view - the journal of biological chemistry

THE J O U R N A L OF BIOLOGICAL C H E M I S T R Y (c> 1991 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 266, No. 32, Issue of November 15, pp. 21595-21607,1991 Printed in U.S.A.

The Hydrophobic Photoreagent 3-(Trifl~oromethyl)-3-rn-([~~~I] iodopheny1)diazirine Is a Novel Noncompetitive Antagonist of the Nicotinic Acetylcholine Receptor*

(Received for publication, June 10, 1991)

Benjamin H. White$§, Satchiko Howardfl, Saul G . CohenlI, and Jonathan B. CohenSII From the $Department of Anatomy and Neurobiology, Washington University School of Medicine, St. Louis, Missouri 63110 and the llDepartment of Chemistry, Brandeis University, Waltham, Massachusetts 02254

We have shown previously that the lipophilic photoreagent 3 - (trifluoromethyl)3-m-( [ '251]iodophenyl) - diazirine (['251]TID) photolabels all four subunits of the Torpedo nicotinic acetylcholine receptor (AChR) and that >70% of this photoincorporation is inhibited by cholinergic agonists and some noncompetitive antagonists, including histrionicotoxin (HTX), but not phencyclidine (PCP; White, B. H., and Cohen, J. B. (1988) Biochemistry 27, 8741-8751). We have now examined the effects of nonradioactive TID on (a) AChR photoincorporation of ['251]TID, ( b ) AChR-mediated ion transport, and (c ) AChR binding of several cholinergic ligands. We find that TID inhibits ['2sI]TID photoincorporation into the AChR to the same extent as carbamylcholine. The saturable component of ['251]TID photolabeling is half-maximal at 4 p~ ['251]TID with 0.5 mol specifically incorporated per mol of AChR after 30 min photolysis with 60 PM [1251]TID. Repeated labeling of membranes at a fixed ['261]TID concentration gave results consistent with a maximal incorporation of one ['251]TID molecule per AChR. Nonra- dioactive TID also noncompetitively inhibits agonist- stimulated "Na+ efflux from Torpedo vesicles with an ICs0 of 1 p ~ . Furthermore, TID inhibits allosterically the binding of [3H]HTX, decreasing its affinity for the AChR 5-fold both in the presence and absence of agonist. In contrast, TID has little effect on [3H]PCP binding in the absence of agonist but completely inhibits it in the presence of agonist. TID enhances the cooperativity of [3H]nicotine binding. [1251]TID is thus a photoaffinity label for a novel noncompetitive antagonist binding site on the AChR that is linked allosterically to the binding sites of both agonists and other noncompetitive antagonists. The ['2sI]TID site is presumably located within the central pore of the AChR.

Pharmacological characterization of compounds that block the agonist-induced permeability response of the nicotinic

*This research was supported by United States Public Health Service Grant NS19522 (to J. B. C.) and by Contract DAMD 17-87- C-7170 (to S. G. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 Supported in part by the Washington University Center for Cellular and Molecular Neurobiology.

( 1 To whom correspondence should be addressed: Dept. of Anatomy and Neurobiology, Washington University School of Medicine, 660 S . Euclid Ave., St. Louis, MO 63112. Tel.: 314-362-3539.

acetylcholine receptor (AChR)' has distinguished two classes of antagonists: competitive antagonists, like d-tubocurarine and a-bungarotoxin, which block activation by binding to the two agonist binding sites on the AChR, and noncompetitive antagonists which exert their effects at sites distinct from the agonist binding sites (for reviews see Taylor et al., 1983; Popot and Changeux, 1984, and Galzi et al., 1991). Efforts to determine the sites of action of noncompetitive antagonists have focussed on a heterogeneous group of compounds that bind to the AChR with relatively high affinity ( K D = p ~ ) in an apparently mutually exclusive manner (Heidmann et al., 1983; Cohen et al., 1986, and references therein). This group con- tains structurally diverse aromatic amines including several local anesthetics, phencyclidine, meproadifen, and the phe- nothiazine derivative chlorpromazine. The spiropiperidine histronicotoxin (HTX) and the detergent Triton X-100 are also included. Certain structural features, such as positive charge and bulky aromatic or aliphatic constituents, tend to characterize this group but are not universal. Study of the binding stoichiometry of these high affinity noncompetitive antagonists (NCAs) has proved difficult, given their lipophil- icity and propensity to bind with low affinity to multiple sites, but where it has been examined carefully results indicate high affinity binding to a single site on the AChR (Heidmann et al., 1983; Cohen et al., 1985). This fact, coupled with the apparently mutually exclusive manner of their binding, has led to the concept of a single high affinity NCA binding site on the AChR.

Although most NCAs bind preferentially to the noncon- ducting, desensitized state of the AChR characterized by high affinity for agonist, stabilization of this state does not appear to be their primary mode of action. Not all NCAs, for example, stabilize the state to the same extent and some, like tetracaine, do not stabilize it at all (Boyd and Cohen, 1984). Evidence from affinity photolabeling studies with the NCA chlorpromazine indicates that NCAs probably bind to a site directly within the ion permeation pathway (reviewed in Galzi et al., 1991).

Electron microscopic imaging of the AChR indicates that its subunits are arranged pseudo-symmetrically around a cen-

The abbreviations used are: AChR, nicotinic acetylcholine receptor; ["'I]TID, 3-(trifluoromethyl)-3(m-['25I]iodophenyl)diazirine; NCA, high affinity noncompetitive antagonist of the AChR; [3H]AD, ["Hladamantane diazirine; PTA, phenyltrimethylammonium; 43K protein, the basic membrane-bound 43 kilodalton protein of Torpedo postsynatpic membranes; a-BgTx, a-bungarotoxin; Hlo-HTX, DL- decahydro(penteny1)histrionicotoxin; HI2-HTX, perhydrobistrioni- cotoxin; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; TPS, Torpedo physiological saline (250 mM NaC1, 3 mM CaCl,, 2 mM MgC12, 5 mM sodium phosphate, pH 7.0); V8 protease, Staphylococcus aureus V8 protease.

21595

TID: A Novel Nicotinic AChR Antagonist 21597

FIG. 3. Inhibition of ['251]TID photoincorporation into the AChR by nonradioactive TID. A, membranes (1 mg/ml) equilibrated with 1 p~ ['2sI]TID were photolysed 30 min in the presence or absence of a 100-fold excess of nonradioactive TID, and 15-118 samples were electrophoresed on an 8% polyacrylamide gel. Proteins were visualized by Coomassie Blue stain (lane 1 ) and ['251]TID incorporation was analyzed by autoradiography (lanes 2 and 3, 103-h exposure). AChR subunits and the Na'/K'-ATPase a-subunit (LYNK)

are indicated. B, membranes (1 mg/ml) equilibrated with 1.1 p~ ['"I] TID were divided into equal aliquots and isotopically diluted with nonradioactive TID to the total TID concentrations indicated. After photolysis for 30 min in the presence (W) or absence (A) of 0.1 mM carbamylcholine, 20-pg samples were subjected to SDS-PAGE on 12% minigels and AChR subunits were visualized by Coomassie Blue stain. ['""]ID incorporation into the AChR was analyzed by coincidence counting of excised gel pieces spanning the AChR subunits. The inhibition data in the absence of carbamylcholine was modeled by a hyperbolic function (solid curue) with values of R and K equal to 0.85 p~ (the AChR concentration) and 3.5 p ~ , respectively. A constant level of nonspecific labeling, equal to the average value of ["'IITID incorporation observed in the presence of carbamylcholine (8179 dpm, solid line), was assumed.

lower mobility than the 43-kDa protein and one with slightly higher mobility than the a-subunit, incorporated ['251]TID in a carbamylcholine-sensitive fashion. These bands represent degradation products of the y- and &subunits, respectively (White and Cohen, 1988). In addition, both probes labeled in a carbamylcholine-insensitive manner a 90-kDa protein previously identified as the transmembrane a-subunit of the Na+/K+-ATPase (White and Cohen, 1988). Other labeled proteins, including a band prominently labeled by [3H]AD and migrating below the AChR a-subunit, were not identified.

Saturability of ['251]TID Phtoincorporation-Carbamyl- choline's failure to inhibit [3H]AD photolabeling of the AChR indicates that agonists do not substantially increase the reactivity or amount of AChR mass exposed to lipid. To confirm that the agonist-sensitive component of ['251]TID photolabeling results from reversible, saturable binding of ['251]TID to the AChR, we examined the ability of nonradioactive TID to inhibit ['251]TID photoincorporation (Fig. 3). Such inhibition would be expected if ['251]TID is photoaffinity labeling specific binding sites on the AChR.

Membranes photolabeled with 1 PM ['251]TID in the presence of a 100-fold excess of nonradioactive TID showed sharply reduced incorporation of radioactivity into the AChR subunits and their associated degradation products (Fig. 3A). Photoincorporation into the Na+/K'-ATPase a-subunit was not similarly affected (Fig. 3A, lanes 2 and 3; Table I), nor was incorporation of [3H]AD into AChR subunits (Table I).

To investigate the concentration dependence of this effect we examined AChR photolabeling over a range of isotopic ['TITID dilutions both in the presence and absence of carbamylcholine (Fig. 3B). Membranes pre-equilibrated with a fixed concentration of [1251]TID (1 p ~ ) and increasing concentrations of nonradioactive TID (0-100 PM) were irradiated in the presence or absence of carbamylcholine and subjected to SDS-PAGE on 12% minigels. The regions containing AChR subunits were excised and counted. ['251]TID photoincorporation into the AChR in the presence of carbamylcholine was unaffected by TID at all concentrations. In contrast, [1251] TID photoincorporation in the absence of carbamylcholine was strongly inhibited by TID in a dose-dependent manner,

characterized by a single hyperbolic function ( K = 4 p ~ ) . The maximal inhibition was 80%, equal to the inhibition produced by carbamylcholine. The results demonstrate the presence of two components of ['251]TID photoincorporation into the AChR. a saturable component resulting from photoaffinity labeling of the AChR and a nonsaturable component presumably resulting from labeling of AChR regions exposed to the lipid phase. These two components can be identified with the agonist-sensitive and agonist-insensitive components of [1251] TID photolabeling, respectively.

Localization of the Saturable Component of ['251]TID Pho- toincorporation on the AChR a-Subunit-We have shown previously that ['251]TID photolabeling of the AChR a-subunit is restricted to two cleavage fragments generated by V8 protease (White and Cohen, 1988). These fragments have apparent molecular masses of 10 (V8-10) and 20 kDa (V8-20). Photolabeling of V8-20, which includes the putative membrane spanning regions M1, M2, and M3, is inhibited by agonist, whereas photolabeling of V8-10, which includes the region M4, is not. We used V8 protease to map the location of the saturable component of ['251]TID photoincorporation in the a-subunit and to examine the combined effects of agonist and TID on the photolabeling of each V8 fragment (Fig. 4). Membranes preincubated with a fixed concentration of ['251]TID (1 p ~ ) were labeled in the presence and absence of 60 p~ TID, both with and without 100 p~ carbamylcholine, and electrophoresed on an 8% polyacrylamide gel. Excised a- subunit bands were subjected to V8 proteolysis as described under "Experimental Procedures," and ['251]TID photoincorporation into the resulting V8-20 and V8-10 fragments was determined by y-counting of the excised gel bands. In the absence of carbamylcholine, 60 p~ TID inhibited approximately 80% of the ['251]TID photoincorporation into the V8- 20 fragment while slightly enhancing labeling of V8-10. In the presence of carbamylcholine, nonradioactive TID did not significantly alter the level of photoincorporation into either fragment.

Stoichiometry of Saturable ['251]TID Photoincorporation- To estimate the number of sites of saturable ['251]TID photoincorporation, we examined the molar incorporation of [1251] TID into the AChR as a function of either increasing isotopic dilution for a single 30-min irradiation (Fig. 5A) or repeated

- E 1000 e e

z:

v

750 4 m + Carb/TID

? 500

250 - L 7

- 0 N Y

V8-20 V8-10 FIG. 4. Proteolytic mapping of the site of saturable [lZ5I]

TID photoincorporation in the AChR a-subunit using Stuph- ~ Z O C O C C U S uureus VS protease. Aliquots of membranes (2 mg/ml) equilibrated with 1 p~ ['251]TID were photolysed for 15 min in the absence of ligands (solid bars) or in the presence of either 100 pM carbamylcholine (open bars), 60 p~ TID (right hatch), or both (left hatch). Samples were then subjected to SDS-PAGE on an 8% gel. The a-subunit bands were excised after identification by Coomassie Blue staining and transferred to the wells of a 15% mapping gel where each was overlaid with 3 pg of S. aureus V8 protease. After electrophoresis, the proteolytic fragments were identified by Coomassie Blue stain, and ['251]TID incorporation was assessed by autoradiography. After verifying that photoincorporation was restricted to the V8-20 and V8-10 fragments, these bands were excised from the gel and counted by y-counter. The range of the data from two experiments is indicated by the error bars.

21598 TID: A Novel Nicotinic AChR Antagonist

0 20 40 60 80 100 [ 1251]TlD (pM)

B1

Number of Labehngs

FIG. 5. Stoichiometry of the agonist-sensitive component of ['251]TID photoincorporation into the AChR. Agonist-sensitive incorporation of ['2'I]TID into the AChR, calculated by subtraction of the molar ["'II]TID incorporation in the presence of carbamylcholine from that in its absence, was determined as a function of increasing ["'I]TID concentration ( A ) and repeated photolabeling at fixed ['Y'I]TID concentration ( B ) . Molar ["'I]TID incorporation was calculated, in both cases, directly from the measured disintegration/ min of a sample and the estimated specific activity of [12'I]TID used in photolabeling. Labeled AChR was isolated and quantified in one of three different ways: 1) gel slices spanning the AChR subunits were excised from polyacrylamide gels after staining with Coomassie Blue, and the amount of AChR present was estimated from the known amount of protein loaded on the gel (15 or 20 pg) coupled with the known concentration of ACh binding sites per mg protein in the sample (A, O), 2) AChR was purified on an acetylcholine affinity column and the AChR content of purified samples was determined from direct composition analysis together with the known amino acid composition of the Torpedo AChR (V), and 3) affinity-purified AChR whose molar concentration had been determined by composition analysis was subjected to SDS-PAGE to remove residual noncovalently incorporated ["'I]TID and gel slices spanning the AChR subunits were excised (B). Photolabeling was carried out as follows. A, membranes (1 mg/ml) pre-equilibrated with 1 p~ ['251]TID and further equilibrated with 0-100 WM nonradioactive TID were irradiated in the presence or absence of carbamylcholine for 30 min, a length of time previously shown to produce maximal incorporation of ["'I]TID into the AChR (White and Cohen, 1988). Inset shows the total molar ["'IITID incorporation into AChR in the presence (0) and absence (0) of carbamylcholine determined by the gel slice technique (without affinity purification) described above. 8, membranes (1 mg/ml) equilibrated with 13 p~ ["'I]TID were divided into two sets: one received carbamylcholine (0.1 mM final) whereas the other did not. Both samples were photolabeled 15 min and then washed as described under "Experimental Procedures" to remove unreacted ['"SIITID and free photolysis products. After washing, aliquots were removed for analysis, and the remaining membranes from each set were again equilibrated with [1251]TID and photolysed. Three 15-min rounds of photolysis, followed by washes, were carried out in parallel on the + and - carbamylcholine-treated membranes. The ratio of concentrations of [12'I]TID to protein varied from 12 to 13 between rounds, but was similar for the two sets of membranes a t each labeling. The theoretical specific molar incorporation (0) was calculated from the measured concentrations of ["'IITID and AChR at each labeling and from the KD (4 p ~ ) and maximal specific photoincorporation (0.5) derived from the experiment in A. The maximal specific photoincorporation was adjusted to 0.4 to compen- sate for the shorter labeling period (15 uersus 30 min) in this experiment (cf. White and Cohen, 1988).

irradiation (followed by removal of photolysis products) at a fixed concentration of ['251]TID (10 p ~ , Fig. 5B). We have shown previously that at a fixed concentration of [1z51]TID irradiation for longer than 30 min results in no further incor-

poration, presumably due to complete photodecomposition of ['251]TID (White and Cohen, 1988). For each method of labeling, [1251]TID incorporation was determined for AChR isolated by three different techniques: (a) excision of gel pieces spanning the AChR subunits, ( b ) affinity purification, and ( c ) excision of gel pieces after electrophoresis of affinity- purified AChR to remove noncovalently bound ['*'I]TID. The third technique was designed to guard against possible errors arising from radioactivity noncovalently associated with the affinity-purified AChR (possibly bound to annular lipid) that could be removed by SDS-PAGE. For the first technique, the molar amount of AChR in each gel slice was estimated from the known concentration of [3H]acetylcholine binding sites in the material subjected to electrophoresis. For the second two techniques, AChR in each sample was quantified directly by composition analysis. In all cases, the absolute incorporation of ['*'I]TID was determined by the coincidence counting method (Horrocks, 1975) together with the known specific activity of the [1251]TID isotopic dilutions.

The inset of Fig. 5A shows the total molar incorporation of ['251]TID into the AChR as a function of increasing isotopic dilution, as estimated by the first technique described above. Incorporation in the presence of carbamylcholine (open circles) increased linearly with isotopic dilution, as would be expected from Fig. 3B. Nearly two mol of [1251]TID were incorporated per mol of AChR at 100 p~ ['251]TID. After an initial nonlinear rise, ['251]TID incorporation in the absence of carbamylcholine paralleled that seen in the presence of agonist. The linearity of photoincorporation in the presence of agonist and the observation that photoincorporation at saturating concentrations of nonradioactive TID appears to equal that observed in the presence of agonist (Fig. 3B) permitted use of the agonist-insensitive component of photolabeling as a measure of nonspecific photoincorporation.

The saturable component of [1251]TID incorporation into the AChR, calculated by subtraction of the agonist-insensitive incorporation from the total incorporation a t each concentration, is shown in Fig. 5A for all three techniques. The results are similar in all cases and indicate that 30 min of irradiation results in a maximal specific incorporation of between 0.4 and 0.5 mol of ['251]TID/mol of AChR. An estimate of incorporation at concentrations higher than 50 p~ was difficult to obtain because of errors inherent in estimating molar incorporation a t large isotopic dilutions, but regression analysis of the combined data using a simple hyperbolic binding function indicated saturation at 0.48 (k0.08) mol of ["51]TID/mol of AChR and 50% maximal incorporation at 4.3 (k2.8) pM [1251] TID. That specific photoincorporation did not, in fact, satu- rate at 0.48 mol of [1251]TID/mol of AChR was indicated by control experiments in which an aliquot of the membranes labeled at 100 p~ ['2sI]TID were washed to remove photolysis products and labeled a second time with 0.5 p~ ['251]TID in the presence and absence of 100 p~ carbamylcholine. These experiments showed that additional agonist-inhibitable incorporation occurred during the second round of labeling, indicating that not all ['251]TID binding sites were blocked by the initial round of photoincorporation.

As an alternative means to quantifying the maximal specific photoincorporation of ['2sII]TID into the AChR, we quantified the specific molar incorporation of ['251]TID into the AChR after one, two, and three sequential rounds of photolysis in the presence of -10 p~ ['2'I]TID. Because unincorporated photolysis products were a potential cause of reduced efficiency of specific ['251]TID photoincorporation (by displacing bound ['251]TID), these were removed after each round of photolysis by washing the membranes. Although it was desir-


able to repeatedly photolabel membranes for 30 min at as high a ['2sII]TID concentration as possible to promote maximal specific incorporation, the need to limit the isotopic dilution of the ['251]TID and limit the exposure of the membranes (1 mg/ml) to UV irradiation placed practical constraints on the attainable ['''I]TID concentrations and irradiation times. Three 15-min periods of irradiation at -10 pM ['251]TID were found to provide an adequate signal-to-noise ratio without resulting in overt membrane damage and were thus selected as conditions for this experiment. The observed specific photoincorporation, as estimated by the three techniques described above, agreed well with each other and attained a value of 0.60-0.65 mol of [1251]TID specifically incorporated per mol of AChR. Based upon the kinetics of photoincorporation of ['"IITID under the labeling conditions used (White and Cohen, 1988) and the results shown in Fig. 5A concerning the concentration dependence ( K D = 4 p ~ ) and maximal specific incorporation after 30-min irradiation (0.5 mol of ['Z51]TID/mol of AChR), it is possible to calculate the specific photoincorporation expected after each 15-min round of irradiation. The calculated incorporation assuming one site per AChR is shown in Fig. 5B (open diamonds) and agrees rea- sonably well with the observed specific incorporation. Calcu- lations assuming five sites of incorporation, each of which is labeled at one-fifth efficiency, predict specific incorporation under these conditions of 0.3 mol of ['251]TID/mol of AChR after one round of labeling, but 0.8 mol/mol after three rounds.

Inhibition of **Na+ Efflux from AChR-rich Membranes by TID-To test the possible effects of occupancy of the TID binding site on AChR function, we examined the effects of TID on "Na+ efflux from well sealed Torpedo postsynaptic membranes in response to the partial agonist phenyltrimethylammonium (PTA). The total releasable "Na+ content of the vesicles was assayed using the ionophore gramicidin.

Initial experiments assessed the effect of TID alone on '"a+ release from Torpedo vesicles. Because TID is a highly lipophilic compound, with a partition coefficient in Torpedo membranes of 1.5 x lo5 pl/mg (White and Cohen, 1988), higher TID concentrations might be expected to disrupt the integrity of bilayer. These experiments showed that at TID concentrations less than 10 p ~ , at which the estimated TID to lipid ratio in the membranes is approximately 1:5, less than 10% of the total releasable "Na+ content of the vesicles leaked out. Concentrations in this range did, however, significantly diminish the maximum response to PTA (Fig. 6). At 2 p~ TID, the maximum response (measured as a percentage of the gramicidin-induced efflux) was inhibited by approximately 65%. Inhibition of **Na+ efflux by PTA itself at concentrations above 1 mM is evident, as noted previously by Forman et al. (1987). Full dose-response experiments showed that TID inhibition of the "Na' efflux induced by 1 mM PTA had an of 1 p~ (Fig. 7, solid circles). In contrast, the noncompetitive antagonist PCP inhibited less potently under similar experimental conditions, having an ICso of approximately 10 p~ (Fig. 7, solid squares).

Effects of TID on PHIPCP Binding to Torpedo Mem- branes-Previous photolabeling results indicate that ['"I] TID cannot bind to either the agonist or the known noncompetitive antagonist binding sites, since ligands for each of these sites exist that fail to inhibit photoincorporation substantially. The inhibition of ['251]TID photolabeling by agonists and noncompetitive antagonists like HTX suggests, however, that the site photoaffinity labeled by ['251]TID must interact with the binding sites for these ligands. We therefore used centrifugation assays to examine the effects of TID on

- loo I 0 Nd TI0 ' rn 0.7 pu no

-20 - 6 5 4 3 2

-log [PTA] (M) FIG. 6. TID inhibition of agonist-induced "Na+ efflux from

Torpedo vesicles. 22Na'-loaded vesicles (0.08 mg/ml, 0.11 PM ACh binding sites) were treated with 0 PM (O), 0.7 pM (H), or 2 FM (v) TID and then exposed to PTA at the indicated concentrations for 20 s. PTA-stimulated efflux of *'Na+ was measured by filtration as described under "Experimental Procedures" and compared with the gramicidin-induced "Na+ efflux. The gramidicin-induced release of "Na+ in these experiments typically ranged from 400 to 500 cpm, with a background of -350 cpm retained on the filter in the presence of gramicidin. The error bars indicate standard deviations for four determinations.

h H

0 4 L

0 d V K x 1

v

d .c(

2 ld z

N N

. . m ' 7 6 5 4

-log [ligand] (M) FIG. 7. Inhibition by TID and PCP of PTA-induced "Na+

efflux from Torpedo vesicles. '*Na+ efflux from Torpedo postsynaptic vesicles in response to treatment with 1 mM PTA was assayed as described in Fig. 6 in the presence of increasing concentrations of TID (0) or PCP (W). Vesicles were incubated with TID for 6 min and with PCP for 10 min before PTA-induced "Na+ efflux was measured. The efflux is shown as a percentage of the PTA-induced efflux in the absence of TID or PCP. The "Na+ released by PTA varied from 200 to 400 cpm in these experiments, representing roughly 65% of the counts/min released by gramicidin. Background retention of "Na+ in the presence of gramicidin was -300 cpm. The solid lines indicate nonlinear least squares fits of the data points to functions of the form: %flux = 100 X (1 - [TID]/([TID] + K ) ) , where [TID] is the total concentration of TID. The error bars indicate standard deviations for three determinations.

the binding of radiolabeled cholinergic noncompetitive antagonists and agonists.

[3H]PCP binding was measured by centrifugation in the presence and absence of carbamylcholine using membranes (1 pM ACh binding sites) equilibrated with 5 nM [3H]PCP and increasing concentrations of TID (0-170 p ~ ; Fig. 8). [3H] PCP binding was higher in the presence of carbamylcholine than in its absence because PCP binds preferentially to the desensitized state of the AChR (Heidmann et al., 1983). Nonspecific [3H]PCP binding was defined as the level of binding observed in the presence of 200 p~ meproadifen, another noncompetitive antagonist. In the absence of agonist TID did not significantly alter [3H]PCP binding even at the highest TID concentration used (Fig. 8, solid triangles). In contrast, in the presence of agonist TID inhibited specific [3H]PCP binding by >98% (Fig. 8, solid squares). The con-


m 6 5 4

-log [TID] (M)

FIG. 8. Effects of TID on the binding of ['HIPCP in the presence and absence of carbamylcholine. Membranes (0.7 mg/ ml, 1.0 p~ ACh binding sites) containing 5 nM ['HIPCP were equilibrated 2 h in the dark with increasing concentrations of nonradioactive TID in the presence (M) or absence (A) of 0.1 mM carbamylcholine and then bound ["HIPCP was determined by centrifugation. The dashed line indicates nonspecific ['HIPCP binding in the presence of 200 p~ meproadifen with (13) and without (A) carbamylcholine at 0 and 170 p~ TID. The ["HIPCP binding data in the presence of carbamylcholine was fit to an inhibition binding function of the form bound = A + R X free/(K X (1 + I / K J + free), where bound and free represent the specifically bound and free concentrations of ['HIPCP, respectively, A is the nonspecific ['HIPCP binding (0.2 nM), R is the AChR concentration (0.5 p ~ ) , I is the free concentration of TID, and K and K, are the dissociation constants for ['HIPCP and TID binding, respectively. Free was calculated independently, from the known total and bound ['HIPCP concentrations, and I was approximated by the total TID concentration. Values of K = 0.7 p~ and K, = 2 p~ were extracted from the fit. Inset, inhibition of ['HIPCP binding by Hlo- HTX. Membranes (0.18 mg/ml, 0.2 pM ACh binding sites) containing 20 nM ["HIPCP were equilibrated with increasing concentrations of HI,,-HTX in the presence (.) or absence (A) of 0.1 mM carbamycho- line and assayed for ["HIPCP binding as described above. The open symbols again indicate nonspecific binding. The IC5,, of HTX was -0.1 p~ in the presence of 100 p~ carbamylcholine and -0.4 p~ in its absence.

centration dependence of inhibition could be modeled as competitive, and a nonlinear least squares fit yielded Ku values of 0.7 and 2 p~ for [3H]PCP and TID, respectively. Although TID did not inhibit ['H]PCP binding in the absence of carbamylcholine, as expected HTX was potent inhibitor of ["HIPCP binding both in the asbence and presence of carbamylcholine (Fig. 8, inset).

We also examined the effect of 100 p~ TID on [3H]PCP binding over a range of concentrations in the presence and absence of agonist. [3H]PCP concentrations were varied from 100 nM to 100 pM in the absence of carbamylcholine and from 10 nM to 20 p~ in its presence. As before, nonspecific binding was determined in the presence of 200 p~ meproadifen. For each condition, specifically bound [3H]PCP is plotted in Fig. 9 according to the method of Scatchard, and the parameters characterizing the binding functions are presented in Table 11. The data in the absence of TID are consistent with previous studies (Heidmann et al., 1983; Oswald et al., 1983) which report low affinity binding sites for ['HIPCP (-5 per AChR) in addition to a high affinity site present at one copy per AChR with a KIj of 1 p~ in the presence of carbamylcholine and 4-6 p~ in its absence. The data in the presence of carbamylcholine of Fig. 9 were determined over a range of ["HIPCP concentrations where low affinity components of binding would not be expected to be evident and were fit well by a single site model giving 0.7 sites per AChR and KO = 1 PM. The data in the absence of agonist were fit well by a two site model with the concentration of the high affinity site constrained to the value obtained in the presence of agonist. The Kl1 of the high affinity site under this condition was 6 p ~ . Consistent with the failure of TID to inhibit the binding of 20 nM ["]PCP in the absence of carbamylcholine (Fig. 8), TID did not substantially change the parameters of ["HIPCP

; 0.1 0 0

0.0 0 2 0.4 0.6 0.8 1.0

Bound [3H]PCP (pM)

FIG. 9. Scatchard plot of specific [3H]PCP binding to Tor- pedo membranes in the presence and absence of both carbamylcholine and TID. Membranes (0.7 mg/ml, 1 p~ ACh binding sites) were equilibrated 2 h in the dark with 100 p~ TID (V), 100 p~ carbamylcholine (H), 100 p~ TID and 100 p~ carbamylcholine (O), or no ligands (A). In the presence of carbamylcholine only, membranes were equilibrated with 0.01-20 p~ [3H]PCP, whereas for all other conditions membranes contained 0.1-100 p~ ['HIPCP. Non- specific binding was determined under all conditions by the addition of 200 p~ meproadifen. Bound and free ['HIPCP were determined by centrifugation assay, and specifically bound ['HIPCP was determined as the difference between the total and nonspecific binding. The solid lines were calculated using parameters determined from regression analysis of the data before transformation for presentation as a Scatchard plot. Nonlinear least squares fits were made to the following equations which treat the specifically bound ['HHJPCP (bound) as a function of the free ['HIPCP (free): 1) bound = R X free/(free + K) (single site fit) or 2) bound = [R1 X free/(free + K,)] + [R2 X free/(free + K2)] (two site fit), where R and R1, and R2 represent binding site concentrations and K, Kl, and Kz represent dissociation constants. For the two-site fits R, was fixed at 0.33 p ~ , the concentration of the high affinity binding site extracted from a single site fit to the 100 p~ carbamylcholine data.

binding under this condition: in the presence of 100 PM TID the high affinity component of [3H]PCP binding had a KO = 7 p ~ . In the presence of both agonist and TID, however, [3H] PCP binding was of such low affinity that different components of binding could not be distinguished, and the data were fit to a single site model (1.2 sites per AChR, KO = 32 p ~ ) .

Effects of TID on fH]HTX Binding to Torpedo Mem- branes-In contrast to its effects on [3H]PCP binding, TID noncompetitively inhibits [3H]HTX binding to Torpedo membranes both in the presence and absence of carbamylcholine (Fig. 1OA). In the absence of carbamylcholine, high concentrations of TID reduced the amount of specifically bound [3H] HTX by approximately 70% with an ICso of 6 p ~ , whereas in the presence of agonist specific binding was reduced by 75% with an IC5o of 10 p ~ . In both cases, inhibition was maximal by 100 p~ TID. The inhibition of [3H]HTX binding by PCP in the presence and absence of carbamylcholine is shown in the inset for comparison (Fig. 1OA). In both the absence and presence of carbamylcholine PCP inhibits specific binding of [3H]HTX by >95% with ICso values of 15 and 1 p ~ , respectively.

To demonstrate that the residual component of [3H]HTX binding at high TID concentrations represented binding to the high affinity binding site, we examined the sensitivity of this component to PCP in the absence of carbamylcholine. Membranes (0.5 p~ ACh binding sites) equilibrated with 14 nM [3H]HTX in the presence and absence of 30 /*M TID were further equilibrated with increasing concentrations of PCP (0-100 p ~ ) and assayed for [3H]HTX binding by centrifugation. ['HH]HTX binding was inhibited by PCP with an IC50 of -10 p~ both in the presence and absence of 30 FM TID (Fig. 10B).

The effects of 100 p~ TID on specific [3H]HTX binding were also examined in the presence and absence of carbamylcholine at [3H]HTX concentrations varying from 25 nM to 12.8 p~ (Fig. 11, Table 11). Comparison of binding in the


'1 6 B i

- 7 6 5 4

-log [PCP] (M) FIG. 10. Inhibition of 13H]HTX binding by TID in the pres-

ence and absence of carbamylcholine. A, membranes (0.35 mg/ ml; 0.5 p~ ACh binding sites) containing 12 nM [3H]HTX were equilibrated 2 h in the dark with increasing concentrations of nonradioactive TID in the presence (.) or absence (A) of 0.1 mM carbamylcholine. The concentration of bound [3H]HTX in each sample was determined by centrifugation. The dashed line again indicates nonspecific binding in the presence of 200 p M meproadifen with (0) and without (A) carbamylcholine at 0 and 170 p~ TID. Inset, inhibition of ["HIHTX binding by PCP. Membranes (0.14 mg/ml, 0.2 p~ ACh binding sites) containing 14 nM [3H]HTX were equilibrated with increasing concentrations of PCP in the presence (.) or absence (A) of 0.1 mM carbamylcholine and assayed for [3H]HTX binding. The open symbols again indicate nonspecific binding. The ICso values of PCP in the absence and presence of 100 p M carbamylcholine were -18 and -1 pM, respectively. B, membranes (0.28 mg/ml, 0.5 p~ ACh binding sites) containing 14 nM [3H]HTX were equilibrated 2 h in the dark with increasing concentrations of PCP in the presence (7) or absence (A) of 30 PM nonradioactive TID. Total and nonspecific binding of [3H]HTX was determined as described above. The estimated IC,, was between 15 and 20 WM for both curves.

presence and absence of TID was complicated by the observation that TID unexpectedly increased the nonspecific binding of [3H]HTX, as was evident both from the component of binding blocked by meproadifen and the slope of the total binding curve asymptote (Fig. 11, inset). The data could be adequately fit, however, by separately defining the nonspecific binding for each curve using the asymptote of each total binding curve and fixing the concentration of [3H]HTX binding sites at the value obtained from a hyperbolic fit of the binding data in the absence of both TID and carbamylcholine. The results were consistent with the observed inhibition of [3H]HTX binding by TID at 12 nM [3H]HTX (Fig. 8): in both the presence and absence of agonist, 100 p~ TID increased the Ku of [3H]HTX 5-fold. The slight decrease in the KO of ["HIHTX in the presence of carbamylcholine is consistent with the preferential binding of [3H]HTX to the desensitized AChR (Elliott and Raftery, 1979, Heidmann et al., 1983).

Effects of TID on PHINicotine Binding to Torpedo Mem- 6ranes"The effects of TID on the binding of the agonist [3H] nicotine (50 nM) were examined both in the presence and absence of 50 p~ PCP (Fig. 12). Although inhibition of binding was observed in both cases, the extent and concentration dependence of inhibition varied. In the absence of PCP, [3H]nicotine binding showed a biphasic response to TID. Initially, [3H]nicotine binding decreased with increasing TID concentrations, with maximal inhibition of -40% at 10- 20 pM. At concentrations greater than 20 p M [3H]nic~tine binding began t o increase, achieving greater than 80% of its

0.2

0.0 0.00 0.05 0.10 0.15

[3H]HTX Bound (pM)

FIG. 11. Scatchard plot of specific ['HIHTX binding to Tor- pedo membranes in the presence and absence of both carbamylcholine and TID. Membranes (0.35 mg/ml, 0.5 pM ACh binding sites) containing concentrations of [3H]HTX from 25 nM to 12.8 pM were equilibrated 2 h in the dark with: 100 ptM TID (V), 100 pM carbamylcholine (B), 100 p~ TID and 100 p M carbamylcholine (O), or no ligands (A). Total concentrations of free and bound [3H]HTX were determined by centrifugation and specifically bound [3H]HTX was determined as described below (data analysis). Inset, variability of nonspecific binding in the presence of TID. The total [3H]HTX binding curves in the presence (V) and absence (A) of 100 p~ TID (in both cases in the absence of carbamylcholine) are shown together with the measured nonspecific binding (open symbols) for these two cases as defined by the addition of 200 p~ meproadifen. Solid curues show fits to the total binding and nonspecific binding in the presence of TID, dashed lines show fits to the binding in the absence of TID. Data analysis, nonlinear regression was first performed on the [3H] HTX binding data for the case in which no other ligands were present. The total concentration of bound [3H]HTX (BT) was fit as a function of the free concentration of [3H]HTX (free) according to Equation I: BT = R X free/(free + K ) + M X free, where R is the concentration of binding sites, K is the dissociation constant, and M is the slope of the asymptote to the binding curve. The value of M derived from this fit was used to calculate the specifically bound [3H]HTX (bound) according to Equation 11: bound = BT - M X free. The value of R derived (0.14 PM) was used to constrain the fits to the data under all other conditions. For each other condition, ET and free were fit to Equation I with R = 0.14 p~ and the derived value of M was used to calculate specifically bound [3H]HTX according to Equation 11. The solid lines were calculated using the values of K derived from the fits to Equation 1.

- 7 6 5 4

-log [TID] (M)

FIG. 12. TID inhibition of ['Hlnicotine binding to Torpedo membranes in the presence and absence of PCP. Membranes (0.18 mg/ml, 0.2 p~ ACh binding sites) containing 50 nM [3H]nicotine were equilibrated 2 h in the dark with increasing concentrations of nonradioactive TID in the presence (.) or absence (A) of 50 p~ PCP. The concentration of bound [3H]nicotine in each sample was determined by centrifugation. The dashed line indicates nonspecific binding in the presence of 100 pM carbamylcholine with (0) and without (A) PCP at 0 and 68 PM TID. Inset, effects of the noncompetitive antagonists PCP, tetracaine, and proadifen on t3H]nicotine binding to Torpedo membranes. [3H]Nicotine binding to membranes equilibrated with increasing concentrations of PCP (O), tetracaine (V), or proadifen (+) was determined under identical conditions as those described above. Nonspecific binding in the presence of 100 p~ carbamylcholine is indicated by the open symbols.

original value (in the absence of TID) at 70 pM TID. In the presence of PCP, TID uniformly inhibited [3H]nicotine binding in an apparently noncompetitive manner with maximal inhibition of approximately 70% at 70 p~ TID. The effects


: o.ly+"+j 0.0

0.00 0.04 0.08 0.12 0.16 0.20

Bound [3H]Nicotine (phi)

FIG. 13. Specific binding of [3H]nicotine to Torpedo membranes in the presence and absence of both TID and PCP. Membranes (0.18 mg/ml, 0.2 p M ACh binding sites) containing [3H] nicotine concentrations between 9 nM and 20 pM were equilibrated 2 h in the dark with: 15 p~ TID (V), 50 p~ PCP (m), 70 p~ TID and 50 p M PCP (O), or no ligands (A). Nonspecific binding was assayed under all conditions by the addition of 100 JLM carbamylcholine. Concentrations of free and bound [3H]nicotine were determined by centrifugation. Specific [3H]nicotine binding in the absence of other ligands was fit to a single hyperbolic equation of the form: bound = R X free/(free + K ) , where R is the concentration of [3H]nicotine binding sites and K is the dissociation constant of binding. All other data were fit to Hill equations of the form: bound = R X free"/(free" + P), where n is the Hill coefficient of binding and K is the nth root of the dissociation constant. The solid lines were drawn from these equations after transformation, with R = 0.17 pM for all curves and values of K = 0.90 and 0.32 for the no ligand (A) and 50 p~ PCP (m) cases, respectively. The value of TLH for the 50 p~ PCP curve (a) is 1.1. Both sets of data representing [3H]nicotine binding in the presence of TID shared a value of nH = 1.4, and a single curve is shown for these cases with K = 0.70 JLM which was within the limits of error of the derived values.

of the NCAs PCP, proadifen, and tetracaine on the binding of 50 nM [3H]nicotine are shown in the inset (Fig. 12). Consistent with their degrees of stabilization of the desensitized state (Boyd and Cohen, 1984; Heidmann et al., 1983), proadifen substantially increases binding while PCP does so to a lesser extent. Tetracaine, by contrast, inhibits binding of the agonist, consistent with its stabilization of a nondensitized state with low affinity for agonist (Boyd and Cohen, 1984).

Based on the results of Fig. 12, we examined the effects on ['HH]nicotine binding (9 nM to 20 pM) of 15 and 70 p~ TID in the absence and presence of 50 p~ PCP, respectively. Analysis of specific [3H]nicotine binding to membranes (0.20 ~ L M ACh binding sites) is presented in Fig. 13. [3H]Nicotine binding in the absence of effectors (solid triangles) appeared noncooperative and could be fit to a single site, with a site concentration of 0.17 pM and a KD of 0.9 pM. 50 p M PCP, consistent with its effects on the binding of other agonists (Heidmann etal., 1983), increases the affinity of specific t3H] nicotine binding. The approximately 3-fold increase in affinity may also be accompanied by slightly enhanced coopera- tively of binding (Fig. 13). Interestingly, enhancement of cooperativity ( n ~ -1.4), rather than lowered affinity, was the principal effect of TID on specific [3H]nicotine binding, both in the presence and absence of PCP. This effect is fully consistent with the inhibition of 50 nM [3H]nicotine binding by TID seen in Fig. 12.

DISCUSSION

The observation that a 100-fold excess of nonradioactive TID inhibits 80% of the AChR photolabeling by 1 p~ [Iz5I] TID demonstrates that in addition to its nonspecific interactions with the AChR of Torpedo postsynaptic membranes, [''"IITID specifically binds to and photoaffinity labels this ligand-gated ion channel. Since excess TID fails to inhibit the labeling of the a-subunit of the Na'/K'-ATPase under these conditions, it is extremely unlikely that the nonradioactive compound is simply saturating the labeling of lipid-exposed

regions of Torpedo transmembrane proteins. The same conclusion can be drawn from the failure of excess nonradioactive TID to systematically inhibit incorporation of [3H]AD into the AChR (Table I). The simplest interpretation of the results is that the saturable component of [1251]TID photoincorporation into the AChR represents photoaffinity labeling, whereas the remainder represents nonspecific labeling from the lipid phase.

The agonist-sensitive component of labeling at 1 p~ [Iz5I] TID is equivalent in magnitude to the saturable component of AChR labeling, indicating that these two components are substantially the same. This conclusion is supported by the observation that agonist-insensitive labeling is not itself inhibited by nonradioactive TID. That the saturable and agonist-inhibitable sites of ['251]TID incorporation are located on the same proteolytic fragment of the AChR a-subunit is also consistent with the identity of these two components of photolabeling. It has been suggested that agonist-sensitive incorporation of ['251]TID into the AChR results from changes in the number or reactivity of amino acid residues exposed to the lipid phase in the agonist-desensitized state (McCarthy and Stroud, 1989; Stroud et al., 1990). This interpretation is unlikely, both in view of the results just described and in view of the failure of [3H]AD to photolabel the AChR in an agonist- sensitive fashion.

The agonist insensitivity of [3H]AD photoincorporation into the AChR has been reported previously by Middlemass and Raftery (1983). In addition, those authors report the insensitivity of [3H]AD labeling to excess nonradioactive ada- mantane diazirine, indicating that this compound does not specifically photoincorporate into the AChR. This is consistent with the observation that 60 p~ TID does not substantially inhibit [3H]AD incorporation into all AChR subunits (Table I). The selective inhibition of [3H]AD incorporation into the a-subunit by both 60 p~ TID and 100 p~ carbamylcholine (Table I) might be evidence for a small component of specific labeling, although further experiments would be nec- essary to verify this.

When specific incorporation of [1251]TID into the AChR was measured as a function of ['251]TID concentration for a 30 min photolysis period, it was found to increase in a concentration-dependent manner up to -0.45 mol of [1251]TID/ mol of AChR at 58 p ~ . Higher [1251]TID concentrations, which could be achieved only by increasing isotopic dilution, could not be reliably studied, but regression analysis of the data at lower concentrations indicated saturation at 0.5 mol of [12sI]TID/mol of AChR with half-maximal specific incorporation at 0.4 p ~ . Further agonist-inhibitable incorporation of ['251]TID into the AChR subunits could be achieved if AChR-rich membranes photolabeled with 100 p~ [1251]TID were washed after photolysis to remove unincorporated photolysis products. This indicated that the observed "saturation" of photolabeling was not due to complete photoincorporation of ['251]TID into the ['2sI]TID binding site(s). The inefficiency of [1251]TID photoconversion provides one possible explanation for the apparent saturation at 0.5 mol of ['251]TID/mol of AChR. Photoactivation of ['251]TID generates the carbene responsible for photoincorporation in only 65% yield (Brun- ner et al., 1980), with an inert diazoisomer of ['Z51]TID (1-m- ['251]iodophenyl-2,2,2-trifluorodiazoethane) produced in -35% yield. During the 30-min course of photolysis the concentration of the inert diazoisomer increases as the [1251]TID concentration declines, presumably to near zero over the 30- min period (White and Cohen, 1988). If the isomer also binds to the ['251]TID site(s), the maximal specific incorporation of ['2sI]TID into the AChR could be limited by competition of


the isomer with unphotolysed ['251]TID for binding. This potential problem could be obviated in a number of ways, including the use of flash photolysis to achieve rapid and complete photoconversion or irradiation at 254 nm which is expected to photolyse both ['*'I]TID and the l-m-['251]iodophenyl-2,2,2-trifluorodiazoethane. The latter technique has the disadvantage of introducing considerable radiation damage to the protein which can complicate interpretation of the results.

An alternative means of avoiding the potential problems of photolysis at increasing ['251]TID concentrations is the repeated photolabeling of membranes at a fixed low dose of ["'I]TID with intermediate washing of the membranes to remove unincorporated photolysis products. Three successive 15-min periods of photolysis at -10 pM ['*'I]TID resulted in the specific incorporation of between 0.60 and 0.65 mol of ['251]TID/mol of AChR. Again, practical considerations made it difficult to achieve saturation experimentally, but the observed nonlinear increase in specific ['*'I]TID incorporation into the AChR over the three labeling periods was inconsist- ent with low efficiency incorporation into five (or more) binding sites per AChR. Furthermore, the results from the stoichiometry experiments involving both increasing ["'I] TID concentrations and repeated photolabeling could be ac- counted for by assuming 50% efficiency of photoincorporation into a single ['*'I]TID binding site on the AChR having a KO of 4 pM. This suggests that the specific photoincorporation of ['*'I]TID seen in the absence of agonist results from ['*'I]TID bound to a single site on the AChR.

Since all AChR subunits are photoaffinity labeled by [1251] TID, a stoichiometry of one specifically incorporated ["'I] TID molecule per AChR implies incorporation into a site in contact with all of them. The centrosymmetric arrangement of the AChR observed by electron microscopy (Brisson and Unwin, 1985, Kubalek et al., 1987) indicates that all subunits come into close proximity only in the region of the central pore, commonly thought to form the ion channel. Thus the site of [I2'I]TID photoincorporation may lie within the AChR pore.

This conclusion is consistent with the observation that nonradioactive TID potently inhibits agonist-induced **Na+ efflux from Torpedo membranes with an IC50 of 1 p~ under the conditions tested. This value was roughly an order of magnitude lower than the corresponding value for PCP tested under similar conditions. Inhibition by TID is clearly noncompetitive, since TID does not compete with the agonist ["Hlnicotine for binding to the AChR. The similarity of the IC60 for inhibition of **Na+ efflux and the half-maximal concentration for saturation of the agonist-sensitive component of photoincorporation (4 p ~ ) is also consistent with a common mechanism underlying inhibition of function and saturable photolabeling. Although the possibility that TID inhibits AChR function by perturbing the lipid environment of AChR in Torpedo vesicles cannot be completely ruled out, this seems unlikely for several reasons. 1) Overt evidence of such perturbation (uiz. "Na+ leakage) was noted only at concentrations in excess of 10 p~ TID. 2) At TID to lipid ratios that substantially inhibit 2*Na+ efflux, there is no evidence of AChR desensitization from the [3H]nicotine binding data. All surface-active agents known to disrupt AChR function strongly promote the desensitized state of the AChR characterized by high affinity for agonist (Forman and Miller, 1989). At 1 pM TID the estimated ratio of TID to lipid in the '*Na+ efflux experiments was less than 1:50. A similar TID to lipid ratio occurs under the conditions of the [3H]nicotine binding experiments at 2 pM TID, at which concentration there is no

evidence for conversion to the high-affinity state for agonist. That such conversion would have been detected is evident from the clear desensitizing effects of proadifen and PCP under the same conditions (Fig. 12, inset). 3) Finally, it is worth noting that perturbation of membrane protein structure by TID has not been reported in other systems where even higher ratios of TID to lipid have been examined. At TID to lipid ratios approaching 1, for example, TID had no effect on the spectral properties of bacteriorhodopsin in purple membranes (Brunner et al., 1985). Future investigation of TID's effects on *'Na+ efflux using AChR specifically photolabeled with TID and conducted in the presence and absence of free TID should help to further elucidate the mechanism of inhibition.

NCAs other than TID have been proposed previously to act by the mechanism of channel block. It is particularly interesting that these compounds bind in an apparently mutually exclusive manner to the AChR and have been suggested to bind to a common site within the channel (Heidmann et al., 1983, Galzi et al., 1991). The effects of TID on the binding of two NCAs, [3H]PCP and [3H]HTX, clearly indicate that TID does not share a common site with these two compounds (summarized in Table 111). In particular, TID noncompetitively inhibits [3H]HTX binding both in the presence and absence of carbamylcholine, indicating that TID and HTX bind to distinct sites on the AChR and can do so simultane- ously. It should be noted that the allosteric modulation of HTX affinity by TID is consistent with the effect of HTX on ['251]TID photoincorporation into the AChR in the absence of carbamylcholine (White and Cohen, 1988). HTX, at concentrations sufficient to fully occupy its binding site, inhibits -70% of the photoincorporation into the AChR. This con- trasts with the 80% inhibition produced by agonists and nonradioactive TID (Table 111).

Similarly, the observation that TID does not significantly affect the binding of [3H]PCP in the absence of carbamylcholine is also consistent with the failure of PCP to significantly inhibit AChR photolabeling by ['251]TID under this condition (White and Cohen, 1988). Surprisingly, the presence of agonist profoundly changes the effects of TID on [3H]PCP binding: TID completely inhibits [3H]PCP binding in the presence of 100 pM carbamylcholine (IC5, = 4 p ~ ) . Again, it seems unlikely that this effect is nonspecific, since no such effects are seen in the absence of carbamylcholine even at TID concentrations as high as 170 p~ when there is one estimated TID molecule in the membranes for every three lipid mole- cules! Also, the concentration dependence of inhibition is not linear as might be expected if lipid perturbation were the cause.

The inhibition data are consistent with a competitive in- teraction between TID and PCP in the presence of agonist, but an allosteric mechanism of inhibition remains possible. In either case the implications for NCA binding are noteworthy. If TID and [3H]PCP compete for binding to the same site in the presence of agonist, then (a) either TID or PCP (or both) change binding sites upon addition of agonist and ( b ) although TID binds competitively with PCP, and PCP apparently binds competitively with HTX, TID does not bind competitively with HTX. If the inhibition of [3H]PCP by TID is allosteric, on the other hand, the assumption that PCP and HTX inhibit each other's binding competitively becomes less firm, particularly given the very different effects of TID on the binding of these two ligands.

In this light, it seems possible that HTX and PCP do not bind to the same site. It may be that in the restricted environment of the AChR ion channel steric effects prevent the

21604 TID: A Novel Nicotinic AChR Antagonist TABLE 111

Summary of interactions between ['2511TID and other cholinergic ligands This table summarizes the effects of several cholinergic ligands on ['251]TID photoincorporation into the AChR

and the effects of TID on the binding of the corresponding tritiated ligands. Effects in both the absence and presence of 100 PM carbamylcholine are indicated. ND indicates that the effects were not determined.

Effect of TID on radioligand bindingb Effect on [1251]TID photoincorporation"

-Carbamylcholine +Carbamylcholine -Carbamylcholine +Carbamylcholine Ligand

TID Inhibits -80% (K , = 4 p ~ ) No effect ND ND HTX Inhibits -70% (ICso = 1 p ~ ) ND Increases K D -5 x PCP Inhibits -20% ND Nicotine Inhibits -80% (IC5,, -2 PM)

Increases KD less than 10% ND Increases cooperativity (n" = 1.1 6 1.4) ND

"The concentration of ['251]TID was 1-2 pM in all experiments, and the maximal inhibition of AChR

The TID concentrations used were 100 PM for [3H]HTX and [3H]PCP binding and 15 p~ for [3H]nicotine

Increases KD -5 X Increases KD greater than 30 X

photolabeling is indicated. Data are from White or Cohen (1988) or this paper.

binding, as described in the legends of Figs. 9, 11, and 13.

simultaneous binding of PCP and HTX to neighboring, but not necessarily identical sites. Both compounds are positively charged and ionic interactions may also prevent their simultaneous binding to neighboring sites. It is further possible that the inhibition is allosteric. Binding of one ligand may distort the AChR channel and prevent binding of other channel-binding ligands to nearby sites. The kinetics of association and dissociation of NCAs are not diffusion controlled except upon transient exposure to agonist (Oswald et al., 1983; Cohen et al., 1986), suggesting that changes in structure are required to accommodate their binding. TID may be able to form ternary complexes with other NCAs within the channel because of its smaller size and electrical neutrality. The identity or difference of the binding sites for NCAs will have to await identification of the individual sites, but the observation that some NCAs photoaffinity label opposite ends of the putative channel-forming region M2 in the a-subunit suggests that they may bind to distinct regions within the ion channel. Chlorpromazine photoaffinity labels a-subunit Ser-248 at the amino-terminal end of M2 (Giraudat et al., 1989)) whereas meproadifen mustard labels Glu-262 at the carboxyl end of M2 (Pedersen and Cohen, 1990). Another noncompetitive antagonist, [3H]quinacrine azide, has been shown to photoincorporate into the M1 region of the a-subunit (DiPaolo et al., 1990), a result that may also reflect the binding of different noncompetitive antagonists to different sites.

Although the TID binding site has yet to be identified, it presumably represents a hydrophobic environment. This is consistent with current models of the AChR pore, which presume that the channel lumen is formed by the apposition of the M2 regions of all subunits (see Galzi et al., 1991 for review). This region, which is presumed to be a-helical, is uncharged and was originally identified as a putative transmembrane region on the basis of its hydrophobicity and length. Electrophysiological evidence for a hydrophobic environment within the AChR ion channel (at least in the open state) also comes from the observation that the permeability of organic cations decreases as a function of increasing hydrophobicity, suggesting a nonpolar region of binding for these compounds (Adams et al., 1981). Also, there is evidence that uncharged compounds, including barbiturates, long-chained alkanols, and volatile anesthetics, can occlude that AChR ion channel (Adams, 1976; Roth et al., 1989; and McLarnon et al., 1986). Indeed, it will be interesting to determine if some of these small lipophilic noncompetitive antagonists of AChR function also interact with the TID binding site.

The markedly different effects of TID on [3H]PCP binding in the presence and absence of carbamylcholine indicate that significant structural changes occur in the region of TID binding upon addition of agonist. This is also clear from the

agonist-sensitivity of ['251]TID photolabeling. How the TID and agonist binding sites interact, however, appears complex. TID has at least two effects on the binding of 50 nM [3H] nicotine. The first is inhibitory and occurs at TID concentrations up to approximately 15 pM. This effect presumably derives from the induction of cooperativity of [3H]nicotine binding observed in full binding curves in the presence of 15 p~ TID (Fig. 13). The second effect, which occurs at TID concentrations greater than 20 pM, involves an enhancement of binding above the inhibited levels. The origin of this effect is not known. Desensitization due to membrane perturbation seems unlikely, since desensitization would also be expected to increase [3H]PCP binding and no such increase was observed. The ability of 50 p~ PCP to suppress the second effect also seems to imply some specificity in the action of TID. The mechanism by which TID induces the cooperativity of [3H] nicotine binding is unknown.

It is noteworthy that the effects of TID on [3H]nicotine binding do not account for the observed effects of agonists on ['"I]TID photolabeling of the AChR (summarized in Table 111). Nicotine, like other agonists, dramatically inhibits AChR photolabeling (data not shown), but the binding data indicate that the mechanism of inhibition of photolabeling must differ from the simple allosteric mechanism deduced for HTX. The observation that TID inhibits [3H]HTX and [3H]PCP binding

= 4-10 p ~ ) in the presence of 100 p~ carbamylcholine implies that TID binds with high affinity to the agonist- desensitized AChR. However, the absence of saturable [lZ5I] TID incorporation into the AChR in the presence of 100 pM carbamylcholine provides no evidence of such binding. The absence of saturable photolabeling in this case is all the more surprising if [ '251]TID is presumed to bind in the AChR pore. One possible explanation is that ['251]TID binds to the desensitized AChR, but photoincorporates with substantially reduced efficiency because the carbene is successfully scavenged by some other reactive molecule close to the binding site. The identity of this molecule is not clear, but it must be small as previous characterization of the distribution of ['251]TID pho- toincorporated in the presence of agonist indicates that incorporation is enhanced in low molecular weight material (White and Cohen, 1988). Perhaps the most congenial can- didate molecule is water which is known to be an extremely effective scavenger of carbenes (Brunner, 1989) and might be expected to be present in the ion channel pore. The relatively high efficiency of photoincorporation of ['251]TID into the AChR in the absence of agonist may thus imply the absence of water from the ['251]TID binding region of the AChR channel in the resting state. Reduced efficiency in the presence of agonist conversely may imply the presence of water in the agonist-desensitized state of the AChR channel.


In summary, we have shown here that TID: (a) is a novel F., and Changeux J.-p. (1987) Biochemistry 2692410-2418 noncompetitive antagonist ofthe AChR and ( b ) photoaffinity G i r d a t , J., G a l 6 J.-L., Revah, F.9 Change"% J.-p., Haumont, p.-

labels what appears to be a sing1e site On the Harter, C., &chi, T., Semenza, G., and Brunner, J. (1988) Bkckm- Y., and Lederer, F. (1989) FEBS Lett. 2 5 3 , 190-198

distinct from the binding site for [3H]HTX. TID thus defines istry 27, 1856-1864 a second high-affinity binding site for noncompetitive antag- Heidmann, T., Oswald., R. E., and Changeux, J.-P. (1983) Biochem- onists of the AChR, and it will be of interest to determine istry 22,3112-3127 whether this site binds other small uncharged compounds, Horrocks, D. L. (lg75) Clin. Chem. 21* 370-375 such as barbiturates or fluorocarbon general anesthetics Huganir, R. L.7 and Rack-, E. (1982) J. B i d Chem. 2579 9372-9378

(Lechleiter and Gruener, 1984)9 that are known to act as Konno, T., Fukuda, K., Kurasaki, M., Bujo, H., Fujita, Y., and Imoto, K., Methfessel, C., Sakmann, B., Mishina, M., Mori, Y.,

nicotinic noncompetitive antagonists. Furthermore, it is Of Numa, s. (1986) Nature 324 , 670-674 interest to determine which amino acid residues are specifi- Imoto, K., Busch, C., Sakmann, B., Mishina, M., Konno, T., Nakai, cally labeled by ['251]TID, since unlike previously used NCA J., B u h H., Mori, y., Fukuda, K., and Numa, s. (1988) Nature

photoaffinity ligands, ['2511T1D preferentially labels a non- Karlin, A,, McNamee, M. G., Weill, C. L., and Valderrama, R. (1976) 335,645-648

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Acknowledgments"We like to thank Dr. Pedersen for Kubalek, E., Ralston, S., Lindstrom, J., and Unwin, N. (1987) J. Cell. macol. 15 , 294-312

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TID: A Novel Nicotinic AChR Antagonist

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