Proc. Natl. Acad. Sci. USAVol. 90, pp. 7308-7311, August 1993Biochemistry

Photoaffinity labeling of Torpedo acetylcholine receptor atmultiple sites

(binding/photolabeling)

SUE-JAN TINE* AND MICHAEL A. RAFTERY*t*Department of Biochemistry, University of Minnesota, St. Paul, MN 55108; and tDepartment of Pharmacology, University of Minnesota Medical School,Minneapolis, MN 55455

Communicated by James F. Bonner, April 26, 1993

ABSTRACT The acetyicholine receptor from Torpedo cal-ifornica electroplax was labeled with the photoaffinity reagentbis(3-azidopyridinium)decane perchlorate. All four receptorsubunits (a, (3, 'y, and 8) were specifically labeled. In thepresence of cholinergic agonists the -, (3-, and &subunitlabeling was decreased significantly, whereas labeling of the asubunit was minimally affected. Full occupancy of the twohigh-affinity sites involving the a subunits in the vicinity ofa-Cys-192-Cys-193 by covalent reaction with bromoacetylcho-line also caused a large decrease of vsubunit labeling by thephotoaffinity reagent and lesser but significant decreases in 1&and 8-subunit labeling. No decrease in labeling of the a subunitwas seen. Labeling of the a subunit could, however, beinhibited by high concentrations of the agonist carbamoylcho-line. We conclude that the binding sites of high-affinity resideat interfaces of the a subunit and other subunits and that thea subunit also contributes to formation of a low-affinity site(s)for cholinergic compounds.

The nicotinic acetylcholine receptor (AcChoR) from Torpedocalifornica electroplax is a pentamer of four homologoussubunits with stoichiometry a2fly8 (1). Many previous stud-ies have demonstrated that a readily reducible disulfide bondinvolving amino acid residues a-Cys-192-Cys-193 (2) is inclose proximity (9-10 A) to a cholinergic ligand-binding site.After thiol-mediated reduction of this disulfide the use ofaffinity alkylating agents, such as the antagonist 4-(N-maleimido)benzytrimethyl ammonium ion (3) or the agonistbromoacetylcholine cation (BrAcCho) (4-6), has been shownto react to form covalent derivatives of the a subunit. Morerecently, on the basis of studies of AcChoR-subunit expres-sion in fibroblasts (7) and on photoaffinity-labeling experi-ments (8) it is likely that ai-y and a-8 subunit interfaces areinvolved in binding of the antagonist d-tubocurarine (d-Tc),placing the location ofthe quatemary ammonium-recognitionsite in this region on one or more of the subunits involved.Similar photolabeling studies with the agonist nicotine haveyielded results involving both the a and y subunits (9). Yetanother study with the antagonist (p-(dimethylamino)ben-zenediazonium fluoroborate; DDF) has yielded similar re-sults (10). The use of the alkylating agent AcCho mustard hasled to identification of the site of labeling by this agent toinvolve the amino acid residue a-Tyr-189 (11), whereasprevious studies with the antagonist lophotoxin yielded sim-ilar results (12). On the other hand, another recent study hasindicated that the site of cationic ligand interaction in thisregion of the AcChoR involves a negatively charged residueon the 8 subunit (13). Taken together, these studies supportthe notion that cholinergic ligands associate with the AcChoRat or close to subunit interfaces of the a subunits with the Ry

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and/or 8 subunits and that the disulfide bond a-Cys-192-Cys-193 is -9 A removed from this locus.The possible involvement of subunits other than the a

subunit has also been indicated by earlier photoaffinitylabeling studies. Use of the bisquaternary compound[3H]bis(3-azidopyridinium)-1,10-decane perchlorate (DAPA)demonstrated that all four AcChoR subunits could be cova-lently labeled by this reagent and that such labeling wasspecific because it was eliminated by use of the nicotinicreceptor antagonist a-bungarotoxin (a-BuTx) (14). Otherstudies of a-neurotoxin association with T. californica Ac-ChoR demonstrated that the a-toxin from Naja naja siamen-sis formed a complex of two a-toxin molecules to oneAcChoR molecule, whereas the a-toxin from Dendroaspisviridis (mambatoxin) formed a complex of four a-toxin mol-ecules bound to one AcChoR molecule (15), suggesting thatmultiple binding sites for cholinergic ligands exist on theAcChoR molecule. Experiments using a different approach,fluorescence spectroscopy of AcChoR covalently labeledwith the fluorophore 4-[N-(acetoxy)ethyl-N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole (NBD), indicated that bindingsites of different affinities for cholinergic ligands can exist onthe AcChoR molecule from T. californica (16-18). Suchbinding sites were shown to have properties different fromthe two sites characterized by 4-(N-maleimido)benzyltri-methylammonium or BrAcCho-labeling studies discussedabove, which could also be detected by direct radioligandbinding (for review, see ref. 19) or by use of a differentfluorescent probe, iodoacetamidosalicylic acid covalentlyattached to the AcChoR to a-Cys-192-Cys-193 after reduc-tion of this disulfide bond (20).Most recently a further indication of the existence of

multiple binding sites for cationic ligands on the AcChoR hascome from both electrophysiological studies (21) andstopped-flow spectroscopic cation transport studies (22, 23)of the effects of the acetylcholinesterase inhibitor physostig-mine (eserine) and related carbamates, which were reportedto activate receptor-induced ion transport in a manner inde-pendent of the classical cholinergic antagonists d-Tc ora-BuTx.

In this communication we describe experiments extendingprevious use of [3H]DAPA (14) as a photoaffinity reagent forthe Torpedo AcChoR. In particular, we address the questionof whether binding sites for cholinergic ligands other thanthose sites at the a&-y and a-S subunit interfaces are a featureof the AcChoR molecule.

EXPERIMENTAL PROCEDURESMaterials. AcChoR-rich membrane fragments were pre-

pared from T. californica electric organs as described (24)and were further purified by alkali extraction to remove

Abbreviations: AcCho, acetylcholine; AcChoR, AcCho receptor;BrAcCho, bromoacetylcholine; DAPA, bis(3-azidopyridinium)-1,10-decane perchlorate; a-BuTx, a-bungarotoxin; d-Tc, d-tubocurarine.

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Proc. Natl. Acad. Sci. USA 90 (1993) 7309

nonreceptor proteins (25, 26). The concentration of a-BuTx-binding sites was measured by the DEAE disk assay (27).Protein concentrations were determined by the method ofLowry et al. (28).Carbamoylcholine, suberyldicholine, and d-Tc were ob-

tained from Aldrich. Reagents for gel electrophoresis werepurchased from Bio-Rad. BrAcCho perchlorate was pre-pared from bromoacetyl bromide, and choline chloride wasprepared according to the method of Chiou and Sastry (29).DAPA was synthesized as described by Witzemann and

Raftery (14). This compound was tritiated by New EnglandNuclear to yield [3H]DAPA. After recrystallization frommethanol the specific activity was 150 mCi/mmol (1 Ci = 37GBq), and the reagent was radiochemically pure as shownspectroscopically and by TLC. Other chemicals were fromstandard sources.

Photoaffnity Labeling. Photolabeling was done at roomtemperature in quartz cuvettes with a path length of 1 cm.Membrane fragments were equilibrated with [3H]DAPA inthe absence or presence ofother ligands in Ca2+-free TorpedoRinger's buffer (250 mM NaCl/5 mM KCI/2 mM MgCl2/20mM Hepes/0.02% NaN3, pH 7.4) in the dark. The solutionswere then irradiated for 2 min with constant stirring by usinga UVGL-25 lamp (Mineralight Lamp, UVP, San Gabriel, CA)on the short-wavelength setting. After irradiation, mem-branes were centrifuged for 30 min in an Eppendorf centri-fuge 5414 (Brinkmann), and the supernatant was discarded.The pellets were resuspended in sample buffer [10%6 (wt/vol)glycerol/3% (wt/vol) SDS/50 mM dithiothreitol/62.5 mMTris HCl, pH 6.8] and denatured by heating at 100°C for 2min. The solutions were centrifuged in a Beckman Airfuge at26 psi (1 psi = 6.9 kPa) for 15 min before gel electrophoresis.Specific labeling was determined either (i) by photolysis of[3H]DAPA in the absence or presence of 20-fold molar excessof a-BuTx-binding sites or (ii) in the absence or presence of20-fold molar excess of unlabeled DAPA.

Gel Electrophoresis. SDS/PAGE was done essentially ac-cording to Laemmli (30), using an 8.75% acrylamide slab gel(1.5mm thick x 17cm long x 13cm wide) and a4% acrylamidestacking gel. Electrophoresis was done at 10 mA in the darkovernight. The gels were then stained with Coomassie blue,destained, and sliced. Each slice was placed in a scintillationvial with 1 ml of2% SDS, and the vials were tightly capped andheated at 50°C overnight. After cooling and addition of scin-tillation fluid, Ecolume (ICN), the radioactivity incorporatedinto individual polypeptides was measured by using a Beck-man LS 3800 liquid scintillation counter.BrAcCho Labeling. Covalent labeling of dithiothreitol-

reduced AcChoR with BrAcCho was done under a nitrogenatmosphere by using Glove Bags (Instruments for Researchand Industry, Cheltenham, PA), and all buffers were thor-oughly deaerated before use. Membrane fragments werediluted to =1 ,uM in a-BuTx-binding sites in the buffer usedby Wolosin et al. (6)-i.e., 150mM NaCl/1.5 mM EDTA/4.5mM NaN3/15 mM Tris HCl, pH 8. Then 0.3 mM of dithio-threitol was added, and reduction was done at room temper-ature for 45 min. Membrane fragments were then diluted andpelleted to remove dithiothreitol by using a tightly cappedcentrifuge tube in a Beckman 35 rotor for 1 hr at 18,000 rpm.The tube was returned to a nitrogen atmosphere, the super-natant was discarded, and the pellet was resuspended in theabove buffer, which had been adjusted to pH 7. Acetylcho-linesterase was inhibited by addition of 50 AM Dursban(Dow). The reduced AcChoR was then treated with 50 AMBrAcCho for 15 min at room temperature, and 5 mM iodo-acetamide was added to alkylate excess dithiothreitol.

RESULTSUpon photolysis of [3H]DAPA in the presence of highlyenriched membrane preparations from T. californica electro-

plax it was shown that photoinsertion of the reagent occurredfor all four AcChoR subunits. The data presented in Fig. 1depict the relative incorporation into each ofthe four subunitsafter correction for nonspecific incorporation by use ofa-BuTx (20-fold excess). The major site of incorporation wasconsistently found to be associated with the y subunit followedby the a, 8, and 8 subunits. This pattern of photoaffinitylabeling is similar to that previously observed using thisreagent (14). In the earlier experiments no studies of inhibitionof the photoaffinity labeling process were conducted, apartfrom a-neurotoxin competition to demonstrate labeling spec-ificity, because subunit homology (1) was not evident at thattime, and it was considered that only the a subunits wereinvolved in cholinergic ligand binding. A major aspect dis-cussed in these early studies centered around the possibilitythat the labeling of subunits other than the a subunit couldresult from specific association of the photoaffinity reagentwith binding sites on the a subunit, with its consequentlabeling, and that the photoactivated species (nitrene) could besufficiently long-lived to diffuse from its binding sites on the asubunits and insert into nearby proteins. In the studies wereport here our interest centered on two aspects of thephotoaffinity labeling process: (i) whether the diffusion pro-cess discussed immediately above is a realistic aspect thatshould be a matter of general concern in such studies and (ii)whether photoaffmity labeling studies can be profitably usedto determine whether the Torpedo AcChoR contains multipletypes of binding sites for cholinergic ligands.

If diffusion of the nitrene generated by photolysis, follow-ing its association with binding sites on the a subunits or ata-subunit interfaces, occurred, then occupancy of those sitesby a cholinergic agonist should prevent specific labeling of allsubunits. To test this hypothesis, we compared photolabelingof AcChoR-enriched membranes with identical membranesthat were covalently labeled with AcCho-i.e., by reactionwith BrAcCho after reduction with dithiothreitol under con-ditions where 2 mol of this alkylating affinity reagent wasincorporated into the a subunits of one AcChoR molecule (6).

800 IE600 -

E&400

200

0

TI

alpha beta gammasubunits

delta

FIG. 1. Specific photolabeling ofAcChoR subunits by [3H]DAPA.AcChoR-rich membrane fragments (5 AM in a-BuTx-binding sites) inCa2+-free Torpedo Ringer's buffer (pH 7.4) were equilibrated with 15,uM [3H]DAPA in the absence or presence of 20-min preincubationwith a-BuTx (50 ,uM) in the dark. Irradiation was done at room

temperature for 2 min by using the short-wavelength lamp. Afterseparation by SDS/PAGE, individual subunits were sliced, and thecpm of [3H]DAPA incorporated into each AcChoR subunit was

determined, as described. The specific labeling is the differencebetween total labeling ([3H]DAPA only) and nonspecific labeling (inthe presence of 20-fold molar excess of a-BuTx). The result shown isthe average of two samples.

Biochemistry: Tine and Raftery

7310 Biochemistry: Tine and Raftery

The results are compared for the two preparations in Fig. 2.It was evident that the data obtained were not consistent withthe notion that all labeling was prevented. Surprisingly, noblockade of a-subunit labeling was seen, whereas inhibitionof label incorporation into the other subunits clearly oc-curred-most notably on the y subunit. Clearly, specificphotoinsertion of the affinity reagent into the a subunit wasunaffected by covalent incorporation of AcCho into bothhigh-affinity sites at subunit interfaces.

In the case that the AcCho-AcChoR structure could differfrom that of AcChoR-agonist complexes under equilibriumconditions by virtue of either the dithiothreitol reduction stepor the covalent incorporation of AcCho, experiments similarto those just described were done by using two agonists,carbamoylcholine and suberyldichloride, under equilibriumconditions where the high-affinity sites for these agonists werefully occupied ([suberyldichloride] = 3 ,uM, Kd = 10 nM;[carbamoylcholinel = 30 ,uM, Kd = 100 nM) but with minimal(or fractional occupancy in the case of suberyldichloride)occupancy of low-affinity sites (Kd carbamoylcholine: 1 mM;Kd suberyldichloride: 1 ,uM) (16-18). The results with theseagonists under equilibrium conditions showed that with car-bamoylcholine, incorporation into the 'y subunit was de-creased by 76%, whereas the level of incorporation into the asubunit decreased by only 16%. A similar result was obtainedwith suberyldicholine (3 j.M), where y-subunit labeling wasdecreased by 73% and a-subunit labeling was decreased by28% (Fig. 3). Taken together with the data of Fig. 2 forinhibition in the case of covalent incorporation ofAcCho intothe two high-affinity sites, it is clear that [3H]DAPA labels thea subunits at locations other than the high-affinity sites at ornear the interfaces involving the a subunits.To compare our photolabeling method with other similar

methods recently recorded in the literature we conductedexperiments to study the inhibition of [3H]DAPA labeling byd-Tc. The data presented in Fig. 4 demonstrate that thischolinergic antagonist inhibited photoinsertion of the radio-label into the a, 'y, and 8 subunits with no apparent effect onlabeling of the f3 subunit. These results agree with thosedescribed (8) for [3H]d-Tc-photoinduced labeling of T. cali-fornica and Torpedo nobiliana AcChoR, which were inter-

500 -

400 -

300 -

E

200 -

100 -

* unlabeled AcChR

e BrAcCh-labeled AcChR

0 _alpha beta gamma

subunits

Ea-0 400 -

200-

0alpha beta gamma delta

subunits

FIG. 3. Inhibition of [3H]DAPA labeling of the AcChoR bysuberyldicholine (sub) and carbamoylcholine (carb). AcChoR-richmembrane fragments (5 ;LM in a-BuTx-binding sites) in Ringer's buffer(pH 7.4) were incubated with 15 ,uM [3H]DAPA in the presence of 3AM suberyldicholine or 30 ,uM carbamoylcholine in the dark for 10min. Specific labeling of the samples in the absence (s) and presenceof suberyldicholine (i) or carbamoylcholine (o) is shown.

preted to demonstrate that this ligand binds at or near the a-yand a-8 subunit interfaces with no detectable labeling of the/3 subunit. Significant inhibition of a-subunit labeling was alsoobserved. This is probably due to overlap between the twobisquaternary ligands DAPA and d-Tc.The effects of the carbamoylcholine concentration depen-

dence on [3H]DAPA incorporation into the BrAcCho-labeledAcChoR a subunit were also measured. BrAcCho-labeledmembrane fragments were preincubated for 30 min withvarious concentrations of carbamoylcholine (0, 10-4, 10-3,and 10-2 M) and 0.5 mM glutathione (oxidized) to decrease thenonspecific labeling; then the samples were incubated with 20,uM [3H]DAPA for 10 min before irradiation. With a highconcentration (1 mM) of carbamoylcholine, the incorporationof [3H]DAPA into the a subunits was slightly inhibited. Ahigher concentration of carbamoylcholine (10-2 M) was re-quired to inhibit the photolabeling by [3H]DAPA of the a

800

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E,- 400

200 -

delta

FIG. 2. Specific photolabeling of native and BrAcCho-labeledAcChoR by [3H]DAPA. Five micromolar (in a-BuTx-binding sites)AcChoR-rich membrane fragments (with or without previous BrAc-Cho labeling) in 0.5 mM glutathione (oxidized) and Ringer's buffer(pH 7.4) were incubated with 15 ,uM of [3H]DAPA. Samples wereassayed as described for Fig. 1. The corresponding specific labelingof native receptor (m) and BrAcCho-labeled receptor (X) is shown.The BrAcCho-labeled membrane fragments were prepared as de-scribed.

0-i_

* without d-Tc

* with d-Tc

alpha beta gammasubunits

delta

FIG. 4. Inhibition of [3H]DAPA labeling of AcChoR by d-Tc.AcChoR-rich membrane fragments (5 pM in a-BuTx-binding sites) inRinger's buffer were incubated with 20 ,iM of[3H]DAPA and 0.5mMof glutathione (oxidized). Samples with or without 10 AM d-Tc wereirradiated, and the [3H]DAPA incorporation was determined asdescribed for Fig. 1. The specific labeling of subunits is plotted forthe samples without inhibitor (i) and with 10 ,uM d-Tc (i).

Proc. Natl. Acad Sci. USA 90 (1993)

Proc. Natl. Acad. Sci. USA 90 (1993) 7311

200nI

EXL 100-\

e.O- ' /j

00 4 3 2-log[Carb] (M)

FIG. 5. Inhibition of [3H]DAPA labeling of BrAcCho-labeled asubunits by carbamoylcholine (Carb). BrAcCho-labeled AcChoR-rich membrane fragiments (5 ,uM in a-BuTx-binding sites) werepreincubated with various concentrations of carbamoylcholine and0.5 mM glutathione (oxidized) for 30 min. Samples were thenincubated with 20 ,uM [3H]DAPA for 10 min before irradiation. The[3H]DAPA incorporation into AcChoR was determined as describedfor Fig. 1. Specific [3H]DAPA labeling of the a subunits is plottedversus carbamoylcholine concentration.

subunits to the same extent as the y subunit. The results of thisstudy are shown in Fig. 5. Taken togetherwith the data in Figs.2 and 3 the inhibition of [3H]DAPA labeling by carbamoyl-choline demonstrated that the a subunits contain a bindingsite(s) with an apparent low-affinity for carbamoylcholine.

DISCUSSIONThe specific labeling of all four subunits (a, (3, y, and 8) ofTorpedo AcChoR by [3H]DAPA strongly indicated that sub-units other than the a subunit participate in cholinergic ligandbinding. We observed such extensive labeling patterns whenfirst using this photoaffinity probe (14) but without knowledgeof the extensive subunit homology of the AcChoR subunit (1,31), and our interpretation of the results suggested that theproteins of 50, 60, and 65 kDa were in close proximity to thea subunit, which at that time was considered to singularlycontain a cholinergic ligand-binding site. As noted in theintroduction, recent photolabeling studies with a variety ofradiolabeled ligands, although lacking in chemically photo-labile centers such as azide or diazo moieties, have been usedto draw the conclusion that cholinergic binding sites arelocated at or close to the interfaces of a subunits and some ofthe other subunits. The photolabeling studies we report hereagree with those reported results. The data we obtained (Fig.2) from [3H]DAPA photolabeling of AcChoR preparations,where both high-affinity sites were covalently modified by anAcCho molecule, clearly demonstrate that these high-affinitysites are close to or directly involve subunits other than the asubunit-most notably the y subunit. The second result fromthese particular experiments was that our earlier interpretationof [3H]DAPA binding to such sites, resulting in nitrene diffu-sion to nearby proteins (subunits) with consequent labeling, isnot a matter ofconcern for [3H]DAPA as a photoaffmnity probebecause if such were the case, we should have observed nospecific labeling. Instead, we obtained the unexpected resultthat AcCho occupancy ofboth high-affinity sites had no effecton photoinsertion of the affinity probe into the a subunit.Therefore, inhibition of labeling of the other subunits in thisexperiment strongly supports their involvement in formationofthe high-affinity sites that are, at least in part, formed by thea subunit close to a-Cys-192-Cys-193. This same experimentalso strongly supports the notion that the a subunit contributesto formation of a separate binding site(s). The data shown inFig. 3 are related to those in Fig. 2, but these experiments were

conducted under conditions of equilibrium binding of twoagonists, carbamoylcholine and suberyldicholine. The dataobtained with these two agonists are consistent with those ofFig. 2 in that their inhibitory effects upon occupancy of thehigh-affinity sites mostly involve subunits other than the asubunit, with little inhibition of a-subunit labeling. This label-ing of the a subunit, which as stated above is not inhibited byagonists occupying the two well-documented binding sites,strongly suggests that the a subunits participate in formationof other sites for [3H]DAPA binding and labeling upon pho-tolysis. The data presented in Fig. 5 suggest that these addi-tional binding sites represent binding sites of low affinity foragonists such as carbamoylcholine.

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Biochemistry: Tine and Raftery