa) andM-type K+ currents (IM) are suppressed by muscarinicagonists (1-4). We have been investigating the intracellularpathways of this dual modulation and show here that bothmuscarinic effects are inhibited when the Ca2+-chelatorbis(2-aminophenoxy)ethane-N, N, N',N '-tetraacetic acid(BAPTA) (5) is applied intracellularly. We then ask whetherthe muscarinic receptors are using changes in intracellularCa2' concentration ([Ca2]i) as a signal or if the BAPTAeffect results from a requirement for minimal free [Ca2+]i forthe signaling pathway or even from a direct effect ofBAPTAitself independent of Ca2+ chelation.

MATERIALS AND METHODSCell Preparation. Male 5- to 6-week-old Sprague-Dawley

rats were anesthetized with sodium pentobarbital and decap-itated. The superior cervical ganglia (SCG) were removedand placed in modified Hanks' solution containing papain (20units per ml). After 20 min at 37°C the papain was replacedby collagenase (590 units per ml)/dispase (8-16 mg/ml) for afurther 45 min, with trituration every 15 min. The cells werecentrifuged and resuspended in L-15 medium/10o fetal bo-vine serum. The neurons varied in appearance and often hadneurites; typically the diameter of the soma was 10-30 ,Am.Neurons were maintained at 4°C, and 1Ca was recorded within10 hr. For IM recording, dissociated neurons were plated oncollagen-coated plastic dishes and maintained up to 32 hr inculture medium at 37°C (5% C02). After 1 day, the neuronswere round and rarely had neurites. The standing outwardcurrent at -30 mV (mostly IM) ranged from 123 to 1424 pA

(n = 75)-much larger than in freshly dissociated neurons,where it was >100 pA in only 7 of 29 cells.Current Recording and Perfusion. Neurons were voltage-

clamped by the whole-cell method (6) at 20-25TC usingpipettes made from hematocrit glass (VWR Scientific) withresistances of 1-2 MQ. Currents were amplified using anAxopatch-iC or a List EPC-7 amplifier and low-pass filteredat 1 kHz. All potentials in the text have been corrected forjunction potentials of -2 mV (0.1 mM BAPTA) or -4 mV (20mM BAPTA). Current was digitized, recorded, analyzed,and plotted with BASIC-FASTLAB (Indec Systems, Sunnyvale,CA). Time given in the figures is time after breakthrough. Tostudy 'Ca, we held the membrane at -80 mV and steppedevery 4 s to -40 mV for 2.5 ms and then to 0 mV for 10 ms(see Fig. lAii). ICa was measured as peak inward currentachieved during the test step minus current recorded laterwhen 100 ,xM Cd2+ was added to the bath. To study IM, theholding potential was -30 mV (where a standing outward IMexists), and the membrane was stepped to -60 mV for 0.5 severy 5 s (see Fig. 2Aii) to close IM channels. When twodifferent intracellular solutions were being compared, wetried to alternate them on every other cell to reduce system-atic bias. Means are given with SEMs. Sample means arecompared by an unpaired Student's t test; P is the probabilitythat the population means are the same. The bath solutionwas constantly flowing at 2-2.5 ml/min through a 100-,ulchamber and was fully changed in 20 s. To avoid stimulatingnicotinic receptors we used the selective muscarnnic agonistoxotremorine methiodide (oxo-M). The usual concentration,10 ,uM, always gave maximal responses. In some ICa exper-iments with 10 mM extracellular tetraethylammonium(TEA), 100 ,M oxo-M was used, as TEA shifted the oxo-Mconcentration-response curve =20-fold to the right (but didnot affect maximum response).

[Ca2J1, Measurement. [Ca2+]i was measured with fura-2loaded into single neurons from the whole-cell pipette (7, 8).[Ca2+], was calculated using the equation:

[Ca2+]i = K*(R - Rmin)/(Rmax -R) [1]

where R is the ratio of the two fluorescence intensities (337nm/380 nm excitation light), and K* is a constant that definesthe calibration curve near resting [Ca2+]j. Rmin was measuredafter dialysis of neurons with a solution of 70 mM EGTA/0.1mM fura-2/50 mM Hepes, pH 7.4; Rmin was 0.676 (the lowestof three measurements). Rm,, was estimated by dialyzingwith standard 0.1 mM fura-2 pipette solution and then lysingthe neuron with a -150-mV step; Rm,, was 104.6 (the higherof two measurements). K* was measured by dialyzing neu-

Abbreviations: BAPTA, bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; TEA, tetraethylammonium; oxo-M, oxotremorine me-thiodide; SCG, superior cervical ganglia; IR, Ca2+ current; IM,M-type K+ current; [Ca2+]j, intracellular Ca + concentration.tPresent address: Department of Pharmacology, St. George's Hos-pital Medical School, London, SW17 ORE, United Kingdom.tTo whom reprint requests should be addressed.

652

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

Proc. NatL. Acad. Sci. USA 88 (1991) 653

rons with a pipette solution of 50 mM EGTA/25 mM CaC12/0.1 mM fura-2/50 mM Hepes, pH 7.48, which has a free Ca2+concentration of 42 nM [calculated according to Blinks et aL(9)]. Similar experiments were done using the Cs/20 mMBAPIA pipette solution with 10mM added Ca2' and 0.1 mMfura-2, which had a free Ca2+ concentration of 143 nM asmeasured on coverslips using an EGTA-calibrated curve. Forthe EGTA solution K* was 2905 nM (n = 5), and for theBAPTA solution, K* was 2963 nM (n = 3). We used 2905 nMfor K*.

Materials. Collagenase (type 1), leupeptin, and EGTA werefrom Sigma. Other compounds were as follows: Oxo-M(Research Biochemicals, Natick, MA), BAPTA, 5,5'-dibromo-BAPTA and 5,5'-dinitro-BAPTA (MolecularProbes), fura-2 (Calbiochem), ATP and GTP (PharmaciaLKB), papain (Worthington), dispase (grade 2, BoehringerMannheim), penicillin/streptomycin, fetal bovine serum, andL-15 medium (GIBCO).

Solutions. Modified Hanks' solution was 137 mM NaCl/0.34 mM Na2HPO4/5.4 mM KCI/0.44 mM KH2PO4/5 mMglucose/5 mM Hepes, pH 7.4. Culture medium was L-15medium/26 mM NaHCO3/30 mM glucose/penicillin at 50units/ml/streptomycin at 50 ,ug/ml/5% rat serum. Externalsolution for measuring ICa was 150 mM NaCl/2.5 mM KCI/5mM CaCl2/1 mM MgCl2/10 mM Hepes/8 mM glucose/500nM tetrodotoxin/1 ,uM propranolol with or without 10 mMTEA bromide, pH 7.4. External solution for measuring IMwas 150 mM NaCl/2.5 KCl/2 mM CaCl2/1 mM MgCl2/10mM Hepes/8 mM glucose, pH 7.3. Pipette solutions were asfollows: Cs or K 0.1 BAPTA, 175 mM CsCl or KCl/5 mMMgCl2/5 mM Hepes/0.1 mM BAPTA; Cs or K 20 BAPTA,115 mM CsCl or KCI/5 mM MgCl2/5 mM Hepes/ 20 mMBAPTA; Cs or K 20 dinitro-BAPTA, 115 mM CsCl or KCl/5mM MgCI2/5 mM Hepes/0.1 mM BAPTA/20 mM 5,5'-dinitro BAPTA; 3 mM Na2ATP/0.1 mM NaGTP/80,Mleupeptin was added freshly each day. Cs- and K-basedsolutions were titrated to pH 7.4 and 7.2, respectively. Themeasured osmolality was 315-330 mOsm for all solutions.

RESULTSIntracellular BAPTA. Fig. 1 compares muscarinic suppres-

sion of ICa 5 min after breakthrough with pipettes containinglow or high BAP1TA. With only 0.1 mM BAPTA (Fig. LA),oxo-M reduced peak ICa by 96% (84 ± 2% in 25 cells). Whenoxo-M was washed out, recovery exhibited fast and slowphases and was sometimes incomplete. With 20 mM BAPTA(Fig. 1B), oxo-M reduced ICa by only 37% (46 ± 4% in 16cells). On wash-out, recovery showed only one phase. PeakICa at 0 mV was 1.8 ± 0.2 nA for both 0.1 mM (n = 25) and20 mM BAPTA (n = 16). Mean series resistance and cellcapacitance were 3.7 ± 0.2 MfQ and 3.5 ± 0.4 Mfl, and 36 ±2 pF and 36 ± 2 pF. Norepinephrine also suppresses ICa in ratSCG neurons (10), but the modulation was not affected byintracellular BAPTA: norepinephrine suppressed Ica by 45 ±8% (n = 11) and 40 ± 9o (n = 8) for 0.1 mM and 20mMBAPTA, respectively.

Fig. 2 compares oxo-M suppression of IM in two neurons.With 0.1 mM BAPTA in the pipette (Fig. 2A), oxo-M abol-ished IM (mean suppression 82 ± 3% in 10 cells), and partialrecovery was observed on wash-out. Unlike frog sympatheticneurons (7, 24), these rat neurons did not show overrecoveryofIM when agonist was removed. With 20 mM BAPTA (Fig.2B), oxo-M had almost no effect on IM (mean suppression 15+ 8% in 9 cells). At 12 min after breakthrough, 4m at -30 mVwas 378 ± 50 pA (n = 12) and 425 ± 81 pA (n = 9) for 0.1 mMand 20 mM BAPTA experiments, respectively. Series resis-tance and cell capacitance were 5.6 ± 0.5 MO and 5.2 ± 0.7MO, and 58 ± 6 pF and 54 ± 8 pF. Norepinephrine did notmodulate IM (n = 6). Hence, muscarinic modulation ofIca is,

A (i)

a)rr.

5 6 7 8 9Time, min

B (i)

a)C.

U

3 ' Oxo-M Cd2+5 6 7 8 9

Time, min

(ii) Oxo-M

r.-21-

U 1 ..Control-4

. OmV-80 m T

0 10Time, ms

(ii)0 - -

Oxo-M^ -2 I-0 .. Control

-4OmV

-80 m

0 10Time, ms

FIG. 1. Intracellular BAPTA inhibits muscarinic suppression ofICa. ICa was recorded from freshly dissociated SCG neurons using aCs-based pipette solution and the Ica external solution without TEA.[A(i) and B(i)] Peak ICa values at 0 mV. [A(ii) and B(ii)] Currents forthe control and during oxo-M, as labeled in A(i) and B(i). (A) Pipettesolution contained 0.1 mM BAPTA; 10 ,M oxo-M was applied, asindicated by the horizontal bar, and Cd2+ (100 tiM) was appliedsubsequently (cell R702). (B) Pipette solution contained 20 mMBAPTA; 10 ,uM oxo-M and 100 AM Cd2+ were applied, as indicated(cell R800).

at most, half sensitive to BAPTA, whereas modulation of IMis almost entirely sensitive.Ca2' Measurements with a Low Level of Buffer. Our initial

supposition was that oxo-M causes a rise of [Ca2+]i that is asignal coupling muscarinic receptors to Ca channels or Kchannels, and BAPTA acts by attenuating this signal. With0.1 mM fura-2 in the pipette, the measured resting [Ca2+]j inseven freshly isolated cells at -80 mV was 72 ± 11 nM, closeto values reported for rat (67 nM) and frog (60-79 nM)sympathetic neurons (7, 11, 12). In the experiment of Fig. 3Aour usual 10-ms depolarizing steps were applied every 4 s,and the small voltage-gated Ca2" currents caused tiny tran-sient [Ca2+]1 increases of =5 nM. When oxo-M was applied,the mean [Ca2+]j at first continued a slow upward drift, butlater, when Ca2+-currents became strongly suppressed,[Ca2+li drifted slowly downward, as if Ca2+ entry throughvoltage-gated Ca channels contributed to the level of [Ca2+]J.Norepinephrine, which causes a BAPTA-insensitive sup-pression of ICa, caused a similar loss of the small Ca2+transients and decreased [Ca2+]i (n = 2). In the experiment ofFig. 3A, 0.1 mM BAPTA/0.1 mM fura-2 were in the pipette.In five other experiments using only 0.1 mM fura-2, oxo-Mcaused normal ICa suppression but again no detectable Ca2"rise. In one of these experiments, depolarizing steps wereturned off, enabling us to look for smaller Ca2+ signals.Oxo-M caused only a slight, steady rise in [Ca2+], from 41 to42.5 nM. (ICa was tested before and after application ofoxo-M, and suppression was 91%). Thus, we have beenunable to find a significant Ca2' signal induced by agonists infreshly isolated SCG neurons, and ifone exists, it must be <2nM when averaged over the image of the cell.Ca signals could, nevertheless, be elicited by other means.

Fig. 3B shows that single depolarizing pulses of increasingduration generated progressively larger Ca2+ transients thatdecayed with a 5-s time constant. Each increment in [Ca2+],was proportional to the integral of ICa evoked by the pulse

Neurobiology: Beech et al.

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

Proc. Natl. Acad. Sci. USA 88 (1991)

A(i)600 Oxo-M

(ii)

~~~~~~~600

4001 A- <4<.~200. 0Control

t~ -Oxo-M~~~~~-200

-LJ30 mV~

12 13 14 15 16Time, min

B (i)Oxo-M

400

<200

C 200

12 13 14 15 16Time, min

-oumvUViAV

0 0.5 1Time, s

(ii)

-200

3L__F-30-mV-60 mV0 0.5 1

Time, s

FIG. 2. Intracellular BAPTA inhibits muscarinic suppression ofIM. IM was recorded from cultured SCG neurons using a K-basedpipette solution and the IM external solution. [A(i) and B(i)] Meancurrent at -30 mV over 50-60 ms before each test step (Upper), andthe time-dependent current at -60 mV measured as the differencebetween current 10-20 ms after beginning of voltage step and currentat end of the step (Lower). [A(ii) and B(ii)] Currents for control andduring oxo-M, as labeled in A(i) and B(i). Na' current and A-type K+current developing on return from -60 to -30 mV have beenattenuated by omitting "10 ms of the record. (A) Pipette solutioncontained 0.1 mM BAPTA; 10 1LM oxo-M was applied as indicated(cell M267). (B) Pipette solution contained 20 mM BAPTA; 10 uMoxo-M was applied as indicated (cell M252).

with a proportionality factor of 0.88 nM/pC. Referring theCa2+ entry to the volume of the cell (assuming a sphere withan area equivalent to the 29-pF capacitance) gives a propor-tionality factor of 2.6 nM rise per uM Ca2+ entry, whichmeans that the total buffering capacity of the cytoplasm,including the fura-2, is equivalent to that of 140 AM BAPTA.Bath-applied caffeine also produced an obvious rise of [Ca2+](n = 3). Fig. 3C shows a transient increase to 220 nM abovethe base-line of 65 nM, which then fell to below the controllevel. Similar caffeine-induced Ca2+ transients have beenreported before in rat and frog sympathetic neurons (7, 11,12).Ca2+ Measurements with 20 mM Buffer. If oxo-M does not

make Ca2+ signals, how does BAPTA disrupt signal flowfrom muscarinic receptors to Ca channels and IM channels?As expected, when the pipette solution contained 20 mMBAPTA, the measured [Ca2+]i fell to low levels (12 ± 2 nM,n = 3); Ca2+ variations were highly damped, and 10 mMcaffeine induced no detectable [Ca2W]i rise (n = 2). Depolar-izing test pulses of increasing duration elicited ICa, like thatin Fig. 3B, but now without a detectable [Ca2+]j rise (arrowsin Fig. 4C Lower). We reasoned that perhaps low resting[Ca2+] rather than buffering per se is inhibitory. Even ifCa2+were not a signal, some steps in coupling might be inhibitedin its absence. Therefore, we raised [Ca2W]i by including 10mM CaCl2 with the 20 mM BAPTA in the pipette. In thesecells, measured [Ca2+], was 143 ± 3 nM (n = 6) and stillpowerfully buffered. Thus we could detect no further in-crease of [Ca2+Ji with 10 mM caffeine (n = 1) or with theincrementing test-pulse protocol (arrows in Fig. 4C Upper).Nevertheless, with 10 mM CaCl2/20 mM BAPTA in the

u -0.8-

4

A

:460-

c) 40 -

O T

Control

Oxo-M

Time, min

Time, min

Caffeine

B

110 T

= 90- .*"

+ 70] .501-

C300 -

-: 200--r .dio~

9 10 11 12Time, min

FIG. 3. oxo-M does not elicit a net change of [Ca2+]i. [Ca2+]J wasmeasured by using fura-2 in freshly isolated neurons. ICa wasmeasured simultaneously with the protocol of Fig. 1, except thatexternal solution with TEA was used. (A) The Cs-based pipettesolution contained 0.1 mM fura-2/0.1 mM BAPTA. [Ca2+]i wasmeasured every 0.5 s, and the points are joined to give a continuousrecord (Upper). Leak current at -80 mV (-) and uncorrected peakICa at 0 mV (.-) were measured every 4 s (Lower). (Inset) Currentsfor the marked dots; oxo-M (100 A.M) was applied as shown (cell Fl).(B) The Cs-based pipette solution contained 0.1 mM fura-2 and noBAPTA. (Upper) The cell was held at -80 mV, and ICa was evokedby stepping at 0.1 Hz to 0 mV for 10, 20, 30, 40, and 50 ms. (Lower)[Ca2+]i was measured simultaneously (as in A). ICa is shown on afaster time base than the [Ca2+li measurement (cell F5). (C) TheCs-based pipette solution contained 0.1 mM fura-2. The cell was heldat -80 mV and stepped to 0 mV for 10 ms every 4 s (as in A), but onlythe (Ca2+], measurement is shown; 10 mM caffeine was applied asshown (cell F7).

pipette, suppression ofIM was restored to control levels (81%compared with 82% for 0.1 mM BAPTA control, Fig. 4B).The suppression of ICa was only partially restored (Fig. 4A),

654 Neurobiology: Beech et al.

-1.2 - .

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

Proc. Natl. Acad. Sci. USA 88 (1991) 655

B

0.1 20 Ca NO2

C D

._cl

&

100 140

20

F-I I I

120 160

Time, s

Q

04.

C.)C0u

0 5 10 15Time, min

FIG. 4. Ca2+ buffer experiments. (A) Mean suppression of ICa 5.5 min after breakthrough with Cs-based pipette solutions containing 0.1 mMBAPTA/0 Ca2+ (0.1) (n = 25), 20 mM BAPTA/0 Ca2+ (20) (n = 16), 20 mM BAPTA/8-10 mM Ca2+ (Ca) (n = 10), or 20 mM dinitro-BAPTA/0Ca2+ (NO2) (n = 6). (B) Mean suppression ofIM 12 min after breakthrough with K-based pipette solutions containing (as in A) 0.1 mM BAPTA/0Ca2+ (n = 10); 20mM BAPTA/0 Ca2+ (n = 9); 20 mM BAPTA/10mM Ca2+ (n = 6); 20 mM dinitro-BAPTA/0 Ca2+ (n = 4). (C) [Ca2+]i measuredwith 20 mM BAPTA/10 mM Ca2+ (Upper) or 20 mM BAPTA and no added Ca2+ (Lower). Arrows mark where depolarizing pulses of 10, 20,30, 40, and 50 ms were applied (as in Fig. 3B). For 20 mM BAPTA with Ca2+ test pulses gave 11, 26, 39, 50, and 61 pC of Ca2+ influx, andfor 20 mM BAPTA without Ca2+ they gave 7, 14, 20, 24, and 28 pC. (D) Dialysis of neurons with fura-2 or BAPTA. Fura-2 emission intensitiesfrom the two wavelengths were summed. For 19 experiments, each record was normalized to the maximum intensity or to the amplitude of afitted exponential when a plateau was not achieved. All defined points were then summed and divided by the number of experiments for eachpoint (always >10). The average points (.* ) were plotted against time after breakthrough and fitted by a least-squares method with a singleexponential plus a constant (T = 399 s, - - -). The estimated rates of dialysis for BAPITA (see text) are also shown as single exponentials (-)with the time constants 362 s (line a) and 919 s (line b) for freshly dissociated neurons and cultured neurons, respectively. Arrows indicateestimated BAPTA concentrations in the neurons when oxo-M was applied.

rising to 62 + 6% (n = 10), which is greater (P < 0.05) thanthe 46% with 20 mM BAPTA alone and significantly less (P< 0.0005) than the 84% with 0.1 mM BAPTA. The experi-ments on ICa included three cells with 8 mM CaCl2 and sevenwith 10 mM CaCl2 added to the BAPTA, with a range of[Ca2+], from 100 to 160 nM.As suppression of 'Ca was not entirely restored by raising

the buffered [Ca2+], to a more physiological level, we con-sidered whether BAPTA-like molecules inhibit signaling in-dependent of their ability to chelate. We tested EGTA, ahigh-affinity buffer, and two BAPTA derivatives that arelow-affinity buffers, 5,5'-dibromo BAPTA [Kd = 1.5 ,uM (13)]and 5,5'-dinitro-BAPTA [Kd = 7 mM (13)]. With 10 mMEGTA alone, measured [Ca2+], was 17 and 23 nM in two cells,and suppression of ICa by oxo-M was 54 ± 6% (n = 4), andwith 5 mM CaCl2/10 mM EGTA, [Ca2+] was 159 nM for twocells, and suppression was 89o and 69% in the same cells. Intwo experiments we tried 20 mM dibromo-BAPTA alone.[Ca2+], was 73 nM and 44 nM, but unfortunately the com-pound also nearly eliminated ICa, so we could not studysuppression. Dinitro-BAPTA should not chelate Ca2+ nearthe resting level, but the deep orange color of 20 mMsolutions absorbed the emission light from fura-2, and wecould not check [Ca2+],. Nevertheless, currents could still berecorded. ICa was 25.8 ± 2.3 pA/pF (n = 4) and 51.7 ± 3.5pA/pF (n = 23) for cells dialyzed with 0.1 mM BAPTA/20mM dinitro-BAPTA or 0.1 mM BAPTA alone, and suppres-sion OfICa by oxo-M was 57 ± 7% (n = 6) versus 84% in thesetwo conditions (Fig. 4A). IM at -30 mV was 7.2 ± 1.7 pA/pF(n = 4) versus 6.8 ± 0.9 pA/pF (n = 12) with these solutions,and suppression of IM was 76% versus 82%.

Estimates ofBAPTA Concentration. The cell cytoplasm willnot have equilibrated fully with the pipette solution at thetime of oxo-M applications. We estimated the rate of dialysiswith BAPTA by using our measurements on fura-2. Byaveraging 19 records we found that fura-2 entered cells witha time constant (Tf) of 399 s (Fig. 4D). According to Pusch andNeher (14), the corresponding time constant (Tb) for BAPTAdialysis into the same cells would be Tf(Mb/Mf)'13, where Mis the molecular weight. Thus Tb would be -362 s in freshly

dissociated neurons. In the cultured cells used for IM, Tb

would be longer (=919 s) because of larger cell size and seriesresistance, Rs (the value is proportional to RSC3/2, where Cis the membrane capacitance). We calculate that, on average,the concentration of BAPTA in the cell would be 59o and54% of that in the pipette for freshly dissociated neurons(5.5-min dialysis) and cultured neurons (12-min dialysis),respectively. Thus, for 20 mM BAPTA experiments, freshlydissociated neurons (Ica) would, on average, contain 12 mMBAPTA, and cultured neurons (IM) would contain 11 mMBAPTA at the time oxo-M was applied-i.e., the buffercapacity of the cytoplasm would be raised 100-fold above itsnormal value.

DISCUSSION

We have not detected any agonist-related [Ca2+], signal infreshly isolated SCG neurons in response to oxo-M ornorepinephrine. Nevertheless, the muscarinic (but not theadrenergic) modulation of currents is sensitive to the inclu-sion of Ca2' buffers in the pipette solution. The action isprimarily on receptor-channel coupling, as the currents are ofnormal size with BAPTA in the pipette. For the modulationof IM we show that the strong effect of chelators is entirelyrelated to a lowering of resting [Ca2+]i and is attributableneither to a damping of [Ca2+]i variations nor to an inhibitoryeffect of BAPTA-like molecules. When [Ca2+], is stronglybuffered but high, a normal large suppression of IM occurs.The modulation of ICa by oxo-M differs in several respectsfrom the modulation of IM. (i) Only half of it is sensitive toBAPTA-like buffers, so a significant modulation persists with20 mM BAPTA or 10 mM EGTA in the pipette. (ii) Bringing[Ca2+], up again by adding CaCl2 to the buffer solutionrestores some of the lost suppression but not all of it. Evenwith raised [Ca2qJ, BAPTA-like molecules seem to interferemoderately with coupling between muscarinic receptors andCa channels. Whatever the mechanism, the results suggestthat solutions with very low concentrations of Ca2' buffersmight be best for routine studies of modulation, and they may

A100 -.

W 50

v:

Neurobiology: Beech et al.

. . . . . .

-rc~

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0

Proc. Natl. Acad. Sci. USA 88 (1991)

explain why we see more modulation of ICa and IM than isreported in most studies.

In the intact rat SCG, inositol 1,4,5-trisphosphate levelsincrease moderately in response to bethanechol, a muscarinicagonist (15). Elsewhere, oxo-M is typically a full agonist atmuscarinic receptors that couple to inositol 1,4,5-trisphos-phate production (16). However, in agreement with theobservations of Wanke et al. (3), we do not observe a Ca2lrise in response to oxo-M in single neurons isolated from ratSCG. It may be that inositol 1,4,5-trisphosphate levels in-crease in cells in the ganglion other than the neurons fromwhich we record or that inositol 1,4,5-trisphosphate levelsincrease too slowly to be related to the fast changes weobserve. Alternatively, there may be a Ca2' rise that isinitiated in a restricted subcellular compartment, appearingonly as a small (<2 nM) net Ca2' rise when measuringaverage [Ca2+], from the entire neuron. However, our ex-periments with high concentrations of Ca2+ buffers argueagainst any Ca2' change being required for muscarinic sup-pressions of ICa or IM. This agrees with the conclusions ofearlier work from this laboratory with IM suppression in frogsympathetic neurons (7), except that there it was argued thatsuppression persists even when [Ca2+]i is very low. Thoseexperiments used 5 mM BAPTA pipettes, larger cells, anddialysis times of 6 min, and because of a different fura-2calibration, probably underestimated the [Ca2+]i achieved,especially at the lowest concentrations. In the present ex-periments, even with 20 mM BAPTA, we saw significant, butreduced, modulation ofIM (55 ± 7%, n = 3) when we dialyzedonly 5-8 min. Therefore, we cannot rule out that modulationof IM in frog is as sensitive to [Ca2+1 as it is in the rat.How might Ca2' dependence of a coupling pathway be-

tween receptors and ionic channels arise? There are manyintracellular enzymes that show a requirement for Ca2+ (17)and for some ofthese, there is evidence that they are involvedin the coupling of receptors to Ca and K channels (18-20).Protein kinase C, for example, appears to couple cholecys-tokinin receptors to Ca channels in Helix neurons, and thisprocess seems to require normal [Ca2+]1 (21). In rat sympa-thetic neurons, however, protein kinase C seems not tocouple muscarinic receptors to Ca channels or M-type Kchannels (3, 22). Other Ca2+-sensitive molecules, such ascalmodulin, calcineurin, or phospholipase A2 could be theorigin of the Ca2+ dependence we observe.We find that ICa suppression is inhibited only partially by

the same concentration of intracellular BAPTA that inhibitsIM suppression almost completely. This could mean eitherthat the coupling pathway to Ca channels is less sensitive to[Ca2+], or that two pathways couple to Ca channels, one Ca2+dependent and one Ca2+ independent. In support of the ideathat there are several pathways, we find that ICa suppressionby norepinephrine is not affected by intracellular BAPTA.We have also found that pertussis toxin blocks muscarinicsuppression of ICa in rat SCG neurons reliably but only

partially (23). IM suppression, however, is insensitive topertussis toxin (4), suggesting that it uses only one of thepathways.

We thank W. Almers for use of his fura-2 apparatus and W. Almersand P. Thomas for much help with [Ca2+]i measurements. M.Leibowitz collaborated in the early development of the SCG prep-aration. L. Miller and D. Anderson assisted us in our work. We alsothank W. Almers, B. P. Bean, M. M. Bosma, K. P. Mackie, A. Tse,L. P. Wollmuth, and J. Yang for reading the manuscript. The workwas supported by National Institutes of Health Grants NS 08174 andAR 17803, a McKnight Neuroscience Research Award, a fellowshipfrom la Fondation Suisse de Bourses de M6decine et Biologie and theMuscular Dystrophy Association, and a Fogarty International Re-search Fellowship F05 TW04577.

1. Constanti, A. & Brown, D. A. (1981) Neurosci. Lett. 24,289-294.

2. Marrion, N. V., Smart, T. G. & Brown, D. A. (1987) Neurosci.Lett. 77, 55-60.

3. Wanke, E., Ferroni, A., Malgaroli, A., Ambrosini, A., Pozzan,T. & Meldolesi, J. (1987) Proc. Natl. Acad. Sci. USA 84,4313-4317.

4. Brown, D. A., Marrion, N. V. & Smart, T. G. (1989) J. Phys-iol. (London) 413, 469-488.

5. Tsien, R. Y. (1980) Biochemistry 19, 23%-2404.6. Hamill, 0. P., Marty, A., Neher, E., Sakmann, B. & Sigworth,

F. J. (1981) Pfluegers Arch. 391, 85-100.7. Pfaffinger, P. J., Leibowitz, M. D., Subers, E. M., Nathanson,

N. M., Almers, W. & Hille, B. (1988) Neuron 1, 477-484.8. Thomas, P., Surprenant, A. & Almers, W. (1990) Neuron 5,

723-733.9. Blinks, J. R., Wier, W. G., Hess, P. & Prendergast, F. G.

(1982) Prog. Biophys. Mol. Biol. 40, 1-114.10. Galvan, M. & Adams, P. R. (1982) Brain Res. 244, 135-144.11. Thayer, S. A., Hirning, L. D. & Miller, R. J. (1988) Mol.

Pharmacol. 34, 664-673.12. Lipscombe, D., Madison, D. V., Poenie, M., Reuter, H.,

Tsien, R. W. & Tsien, R. Y. (1988) Neuron 1, 355-365.13. Pethig, R., Kuhn, M., Payne, R., Adler, E., Chen, T.-H. &

Jaffe, L. F. (1989) Cell Calcium 10, 491-498.14. Pusch, M. & Neher, E. (1988) Pfluegers Arch. 411, 204-211.15. Bone, E. A., Fretten, P., Palmer, S., Kirk, C. J. & Michell,

R. H. (1984) Biochem. J. 221, 803-811.16. Jacobsen, M. D., Wusteman, M. D. & Downes, C. P. (1985) J.

Neurochem. 44, 465-472.17. Kennedy, M. B. (1989) Trends Neurosci. 12, 417-420.18. Armstrong, D. L. (1989) Trends Neurosci. 12, 117-122.19. Dolphin, A. C. (1990) Annu. Rev. Physiol. 52, 243-255.20. Brown, D. A. (1990) Annu. Rev. Physiol. 52, 215-242.21. Hammond, C., Paupardin-Tritsch, D., Nairn, A. C., Green-

gard, P. & Gerschenfeld, H. M. (1987) Nature (London) 325,809-811.

22. Grove, E. A., Caulfield, M. P. & Evans, F. J. (1990) Neurosci.Lett. 110, 162-166.

23. Bernheim, L., Beech, D. J. & Hille, B. (1990) Soc. Neurosci.Abstr. 16, 793.

24. Pfaffinger, P. J. (1988) J. Neurosci. 8, 3343-3353.

656 Neurobiology: Beech et A

Dow

nloa

ded

by g

uest

on

Oct

ober

17,

202

0