a novel photoaffinity ligand for the phencyclidine site of the n

THE JOURNAL OF BIOI.OGICA~ CHEM~~Y 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 265, No. 12, Issue of April 25, pp. 67764781, 1990 Printed in U.S.A.

A Novel Photoaffinity Ligand for the Phencyclidine Site of the N- Methyl-maspartate Receptor Labels a Mr 120,000 Polypeptide*

(Received for publication, October 23, 1989)

Mark S. Sonders$, Peter Barrnettler& Jeffrey A. Lee& Yoshiyasu Kitahara& John F. W. Keanag, and Eckard Weber$Vl From the Vellum Institute, Oregon Health Sciences University, Portland, Oregon 97201 and the SDepartment of Chemistry, University of Oregon, Eugene, Oregon 97403

A radiolabeled photoaffinity ligand has been devel- oped for the N-methyl-D-aspartate (NMDA)-preferring excitatory amino acid receptor complex. [3H]3- Azido-(5S, lOR)(+)-5-methyl-lO,ll-dihydro-5H-di- benzo[u,cZ]cyclohepten-5,10-imine [3H]3-azido-MK- 801 demonstrated nearly identical affinity, density of binding sites, selectivity, pH sensitivity, and pharmacological profile in reversible binding assays with guinea pig brain homogenates to those displayed by its parent compound, MK-801. When employed in a photolabeling protocol designed to optimize specific incorporation, [3H]3-azido-MK-801 labeled a single protein band which migrated in sodium dodecyl sulfate-polyacrylamide gels with Mr = 120,000. Incorporation of tritium into this band was completely inhibited when homogenates and [3H]3-azido-MK-SOl were coincu- bated with 10 pM phencyclidine. These data suggest that the phencyclidine site of the NMDA receptor complex is at least in part comprised of a Mr = 120,000 polypeptide.

The NMDAI preferring glutamate receptor is an excitatory amino acid receptor that is found abundantly in mammalian brain and spinal cord. Much attention has been directed toward this receptor, in part due to its hypothesized roles in such phenomena as learning and memory, regulation of nervous system excitability and neuroprotection (reviewed in Refs. l-4).

Although the pharmacology (l-3) and the electrophysiology (1, 4) of the NMDA receptor have been well characterized, little is known about the structure of this molecule. It is a ligand-gated cation channel which contains at least five in- dependent and functionally interacting binding sites. Two of these are ligand recognition sites: 1) a site for NMDA agonists and competitive antagonists such as glutamate and Z-amino- &phosphonovalerate, respectively, and, 2) a strychnine-in-

*This work was supported by Grant MH40303 (to E. W.) and Grant MH42068 (to E. W. and J. F. W. K.) from the National Institute of Mental Health and by a grant from Cambridge Neuro- Science, Inc., Cambridge, MA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be herebv marked “acluertisement” in accordance with 18 U.S.C. Section 1734 iolely to indicate this fact.

i Present address: Deut. of Pharmacolom. Colleee of Medicine. Uiiversity of California, Irvine, CA 92717. -” -

Yl To whom correspondence should be addressed. ’ The abbreviations used are: NMDA. N-methyl-D-aspartate; MK-

801, (5s, lOE), (+)-5-methyl-lO,ll-dihydro-5~-diben~o[cz,d]cyclo- hept-5,10-imine dizocilpine; PCP, phencyclidine, N-(l-(phenyl) cyclohexyl)piperidine; TCP, jV-(l-(2-thienyl)cyclohexyl)piperidine; SDS, sodium dodecyl sulfate; HPLC, high performance liquid chromatography.

sensitive glycine site at which glycine potentiates the effects of NMDA agonists and at which 7-chlorokynurenate and several other drugs are selective antagonists (reviewed in Ref. 5). Three other sites have been proposed to be associated with the ionophore: 3) a site at which the Mp ion mediates a voltage-dependent blockade on the ion flux, 4) a site at which MK-801 (6, 11, 12) and the psychotomimetic drugs PCP, TCP, ketamine, cyclazocine, and SKF 10,047 (N-allylnorme- tazocine) bind (7) with high aftinity and block ion flux in a use-dependent, non-competitive manner, and 5) a site at which Zn’+ (8) can also block ion flux. Other distinct sites have been proposed for polyamines (9) and the neuroprotec- tive and vasodilatory drug ifenprodil (10).

Numerous groups have employed electrophysiological and biochemical techniques to study the interactions of agents working through the various sites. It is clear that the NMDA and glycine sites can modulate binding at the PCP site, but unequivocal evidence is lacking for a reciprocal effect on the recognition sites by PCP-site ligands. Knowledge about the function of the NMDA receptor has not yielded a compelling model of its structure.

In order to investigate the structure of the NMDA receptor, we have synthesized a radiolabeled photoaffinity ligand based on the drug MK-801, the ligand that demonstrates the highest affinity for the NMDA receptor complex of any yet described (6, 11-13). [3H]3-Azido-MK-801 is chemically inert until ac- tivated by ultraviolet light. The binding site of the unphoto- lyzed ligand can therefore be characterized in the same manner as any other binding site for a reversible ligand. Data presented here demonstrate that the characteristics of [3H]3- azido-MK-801 binding are virtually identical to those of its parent compound and are consistent with its binding to the PCP site. Furthermore, we have performed photoaffinity labeling employing a protocol intended to maximize the specific binding to this site and, consequently, the chances of radiolabeling a constituent protein of the NMDA receptor complex.

MATERIALS AND METHODS

Synthesis of [3H]3-Azido-MK-801 (9)-We had previously reported that 3-azido-MK-801 displayed a potency in inhibiting [3H] MK-801 binding in guinea pig brain homogenates equivalent to that of MK-801 itself (14). Therefore, we sought to develop and investigate a radiolabeled analogue that could be-used to coialently tag the NMDA receptor. Details of the synthesis will be reported elsewhere. Briefly, (+)-MK-801 free base (1, see Fig. 1) was prepared from (+) MK-801 maleate as described (13). Acetvlation of 1 gave 2 which was nitrated (NH4NOa, trifluoro&tic aci; anhydride, %I “C, Ref. 15) in CDC& or CH&N to give a mixture of two major and two minor mono nitro isomers together with small amounts of dinitro com- pounds. Pure 3-nitro isomer 3, m.p. 221-222 ‘C (from toluene, the most polar of the mono nitro isomers), was obtained by preparative thin layer chromatography over silica gel (three elutions with ether).

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Photolabekg of the PCPjNMDA Receptor

1 X = Y = 2 = H; C+)MK-SO1 6 X=Z=Br;Y=NHz 2 X=Y=Z=“;N-*c&y, 7 X,Z=H,Br;Y=NHZ 3 X=Z=H;Y-NOzN-Acetyl 8 X,Z=H,3H;K=NHZ 4 X=Z=RY=NH2 9 X,Z=H,3H;Y*N3 5 X=Z=H;Y=N3

FIG. 1. MK-801 analogues.

In a meeting abstract (14), we had tentatively assigned the nitro group in this isomer to the 7-position. However, a recently completed series of two-dimensional nuclear Overhauser effect 300 MHz NMR spectroscopy experiments indicate that the nitro group is in the 3- position. A mixture of mono nitro isomers containing mostly 3 was next reduced and deacetylated (hydrazine hydrate, NaOH, ethylene glycol, reflux 10 h) to give pure 3-amino-MK-801 (4) after chromatography. Diazotization followed by treatment with NaNs gave azide 5 (MS m/e 262.122Q calculated for &Hi4Na, 262.1218). Bromination (Br?, sodium acetate, CDCb, trifluoroacetic acid) of amine 4 gave a mixture of bromides 6 and 7 which was subjected to catalytic reduc- tion in the presence of ‘HZ gas (Amersham Corp.) to give tritiated amine 8.

[3H]3-Azido-MK-801 (9) was prepared from 8 by diazotization of the precursor with a molar excess of NaNOz (10 mg/ml in 3 N acetic acid) for 15 min on ice in darkness followed by addition of a molar excess of NaN% (10 mg/ml in 3 N acetic acid). After 15 min in the dark, the mixture was diluted with 0.1% trifluoroacetic acid (w/v) and purified by reverse-phase HPLC (octadecyl silica, Vydac 218TP54) with a 0.1% trifluoroaceti~ acid to acetonitrile, 0.1% trifluoroacetic acid gradient (flow 1 ml/min). The HPLC run was moni- tored by measuring 214 nm absorbance. One-ml fractions were col- lected and aliquots assayed for tritium by liquid scintillation spectrometry using Cytoscint ES* (ICN Radiochemicals, Irvine, CA) at an efficiency of -48%. The major radioactive peak eluted at the same position as “cold” 3-azido-MK-801 standard and was easily separable from all other ultraviolet-absorbing and radioactive peaks. The specific activitv of 13H13-azido-MK-801 was estimated to be 43 Ci/mmol by measuring the radioactivity associated with the ultraviolet peaks and interpolating the mass of these peaks from calibration curves generated from coeluting standards. [3H]3-Azido-MK-801 was prepared and purified within 2 days of use in binding assays or for photoaffinity labeling. It was stored at -70 ‘C until use.

Guinea Pig Brain Membrane Preparation-Frozen guinea pig brains were acquired from Biotrol (Indianapolis IN) and orepared as - - - previously described (131, with slight modification. Briefly, frozen brains were homogenized with a PoIytron for I min in 10 volumes of ice-cold 0.32 M sucrose containing a protease inhibitor mixture of 0.1 rnM phenyImethylsulfony1 fluoride, 1 mM NaZEDTA, and 50 pg/ml bacitracin (Sigma) and then centrifuged at 900 X g at 4 OC for 10 min. The supematant was recentrifuged at 13,000 X g for 30 min. After the second supernatant was aspirated, the pellet was resuspended in 10 original volumes of 5 mM Tris-HCl, pH 7.8, including the inhibitor mixture, and recentrifuged. The third supernatant was discarded and the pellet resuspended in 5 original volumes of Tris/ inhibitor buffer. Protein concentration of the homogenate was determined with a Bio-Rad protein assay using thyroglobulin as a standard. Tris/inhibitor buffer was then added to bring the homogenate to a concentration of 3 mg/ml. Aliquots were frozen at -70 ‘C until used.

Binding Assay Protocol-Membranes were thawed and diluted to 125% of the desired final concentration with 5 mM Tris-HCl buffer. pH 7.8, including the inhibitor mixture. All drug standard and tracer solutions were made in this buffer. Incubations were performed by adding 100 ~1 of tracer and 100 ~1 of drug standard or buffer to 800 ~1 of membrane homogenate for a final volume of 1 ml. Assays were terminated by diluting the homogenates with an additional 3.5 ml of 5 rnM Tris buffer and filtering rapidly through Schleicher & Schuell 32 glass fiber paper with a 48Iwell cell harvester (Brandel, Gaithers- burg, MD). Following filtration samoles were immediatelv washed twi& with 5 ml of Tris buffer. Filter paper was treated w&h 0.03% polyethyleneimine for at least 1 h prior to filtration in order to minimize nonspecific adsorption of the radioligand. Filters were dis-

solved in 10 ml of Cytoscint ES* and counted by liquid scintillation spectrometry.

Time course experiments demonstrated that [3H]3-azido-MK-801 binding reached a maximum after 4-6 h at 21 *C. Equilibrium was not attained more rapidly nor was the level of binding stimulated when time course experiments were performed with 10 FM r,-glutamate and 1 pM glycine added to the incubation buffer (data not shown). Tissue dilution experiments showed that specific binding of [3H]3-azido-MK-801 varied linearly with membrane protein concentration in the range of 50-400 pg/ml (data not shown). Therefore, characterization of reversible [‘H]3-azido-MK-801 binding was performed by incubating radioligand with approximately 150 pg of membrane protein/ml for at least 4 h. Nonspecific binding was defined by inclusion of 10 /AM PCP.

Experiments on the pH sensitivity of binding were performed by thawing homogenates, centrifuging them at 30,000 x g for 10 min, and resuspending the pellets in 5 rnk+ Tris (no HCl) with protease inhibitors to a concentration of -150 pg of protein/ml. 20-ml samples of membrane homogenate were then titrated with HCl to pH 6.5,7.0, 7.2, 7.4, 7.6, 7.8, 8.0, and 8.5. Volumes of 980 &assay tube were removed and incubated with 10 ~1 of radioligand and 10 ~1 of water or a 1 mM PCP solution. Assays were terminated as described above. The pH values of the remaining unused samples were remeasured after 2 and 4 h and were found to be stable over the course of the assays.

Saturation analyses were performed with a concentration range of [3H]3-azido-MK-801 between 0.5 and 120 nM. The equilibrium dis- sociation constant (I&), binding capacity (B,&, and apparent Hill coefticient (nn,+r) were calculated by non-linear least squares regression analysis using the program LIGAND (16).

Potencies of drugs to inhibit reversible [‘H]3-azido-MK-801 binding were estimated by interpolating I&, values from semi-log plots of drug inhibition curves. Ki values calculated by the equation KC = I&/(1 + [L*]/&*). All drugs were assayed at least three times in triplicate.

P~t~~inity cling-2~ or 400 pg of membrane protein/ml was incubated with Cl0 nh+ [3H]3-azido-MK-801 (<450,000 cpm) in the dark for 4 h at 21 ‘C. Parallel samples (“NSB”) were always run that were treated identically except for the inclusion of 10 pM PCP in the initia1 incubations. Homogenates were diluted with 3.5 ml of Tris/ inhibitor buffer and filtered with 2.4-cm Whatman GF/B or C discs on a Hoeffer vacuum manifold. The filters were then washed twice with 5 ml of buffer. The filters were not treated with polyethyleneimine in order to minimize the chance of radiolabeling that polymer. In order to activate the azide group, the filters were then placed on a sheet of aluminum foil on top of an ice water bath and irradiated with 366 nm light (500 watt, Sunjet 400T lamp, Electrolux-Kern GmbH, Goettingen) for 15 min at a distance of 15 cm. Filters in batches of two or four were then placed in glass scintillation vials with 1 ml/filter of a 0.5% solution of SDS and placed on a rotary shaker at 21 ‘C for at least 1 h. Supernatants were decanted, the tilters were squeezed with a glass rod, and the pooled supernatants were filtered through a 0.45-pm pore Acrodisc (Gelman Sciences, Ann Arbor). The filtrates were then concentrated by lyophilisation.

The time course of irradiation for photoaf~nity labeling was stud- ied by measuring the incorporation of radioactivity into acetone- precipitable molecules. Photolabeling was performed as described except that the UV exposure time of batches of four filters was varied. Following solubilization of the labeled membranes with 0.5% SDS, 4 volumes of cold acetone were added to the filtrates. The mixtures were incubated at -20 ‘C for >20 min and then centrifuged at 9000 X g for 10 min at 4 ‘C. The pellets were then resuspended in -500 hl of 2% SDS, added to scintillation fluid, and counted.

SDS-Polyacrylumide Electrophoresk-Samples were prepared by addition of 50-100 ~1 of treatment buffer consistina of 125 mM Tris- HCl, pH 6.8, 4% SDS, 20% glycerol, 10% 2-mer~aptoetbanol and bromphenol blue. They were dissolved with gentle shaking and either permitted to stand at 21 -C! for longer than 2 h or else placed in a boiling water bath for 2 min. These samples were then transferred to Eppendorf microcentri~ge tubes and centrifuged at 8000 rpm for 10 min to pellet out insoluble and pa~icula~ matter.

Samples were electrophoresed on discontinuous 1.5 mm 0.1% SDS- polyacrylamide slab gels according to the method of Laemmli (17), with modifications. Stacking gels (2 cm) were composed of 4% acrylamide monomer (the crosslinker N,N’ methylene-bis-acrylamide was 2.7% of all monomer concentrations). Resolving gels (12 cm) were comprised of various monomer concentrations between 5 and 10%. Gel and buffer components were acquired from Bio-Rad and were of


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6778 Fhot~~beli~~ of the PCP~~M~A Receptor

the highest grade available. Electrophoreses were performed in a Hoeffer SE400 gel apparatus using 30 mA constant current for 4 h. All gels included two lanes of molecular weight standards (Sigma) comprised of 5 pg each of rabbit muscle myosin (iVZr 205,~), @- galactosidase (Z& 116,000), rabbit muscle phosphorylase b (Ii& 97,400), bovine albumin (Mr 66,000), egg albumin (ZVZ~ 45,000) and carbonic anhydrase (ZV& 29,000).

Following electrophoresis, gels were stained for 20 min with a 0.125% (w/v) Coomassie Blue R-250 solution containing 50% methanol and 10% acetic acid (v/v) and destained with 50% methanol, 10% acetic acid. Thereafter gels were treated for fluorography by incubating them in En3Hance (Du Pont-New England Nuclear) for 90 min, transferred into 5% glycerol for 30 min, and dried. Calibration equations were generated for each gel using Cricket Graph (Cricket Software, Malvern, PA) to perform linear regressions of logI Mr uersus RF of the stained standards. Correlation coefficients were r 2 0.94 for all gels. Gels were apposed to Kodak X-Omat AR film for 7- 12-weeks at -70 -C.

Drugs: (+) and (-)-MK-801 were synthesized and purified by procedures previously published (13). MK-801 maleate was also a gift from Merck (West Point, PA). The (-)-isomer was also purchased from Research Biochemicals (Natick, MA). Dexoxadrol-HCl and levoxadrol-HCl were gifts of Dr. James Woods. PCP-HCl, cyclazo- tine, and SKF 10,047 stereoisomers were acquired through the Na- tional Institute on Drug Abuse Research Technology Branch. Keta- mine-HCl stareoisomers were gifts of Parke-Davis (Ann Arbor, MI).

RESULTS AND DISCUSSION

Characterization of Reversible [3H]3-Azido-MK-801 Bind- ing-We have described the development and use of [3H]3- azido-MK-801, a novel photoaffinity ligand for the high affinity PCP-binding site of the NMDA receptor. Its characteristics of reversible binding are consonant with those that give its parent compound, MK-801, the ability to bind to the NMDA receptor with higher affinity and selectivity than any other ligand yet described.

The reversibIe binding of [3H]3-azido-MK-801 displayed a strong pH dependence in 5 mM Tris buffer, similar to that observed for [3H]MK-801 binding in guinea pig brain membrane homogenate (13). Utilizing a concentration of 2.2 nM [3H]3-azido-MK-801 (-100,000 cpm) and 150 pg protein/ml, nonspecific binding (defined by 10 pM PCP) remained at 0.4% of total counts over the pH range 6.5 to 8.5. In contrast, specific binding increased linearly from 0.7% at pH 6.5 and 7.0 to 3.2% at pH 8.0, and declined to 2.9% at pH 8.5. We previously reported that [3HJMK-801 binding reached a maximum at pH 7.6-7.8 (13).

Equilibrium saturation experiments with concentrations of [3H]3-azido-MK-801 up to 120 nM indicated that the ligand binds saturably (Fig. 2). Analysis of the saturation data with LIGAND indicated that the data is best fit to a single site model with an apparent KD of 3.4 nM, a Bmax of 1.12 pmol/mg membrane protein, and an Knapp of 1.00 (mean values from two experiments, tubes in quadruplicate), These figures are in reasonably good agreement with ones derived from [3H] MK-801 saturation binding experiments: KD = 2 nM and &,,aX = 1.28 pmol/mg (13).

The pharmacological profile of sites labeled by 2 nM [3H]3- azido-MK-801 appears quite similar in rank order and abso- lute potencies to the high affinity Pep-binding site of the NMDA receptor (Table I). This site has been well characterized in rodent brain with [3H]TCP and the more selective and higher affinity [3H]MK-801 (l-3, 6, 7, U-13). Its hallmarks are a strong stereoselectivity for dexoxadrol over levoxadrol, somewhat weaker selectivity for D- over r,-ketamine, (-)-over (+)-cyclazocine, (+)- over (-)-SKF 10,047, (+)- over (-)- MK-801, and a rank order potency of MK-801> TCP > PCP > ketamine. The correlation coefficient for the K< values of eight drugs numbered in Table I for inhibition of [3H]3-azido- MK-801 and [3H]MK-SOl binding is 0.92 (p C 0.001).

0 20 40 60 80 100

pmol [3H]3-N34#K-801

FOG. 2. Saturation analysis with ]3H]3-azido-MK401. Ab- s&sa, quantity of [‘H]3-azido-MK-801 added/assay tube. Tubes were incubated in tha dark with tracer and 0.4 mg of protein in 1 ml. Total binding (X- X), nonspecific binding (10 KM PCP included) (w), and specific binding (A-A) are shown for a single representative experiment, tubes in quadruplicate. Znset, Scatchard transformation of data. Mean values determined using LIGAND were KD 3.4 nM and Bmex 1.12 pmol/mg protein.

TABLE I Potencies of drugs inhibitiw reuersibfe pH]3-azida-MK-801, PHI

MK-801, and pH]TCP binding to guinea pig brain ~~ogenates

1. (+)-MK801 (-)-MK801

2. TCP 3. Dexoxadrol

Levoxadrol 4. PCP 5. (-)-Cyclazocine 6. (+)-Cyclazocine 7. (+)-SKF 10,047 8. (-)-SKF 10,047

B-Ketamine L-Ketamine rrL-Ketamine

[3H]3-Azido-MK801 [‘H]MK801’ [‘H]TCP’

nhf

1.5 k 0.3 2.0 * 0.3 15 * 1.4 3*7 E?E 0.3 5.7 Lk 0.5 12 z!z 0.3 27 zk 2.2 9.3 zk 1.3 15 * 1.4 50 zk 2.5

z=.lOoo >lOOO 18.7 zt 1.3 45 2 6.3 80 * 4.1 28.7 & 3.3 15 zk 3.2 46 k 4.6 116 & 23 160 z!z 20 327 k 32 120 k 8 210 * 12 313 zk 22 353 & 52 260 zk 16 587 * 11 467 AZ 47

38’70 k 400 940 2 25 967 3172

r = 0.92 D c 0.01

a & values taken from Ref. 13. For each drug n 2 3 determinations, assays performed in triplicate.

P~toaffinity libeling-The protocol we have employed to photoaffinity label guinea pig brain membranes with [3HJ3- azido-MK-801 is one designed to preserve the high specificity demonstrated in reversible binding. Fig. 3 illustrates results that are typical of those observed in five other gels. The saturation binding data (Fig. 2) show that nonspecific binding increased linearly with [3H]3-azido-MK-801 concentration while specific binding clearly did not. We therefore chose to limit the ligand concentration in photoaffinity labeling experiments to not exceed 20 nM since higher concentrations would only marginally increase the amount of ligand bound to NMDA receptors but substantially increase the ligand bound


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Photolabeling of the PCP/NMDA Receptor 6779

205

116 - 97.4 -

-

45

29

-

FIG. 3. Fluorogram (left) of Coomassie Blue-stained slab gel (right) of [3H]3-azido-MK-801-labeled guinea pig brain homogenates. 7.5% acrylamide, 0.1% SDS gel. Orciimte, ikf* of standard proteins in 1000 s. Samples were prepared from 4 (4F) or 2 (2F) UV- irradiated filters through which l-mieach of incubation mixture (0.4 mg of protein, 10 nM [‘H]3-azido-MK-801) had been filtered. NSB lane samples were incubated with 10 pM PCP.

to pharmacologically irrelevant sites. Fluorograms of gels exhibited single bands which migrated slightly ahead of the ,&galactosidase marker. These bands were completely absent in parallel NSB lanes. Interpolation of the Mr of the radiolabeled bands by regression equations derived from the standards gave a value of 120,000 ? 3,000 (mean of six gels k SE.; range 114,000-125,000). The Mr = 120,000 bands on fluorograms did not correspond to densely staining Coomassie bands. Additionally, we observed broad, heavy bands on the fluorograms which migrated ahead of the dye fronts; these were, to variable degrees, diminished in NSB lanes. There was no apparent Coomassie staining on the gels ahead of the tracking dye.

Two lines of evidence suggest that the radioactive bands visualized in fluorograms result from photoactivation of the azido group of [3H]3-azido-MK-801 and covalent linkage to macromolecules. In one gel (not shown), lanes which con- tained “total binding” and NSB samples treated as described displayed the typical pattern on a fluorogram. However, in a parallel lane, a non-irradiated sample that had been otherwise treated as a “total binding” sample did not expose the film either at the Mr = 120,000 position nor ahead of the tracking dye. The acetone precipitation protocol showed that the specific incorporation of [3H]3-azido-MK-801 into precipitable molecules was absolutely dependent on ultraviolet exposure of filtered homogenates. Fig. 4 illustrates this dose dependence.

Corroboration for the hypothesis that a polypeptide of Mr = 120,000 is a component of the NMDA receptor comes from two groups who have performed molecular target size analysis of ligand binding proteins by radiation inactivation of rat brain cortical membranes. Using a [3H]MK-801 binding assay as an indicator of target destruction, Wong and Nielsen (18) derived a value of 128,000 & 9,000 Da as the molecular mass of the ligand-binding protein. Honor6 and collaborators (19,

20000

-0 . n Total Binding a

5 . q Nonspecific

*E .-

: 10000 -

k

0 2 5 10 20

Exposure Time (min)

FIG. 4. Dependence on UV exposure time of incorporation of [3H]3-azido-MK-801 into acetone-precipitable molecules. Batches of four filters (total binding, average 10,200 cpm/filter; nonspecific binding average 2,426 cpm/fYter) were irradiated for the times indicated, then solubilized, precipitated with cold acetone, and the pellets counted.

20) have performed a more extensive study utilizing four radioligand binding assays aimed at three discrete sites of the NMDA receptor complex. They derived a value of 118,000 k 10,000 Da for the [3H]TCP-binding protein. Furthermore, they reported that the weights of the [3H]glutamate- and [3H] glycine-binding proteins are 121,000 & 17,000 and 115,000 ? 10,000, respectively. The value of 209,000 & 19,000 derived for the competitive antagonist [3H]CPP-binding protein de- viated from the other three and might possibly reflect hypothesized NMDA receptor heterogeneity (2).

Interpretation of these data is not entirely straightforward. The radiation inactivation technique is thought to indicate the molecular mass of a “functional unit,” the function in these cases being radioligand binding (21, 22). One cannot distinguish u priori whether a size estimate determined by binding assay is that of a single polypeptide or of an entire hetero-oligomeric receptor complex associated with the binding site. We observed the [3H]3-azido-MK-801-labeled 120,000 band under reducing and denaturing electrophoresis conditions, and we expect that disulfide-linked or noncova- lently associated subunits of a multiprotein complex would have been separated. Our results are consistent with the hypothesis of Honor6 and co-workers (20) that the PCP, glutamate, and glycine sites of the NMDA receptor complex may all reside on a single Mr - 120,000 polypeptide. However, our results are not inconsistent with the less parsimonious idea that two or more polypeptides in the same size range may contain the radioligand-binding sites. It is worth noting that in reports of solubilization of [3H]MK-801 or [3H]TCP binding activity, radioligand binding can be modulated by glutamate and/or glycine in a manner similar to that observed with membrane homogenates (23,24). These data also do not distinguish between the solubilization of an oligomeric complex or of a single polypeptide on which the three binding sites reside.

Haring and co-workers (25-27) and Sorensen and Blaustein (28) have previously attempted to photoaffinity label the high affinity PCP-binding site in rat brain membranes with the ligand [3H]-m-azido-PCP. Both groups reported the labeling of several polypeptides, among them a band of Mr 90,000- 95,000 for which radioactive incorporation could be largely inhibited by coincubation with 1 mM TCP. Both groups also reported that the radiolabeling of other smaller polypeptides could be, to varying degrees, inhibited by PCP or its conge- ners. Although we employed guinea pig brain membranes in our photolabeling protocol, species differences probably do not account for the discrepant results, since we have also


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6780 Photolabeling of the PCP/NMDA Receptor

observed a fluorographic band of Mr - 115,000 in an experiment in which [3H]3-azido-MK-801 was used to photolabel rat cortical and hippocampal membranes in solution (details to be published elsewhere). Radiolabeling of this band too was completely inhibited in the presence of 10 pM PCP.

It is possible that [3H]-m-azido-PCP labeled a set of proteins different from the NMDA receptor complex. [3H]-m- Azido-PCP is inferior as a photoaffinity ligand to [3H]3-azido- MK-801 in a number of ways. The specific activity of [3H]- m-azido-PCP was less than half, and the affinity was nearly 50-fold lower than the values for [3H]3-azido-MK-801 (25- 28). These parameters necessitated the use of relatively high concentrations (10e7 to 10m6 M) for photoaffinity labeling. Since nonspecific binding of radioligands tend to increase as a linear function of concentration, the necessity of using comparatively high concentrations of [3H]-m-azido-PCP en- tails a loss of binding specificity. The parent compound PCP, is, itself, much less selective than MK-801 and is known to bind to numerous sites in brain with KD or Ki values in the range of lOma to 10m5 M (e.g. Refs. 2, 7, 29, 30). Perhaps for these reasons, photoaffinity labeling with [3H]-m-azido-PCP does not yield the same results as with [3H]3-azido-MK-801.

Utilizing radiation inactivation and immunological techniques, other groups have identified 70-75-kDa glutamate- binding proteins in the rat (31-33) and cockroach (34) nervous systems. The pharmacological profile of these binding protein is quite distinct from that expected for the NMDA receptor. Similarly, Voukelatou et ul. (35) have recently reported the cross-linking of [3H]glutamate to a Mr 45,600 protein in chick brain. They report, however, that coincubation with 100 pM NMDA inhibits incorporation of radioactivity only by 19%.

Characterization of the reversible binding of [3H]3-azido- MK-801 and experiments on the incorporation of tritium into acetone-precipitable molecules seemed to suggest that it would be an ideal photoaffinity ligand for the NMDA receptor, however, fluorograms indicated that relative incorporation into proteins was comparatively low. The nature of radiolabeled material migrating ahead of the tracking dye is not known, although the fact that it does not stain with Coomassie Blue might be consistent with it being a lipidic membrane constituent associated with the MK-801 binding site. Reports on the use of some other radioactive azido-photoaffinity de- rivatives described that although their reversible binding was consistent with their binding to proteinaceous receptor, on photolysis non-proteins were radiolabeled (36).

Until recently, all ligand-gated ion channels were thought to exist as hetero-oligomers belonging to a single superfamily. All subunits for the nicotinic acetylcholine, GABA*, and strychnine-sensitive glycine receptors whose primary struc- tures have been elucidated have sizes falling in the range of 40-67 kDa, share significant sequence homology as well as a motif of four putative transmembrane regions (37-40, but see Ref. 41). In contrast stand the recent reports by Kaupp and co-workers (42) and Hollmann and colleagues (43) describing the cloning of novel polypeptides which serve as the minimal functional units of two mammalian ligand-gated ion channels. Neither sequence displays more than slight sequence homology to known receptor proteins. Kaupp et ul. (42) predict the Mr of the unmodified polypeptide forming a cyclic GMP-gated cation channel in bovine retina to be 79,601. Hollmann et al. (43) have expressed a kainate-preferring excitatory amino acid receptor from rat brain. The predicted weight of the unglycosylated polypeptide is Mr = 99,769, substantially larger than the sizes of other previously identified superfamily members. This value is also well matched by immunological

detection of a putative kainate-binding protein from rat brain of Mr = 99,000 (44).

The NMDA receptor complex may share structural homology with the kainate receptor inasmuch as they share some pharmacological cross-reactivity (l-4) and the Mr estimates derived in this study with [3H]3-azido-MK-801 and those derived by radiation inactivation (18-20) are fairly similar to that of the cloned polypeptide. At this point, almost nothing is known about the quarternary structure of the excitatory amino acid receptors. It remains to be determined whether the multiple functionalities of these receptors, particularly of the NMDA receptor complex, reside on a single channel- forming polypeptide or whether they are distributed among several subunits. Likewise, it is intriguing to consider what structural commonalities may exit between these receptors and other proteins of comparable molecular weight which also form regulated transmembrane channels, such as the ATP- sensitive K+ channel (45, 46) and the P-glycoprotein family implicated in multidrug resistance and transmembrane trans- port (reviewed in Ref. 47).

Acknowledgments-We thank Michael Rhodes for the synthesis of (+)-MK-801 and Nam Hoang for his assistance in binding assays.

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M S Sonders, P Barmettler, J A Lee, Y Kitahara, J F Keana and E Weberreceptor labels a Mr 120,000 polypeptide.

A novel photoaffinity ligand for the phencyclidine site of the N-methyl-D-aspartate

1990, 265:6776-6781.J. Biol. Chem.

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