Demonstration and Affinity Labeling of a Stereoselective Binding Site for A

Home , Benzomorphan, Metazocine

Proc. Nati. Acad. Sci. USA Vol. 82, pp. 940-944, February 1985 Neurobiology Demonstration and affinity labeling of a stereoselective binding site for a benzomorphan opiate on acetylcholine receptor-rich membranes from Torpedo electroplaque (cholinergic receptor/ion channel/photoaffinity labeling/N-aliyl-N-normetazocine/phencyclidine) ROBERT E. OSWALD, NANCY N. PENNOW, AND JAMES T. MCLAUGHLIN Department of Pharmacology, New York State College of Veterinary Medicine, Cornell University, Ithaca, NY 14853 Communicated by R. H. Wasserman, October 3, 1984

ABSTRACT The interaction of an optically pure benzo- Benzomorphan opiates have been shown to inhibit the morphan opiate, (-)-N-allyl-N-normetazocine [(-)-ANMC], binding of [3H]PCP to central nervous system synaptic mem- with the nicotinic acetylcholine receptor from Torpedo electro- branes (15, 16) and to modify the activity of serum cholines plaque was studied by using radioligand binding and affinity terase (17). Some opiate derivatives, in particular the benzo- labeling. The binding was complex with at least two specific morphans, can inhibit the binding of [3H]perhydrohistrioni- components having equilibrium dissociation constants of 0.3 cotoxin and [3H]PCP to the Torpedo AcChoR (18, 19). In the jiM and 2 jiM. The affinity of the higher affinity component present studies, a radioactive optically pure benzomorphan, was decreased by carbamoylcholine but not by a-bungaro- (-)-[3H]N-allyl-N-normetazocine {(-)-[3H]ANMC}, was toxin. The effect of carbamoylcholine was not blocked by a- used to measure reversible binding to and affinity labeling of bungarotoxin. In comparison, the affinity of [3Hlphencycli- the Torpedo electroplaque AcChoR. Specific binding was dine, a well-characterized ligand for a high-affinity site for shown to be complex with at least one component having noncompetitive blockers on the acetylcholine receptor, is in- lower affinity in the presence rather than the absence of cho- creased by carbamoylcholine and the increase is blocked by a- linergic effectors. Binding to the high-affinity site was inhib- bungarotoxin. The binding of (-)-[3HJANMC was inhibited ited by PCP with a mechanism that is not competitive. a- by a number of other benzomorphans, with (-) isomers being Bungarotoxin (a-BuTx) had no effect on the decrease in af- 4- to 5-fold more potent than (+) isomers. Phencyclidine inhib- finity induced by cholinergic effectors. UV-catalyzed its the binding of (-)-[3H]ANMC to its high-affinity site by a affinity labeling (20) resulted in the labeling of the 8 subunit mechanism that is not competitive. UV-catalyzed affinity la- and, in some cases, the a subunit. these results may indicate beling indicated that the high-affinity-binding site for (-)- a unique binding site for benzomorphans on the AcChoR. [3H]ANMC is at least partially associated with the 8 subunit. Tryptic degradation of the Torpedo marmorata 8 chain sug- MATERIALS AD METHODS gested that (-)-ANMC labeled a 16,000-dalton COOH-termi- AcChoR-rich membranes were prepared from the electro- nal portion of the subunit. In contrast, 5-azido-[3Hltrimethiso- plaque of Torpedo californica, Torpedo marmorata, and quin, a photoaffinity label of the high-affinity site for noncom- Torpedo nobiliana as described (21). The binding of (-)- petitive blockers, labels a 47,000-dalton N1H2-terminal [3H]ANMC was measured by either a centrifugation (10) or a fragment of the 8 subunit. These results suggest that (-)- filtration assay (22, 23) in a total volume of 125 a.l. The buff- [3HJANMC binds to sites completely distinct from the binding er used in all cases was 50 mM 3-(N-morpholino)propanesul- sites for acetylcholine. The high-affinity-binding site for (-)- fonic acid (Mops)/NaOH and } mM EGTA, pH 7.5 (Mops/ ANMC and that for phencyclidine and 5-azidotrimethisoquin EGTA). are allosterically coupled but are regulated differently and are Cross-linking of (-)-[3H]ANMC and 5-azido-[3H]trimethi- probably physically distinct. soquin (5A[3H]T) induced by irradiation with UV light was performed as described (20, 24) except that Mops/ The nicotinic acetylcholine receptor (AcChoR) from Torpe- EGTA was the buffer. Trypsin degradation was performed do electroplaque is a multisubunit protein that mediates ion as described by Wennogle et al. (25). flux across the cell membrane in response to the binding of Benzomorphan opiates (ANMC, metazocine, pentazo- acetylcholine (AcCho). The receptor complex consists of cine, cyclazocine), etorphine, naloxone, and (-)- four different subunits of 50,116 (a), 53,681 (,B), 56,279 ('y), [3H]ANMC (49.5 Ci/mmol; 1 Ci = 37 GBq) were supplied by and 57,565 (8) daltons (measured by sequence analysis; ref. the National Institute on Drug Abuse. Phenazocine, theba- 1), existing in a stoichiometry of 2:1:1:1 (2-4). These sub- ine, and oxymorphone were gifts of A. E. Jacobson (Nation- units migrate in NaDodSO4/polyacrylamide gels with appar- al Institutes of Health). A. Gero (Hanemann University) ent molecular masses of 40,000, 50,000, 60,000, and 66,000 generously supplied metazocine, pentazocine, cyclazocine, daltons, respectively. A high-affinity-binding site for AcCho 5-ethyl-2'-hydroxy-2-methyl-6,7-benzomorphan, and 5-(m- is associated, at least in part, with each of the a subunits hydroxyphenyl)-2-methylmorphan. PCP was obtained from (reviewed in ref. 5). In addition, a series of compounds, U.S. Pharmacopeial Convention (Rockville, MD), and known collectively as noncompetitive blockers, has been [3H]PCP (48 Ci/mmol), [3H]AcCho (90 mCi/mmol), and 125I1 shown to bind to the AcChoR (6-13). These compounds, labeled a-BuTx (1251-a-BuTx) were purchased from New which include phencyclidine (PCP), histrionicotoxin, chlor- England Nuclear (Boston, MA). 5A[3H]T wag a gift of J. P. promazine, and some aminated local anesthetics, bind to one Changeux (Institut Pasteur, Paris). Live T. californica were high-affinity site per AcChoR monomer (reviewed in ref. 14) purchased from Pacific Biomarine (Venice, CA), and frozen as well as to a variable number of low-affinity sites associat- T. nobiliana were purchased from Biofish Associates ed with the membrane lipids (10, 11). Abbreviations: AcCho, acetylcholine; AcChoR, AcCho receptor; The publication costs of this article were defrayed in part by page charge (-)-ANMC, (-)-N-allyl-N-normetazocine; 5A[3H]T, 5-azido- payment. This article must therefore be hereby marked "advertisement" [3H]trimethisoquin; a-BuTx, a-bungarotoxin; MPTA, [4-(N-malei- in accordance with 18 U.S.C. §1734 solely to indicate this fact. mido)phenyl]trimethylammonium; PCP, phencyclidine. 940 Downloaded by guest on September 28, 2021 Neurobiology: Oswald et aL Proc. NatL. Acad. Sci USA 82 (1985) 941 (Georgetown, MA). Ptirified T. marmorata membranes were a gift of B. Holton and A. Ribera (Institut Pasteur). RESULTS Equilibrium Bindihg of (-)-[3H]ANMC to AcChoR-Rich Membrane Fragments. As illustrated in Fig. 1, the specific binding of (-)-[3H]ANMC to AcChoR-rich membrane fragments from T. californica is saturable. (Identical results were obtained with T. marmorata and T. nobiliana.) The curve drawn through the experimental points represents a nonlinear least squares fit of the data to a two-site model. 0 The equilibrium dissociation constant (Kd) of the high-affini- L.O ty component is 0.36 ± 0.07 MM and that of the low-affinity ~0 component is 2.2 ± 0.4 AM. The high-affinity component o 1.0 comprises between 40% and 50% of the specific binding. The mo total concentration of sites in the preparation shown in Fig. 1 was 0.1 MM, which is within 10% of the number of AcCho binding sites measured by [3H]AcCho binding and a-BuTx binding. This suggests that two binding sites for (-)-ANMC may exist on each 250,000-dalton AcChoR monomer. Both components were lost following heating to 100'C for 3 min. Fig. 2 shows a comparison of the binding of (-)- [3H]ANMC and of [3H]PCP in the presence and absence of carbamoylcholine and a-BuTx. The affinity and number of binding sites for (-)-[3H]ANMC are unaffected by the presence of a-BuTx; however, carbamoylcholine decreases the affinity of the high-affinity component without changing the number of binding sites (Fig. 2A), with an IC50 of 40 ± 8 AM. This effect is not blocked by a-BuTx. Under identical condi- Bound, cpm x 10O3 tions, a-BuTx blocks completely the agonist-induced in- FIG. 2. Binding of (-)-[3H]ANMC (A) and [3H]PCP (B) to Ac- crease in affinity for [3H]PCP and inhibits completely the ChoR-rich membrane fragments (200 nM in I251-a-BuTx sites, 0.31 binding of 125I-a-BuTx. d-Tubocurarine and decamethonium nmol of 125I-a-BuTx sites per mg of prdtein) in the presence or ab- also decrease the affinity of (-)-[3H]ANMC (IC50 values of sence of 0.2 mM carbamoylcholine (Carb) and/or 2 AM a-BuTx us- 47 ± 10 AM and 7.6 ± 1.1 MM, respectively). The effects of ing the centrifugation assay. Assays were performed following a 45- d-tubocurarine and decamethonium are not inhibited by a- min incubation at 25°C. The data are plotted a§ a Scatchard transfor- BuTx. This is in contrast to the binding of [3H]PCP, which mation of the specific binding determined following subtraction of an increased in the of nonspecific binding measured using either 0.2 mM nonradioactive shows affinity presence carbamoyl- (-)-ANMC (A) or 0.2 mM nonradioactive PCP (B). (A) The Kd for choline (EC50 = 0.15 ,uM), d-tubocurarine (EC50 = 0.08 (-)-[3H]ANMC in the presence of carbamoylcholine was 1.9 ,M, and, in the absence of carbamoylcholine, the Kd values were 0.3 AM and 2 ,M. a-BuTx had no effect on binding either in the presence or 3 A absence of carbamoylcholine. (B) The Kd for [3H]PCP was 0.11 ,M 45 S in the presence of carbamoylcholine, 1.0 AM in the absence of carbamoylcholine, 1.6 AM in the presence of a-BuTx, and 1.1 MM in 2 0 the presence of both carbamoylcholine and a-BuTx. co 1 2 -43 MM), and decamethonium (EC50 = 0.1 AM) and a decreased Free cpm x 10 affinity in the presence of a-BuTx. In the case of decametho- o 30 - nium, a decrease in [3H]PCP binding was observed at higher concentrations (IC50 = 7 AM). In the case of PCP, a-BuTx 0C.m 5| S ~ npl O f blocks the increase in affinity induced by carbamoylcholine, fragmens (Total IL B d-tubocurarine, and decamethonium. C Interaction Between Sites for PCP and (-)-ANMC. (-)- co15 ANMC is a competitive inhibitor of [3H]PCP binding both in the presence (Fig. 3D) and absence (Fig. 3C) of 0.2 MM car- e Nonspecific 00 bamoylcholine. The Ki for (-)-ANMC, calculated from Dix- '.C Bound cpm ax1 on plots, was 7.7 MM in the presence of carbamoylcholine and 4.8 MM in its absence. Nonlinear least squares analysis

~~~~~ 2 the 5 of the binding isotherms for in presence 0 (-)-[3H4]ANMC - 5 Free cpm x 10 of carbamoylcholine indicates that the Kd of the high-affinity component is unchanged in the presence of PCP, but the ap- FIG. 1. Binding of (_)-[3H]ANMC to AcChoR-rich membrane parent Bmax is decreased. On the other hand, the Kd of the fragments (300 nM in 1251-a-BuTx sites, 0.31 nmol of "'I5-a-BuTx low-affinity component is increased and the apparent Bmax sites per mg of protein). The centrifugation assay was used following remains unchanged. These data indicate that the high-affini- a 45-mmn incubation at 250C. At this incubation time and tempera- ture, the binding had reached equilibrium at all concentrations test- ty component is decreased by an apparent noncompetitive ed. Nonspecific bindingy was determined in the presence of 0.2 mM mechanism. The low-affinity component in the absence of nonradioactive (-)-ANMC. (Inset A) Expansion of the binding data carbamoylcholine is inhibited competitively by PCP. The at low concentrations, indicated by the box. (Inset B) Plot of the IC50 for PCP was calculated for each concentration of (-)- Scatchard transformation of the "specific" data points, calculated [3H]ANMC and was found to be constant (6.9 + 0.5 MM). In by subtracting the "nonspecific" binding from the "total" binding. the presence of carbamoylcholine, PCP inhibited the binding Downloaded by guest on September 28, 2021 942 Neurobiology: Oswald et al. Proc. NatL Acad Sci. USA 82 (1985) 0.10

0.05

0 0 @ FIG. 3. Binding of (-)- IL. [(3HIANMC (A and B) and 0 c n [3H]PCP (C and D) to AcChoR- 0 3 rich membrane fragments (300 m m nM in I251-a-BuTx sites, 0.32 nmol of 1251-a-BuTx sites per 0.15 mg ofprotein) in the presence (B and D) or absence (A and C) of 0.2 mM carbamoylcholine and varying concentrations of either nonradioactive PCP (A and B) 0.10 or (-)-ANMC (C and D). The centrifugation assay was used following a 45-min incubation at 250C. The data are plotted as 0.05 Scatchard transformations of the specific binding determined following subtraction of nonspecific binding measured using ei- 0 ther 0.2mM nonradioactive (-)- 1 2 3 3 ANMC (A and B) or 0.2 mM Bound (-)[3HiANMC, cpm x Bound 13Hi PCP, cpm x 10-3 nonradioactive PCP (C and D).

of (-)-[3H]ANMC with a Ki of 0.8 jM. The most likely inter- product, with the a subunit labeled relatively greater in the pretation of these results is that the high-affinity-binding site case of T. marmorata. Fig. 4 shows the labeling of peptides for (-)-ANMC is distinct from that for PCP but is allosteri- from T. nobiliana AcChoR-rich membrane fragments. Ap- cally coupled. The concentration of (-)-ANMC required to proximately 60% of the specifically bound radioactivity mea- inhibit [3H]PCP binding and the competitive nature of the sured by a filtration assay was incorporated into the sub interaction between the low-affinity (-)-ANMC site and the unit. Consistent with the reversible binding of (-)- PCP site suggest that these may be the same site. [3H]ANMC (Fig. 2), carbamoylcholine decreases the label- Interaction Between Sites for a-BuTx and (-)-ANMC. The ing of the subunit (Fig. 4, lane B vs. lane F). Again a-BuTx effect of (-)-ANMC on the initial rate of binding of 15 nM has no effect either in the presence (lane I) or absence (lane I-a-BuTx to AcChoR-rich membrane fragments (5 nM in E) of carbamoylcholine. PCP (150 AM) decreased the label- 125I-a-BuTx sites) was measured. The IC 0 for (-)-ANMC ing of the 8 chain (lanes D and H) in proportion with the was 440 ALM, indicating that the sites described in Figs. 1-3 decrease in the reversible binding measured with a filtration are not the high-affinity sites for AcCho and a-BuTx. assay. These results suggest that the binding site(s) for (-)- Stereoselectivity of Benzomorphan Binding. (-) Isomers of [3H]ANMC is associated, at least partially, with the 8 sub- benzomorphans are 4- to 5-fold more potent inhibitors of unit. (-)-[3H]ANMC binding than (+) is6mers (Table 1). This in- The labeling of the T. marmorata AcChoR was studied dicates that, in each case, the (-) isomer exhibits a decrease further by trypsin degradation of the (-)-[3H]ANMC-labeled in the free energy of binding 0.8-0.9 kcal/mol (1 cal = 4.184 membrane-bound AcChoR. T. marmorata was used in this J) greater than the (+) isomer. experiment to allow direct comparisons with previous data Changes in the substituents on the three benzomorphan (25, 26). As described by Wennogle and Changeux (26), the a rings had only minor effects. However, the addition of an- subunit is degraded sequentially into large NH2-terminal other ring and ether linkage in morphine derivatives such as fragments of 38,000, 35,000, and 32,000 daltons, which retain naloxone, etorphine, thebaine, and oxymorphone (data not the site of [3H][4-(N-maleimido)phenyl]trimethylammonium shown) or the removal of the 6,7-aromatic ring in the methyl- ([3H]MPTA) labeling (an affinity label for the AcCho binding morphan resulted in drastically decreased affinity but reten- site). The site of (-)-[3H]ANMC labeling on the a subunit, tion of stereospecificity, with approximately the same ratio like the site of [3H]MPTA labeling, remains with these large of affinities between the (-) and (+) isomers. fragments (Fig. 5). The 8 chain has been shown to be degrad- Photoaffinity Labeling. (-)-[3H]ANMC can be covalently ed sequentially by trypsin into NH2-terminal fragments of cross-linked to the AcChoR by irradiating AcChoR-rich 50,000, 49,000, and 47,000 daltons, which retain the site of membrane fragments with 254-nm light in the presence of labeling for the local anesthetic 5A[3H]T (25). Although an (-)-[3H]ANMC. Slight differences in the labeling patterns increase in radioactivity at level of the tracking dye with were observed for AcChoR-rich membrane fragments pre- trypsin treatment was observed, this is apparently not relat- pared from T. marmorata and from T. nobiliana and T. cali- ed to fragments of the 8 chain because >97% of the radioac- fornica. In general, the 8 subunit was the major cross-linked tivity incorporated into the chain can be recovered in the Downloaded by guest on September 28, 2021 Neurobiology: Oswald et aL Proc. NatL Acad Sci USA 82 (1985) 943 Table 1. Inhibition of (-)-[PH]ANMC binding by opiate derivatives R R N R3R3 N R 2 R2

)1 (+) Optical IC50, R RI R2 R3 Name rotation /IM CH3 OH CH3 CH3 Metazocine (-) 3.2 (+) 25 CHz-< OH CH3 CH3 Cyclazocine (-) 2.6 (+) 9.2 CH3 CH2-CH=C OH CH3 CH3 Pentazocine (-) 2.0 CH3 (+) 9.4 CH2CH2-jZ OH CH3 CH3 Phenazocine (-) 2.2 (+) 8.0 CH3 OH CH2CH3 H 5-Ethyl-2'-hydroxy- (-) 4.0 2-methyl-6,7- (+) 13.7 benzomorphan OH CH3 * H 5-m-Hydroxyphenyl- (-) 31 2-methylmorphan (+) 61 The binding of 50 nM (-)-[3H]ANMC to AcChoR-rich membrane fragments (200 nM in '25I-a-BuTx sites) was measured by using the rapid filtration assay following a 45-min incubation at 25°C. No carbamoylcholine or a-BuTx was present. *The 6,7-benzyl group is absent. NH2-terminal fragments. The labeling of the 8 chain by (-)- DISCUSSION [3H]ANMC does not appear in the NH2-terminal tryptic fragments of the 8 chain (Fig. 5). Instead, the site of (-)- The stereoselective binding of a benzomorphan opiate, (-)- [3H]ANMC labeling is lost upon the cleavage of the 8 chain [3H]ANMC, to AcChoR-rich membrane fragments from Tor- to its 50,000-dalton fragment with the loss of the 16,000-dal- pedo has been demonstrated. Several characteristics of the ton COOH-terminal portion of the subunit. This suggests binding distinguish this ligand from others that bind to the that (-)-[3H]ANMC may label a domain of the 8 subunit that AcChoR (reviewed in refs. 5 and 14). (i) The reversible equi- is distinct from the site labeled by 5A[3H]T. librium binding is complex, with a high-affinity component

it' 132-0

60- 66- Pw, AML = 40- ...... : so4 32-40- 3 w -- .

A BC D E F G H I 1 2 3 4 1 2 3 4 FIG. 4. NaDodSO4 electrophoretic analysis (10% acrylamide) of FIG. 5. Degradation by trypsin of alkaline-treated T. marmorata UV-catalyzed labeling of T. nobiliana AcChoR-rich membranes by AcChoR-rich membrane proteins labeled with either 5A[3H]T (Left) 50 nM (-)-[3H]ANMC (700 nM in '25I-a-BuTx sites, 0.6 nmol of or (-)-[3H]ANMC (Right) (700 nM in 125I-a-BuTx sites). Incuba- 125I-a-BuTx sites per mg of protein). A Coomassie blue stain of the tions with varying concentrations~~~~~~~~~~~~~~~~~...... oftrypsin were for 15 min at 25°C; protein (lane A) is shown next to fluorograms of labeled membranes the reactions were terminated by the addition of diisopropyl fluoro- cross-linked under the following conditions: absence of effectors phosphate to 1 jM. Shown are the fluorograms of NaDodSO4/10% {(-)-[3H]ANMC only} (lane B); 0.15 mM nonradioactive (-)- acrylamide gels of 5A[3H]T-labeled peptides (Left) and (-)- ANMC (lane C); 0.15 mM nonradioactive PCP (lane D); 2 ,uM a- [3H]ANMC-labeled peptides (Right). The concentrations of trypsin BuTx (lane E); 0.2 mM carbamoylcholine (lane F); 0.2 mM carba- were no trypsin (lanes 1), 0.16 ,ug/ml (lanes 2), 1.3 pg/mI (lanes 3), moylcholine, 0.15 mM nonradioactive (-)-ANMC (lane G); 0.2 mM and 10 /g/ml (lanes 4). 5A[3H]T labeling was performed in the pres- carbamoylcholine, 0.15 mM nonradioactive PCP (lane H); and 0.2 ence of 0.2 mM carbamoylcholine. This results in a more intense mM carbamoylcholine, 2 ,uM a-BuTx (lane I). The apparent molecu- labeling than in the absence of carbamoylcholine but produces the lar mass of the receptor subunits is given in kilodaltons (40,000 dal- same labeled tryptic fragments. Molecular masses are given in kilo- tons, a; 50,000 daltons, /8; 60,000 daltons, y; 66,000 daltons, 8). The daltons. The 50,000- and 47,000-dalton bands are fragments of the 8 132,000-dalton band represents a small population of unreduced 8-8 subunit and the 32,000-dalton band is a fragment of the a subunit; t dimer; t indicates the position of the tracking dye. indicates the position of the tracking dye. Downloaded by guest on September 28, 2021 944 Neurobiology: Oswald et aL Proc. NatL Acad ScL USA 82 (1985)

having a Kd of -0.3 uM and a low-affinity component of 2 peptide described by Anderson et al. (31), which contains AM. In the previous report by Heidmann et al. (10), multiple the major antigenic sites on the 8 subunit. components were reported but the low-affinity sites were In conclusion, the benzomorphan (-)-ANMC has been heat insensitive and, except in the case of PCP, were not shown to bind stereoselectively to the nicotinic AcChoR. inhibited by PCP, suggesting a lipid-associated binding site. The high-affinity-binding site is regulated by cholinergic ef- Both low- and high-affinity-binding sites for (-)-ANMC re- fectors in a manner opposite to that of PCP and most other ported here are heat sensitive and inhibited by PCP, suggest- noncompetitive blockers and is allosterically coupled to the ing that the sites are associated with protein rather than lipid. PCP site. Affinity labeling suggests that PCP and other non- (ii) Equilibrium binding simplifies to a single low-affinity competitive blockers interact with a site distinct from that component in the presence of carbamoylcholine and this ef- for (-)-ANMC. These results indicate that benzomorphan fect is not blocked by a-BuTx. (iii) The high-affinity compo- opiates bind to a unique site on the AcChoR. nent is noncompetitively inhibited by PCP and the low-affinity component is competitively inhibited by PCP. (iv) Tryp- The authors thank Prof. Alexander Gero, whose elegant studies of sin cleavage suggests that the portion of the T. marmorata 8 opiate action on serum cholinesterase inspired this work, for supply- subunit labeled by (-)-[3H]ANMC may be associated with a ing a number of the benzomorphans. We also thank Dr. A. E. Jacob- 16,000-dalton COOH-terminal portion of the molecule. The son for providing several opiate derivatives and Drs. B. Holton and results demonstrate a A. Ribera for their gift of purified AcChoR-rich membranes from T. unique, high-affinity-binding site for marmorata. We are grateful to Drs. G. A. Weiland, C. M. S. Few- benzomorphans that is allosterically coupled to the site for trell, S. Simasko, and M. J. Bamberger for helpful discussions, J. noncompetitive blockers (i.e., the PCP binding site). Bigelow and L. Michel for excellent technical assistance, and C. S. The equilibrium binding data are consistent with two bind- Negm for typing and editing the manuscript. This work was support- ing sites per AcChoR monomer. This is in contrast to the ed by grants from the Muscular Dystrophy Association, the Nation- binding of [3H]PCP, which exhibits one high-affinity site per al Institutes of Health (Grant 1 R23 NS 18660-01 Al NEUB), and the AcChoR monomer (10). The regulation of the binding of (-)- Department of Defense (Contract DAAK 11-83-C-0049). [3H]ANMC by effectors differs cholinergic dramatically 1. Noda, M., Takahashi, H., Tanabe, T., Toyasoto, M., Kikyotani, S., from that of [3H]PCP. Carbamoylcholine, d-tubocurarine, Furutani, Y., Hirose, T., Takashima, H., Inayama, S., Miyata, T. & and decamethonium increase the affinity of the AcChoR for Numa, S. (1983) Nature (London) 302, 528-532. [3H]PCP but decrease the affinity for (-)-[3H]ANMC. The 2. Lindstrom, J., Merlie, J. & Yogeeswaran, G. (1979) Biochemistry 18, concentrations of cholinergic effectors required to increase 4465-4470. the 3. Reynolds, J. A. & Karlin, A. (1978) Biochemistry 17, 2035-2038. affinity for PCP (ED50) are lower by a factor of 75-600 4. Raftery, M. A., Hunkapiller, M. W., Strader, C. D. & Hood, L. E. than the IC50 values for inhibition of (-)-ANMC binding. A (1980) Science 208, 1454-1457. decrease in affinity in the presence of carbamoylcholine has 5. Karlin, A. (1980) in The Cell Surface and Neuronal Function, eds. Cot- also been for the local anesthetic tetracaine man, C. W., Poste, G. & Nicolson, G. L. (Elsevier/North-Holland, Am- reported (27, 28). sterdam), pp. 191-260. King and Aronstam (19) reported a slight increase in affinity 6. Eldefrawi, A. T., Eldefrawi, M. E., Albuquerque, E. X., Oliveira, for racemic ANMC in the presence of carbamoylcholine. A. C., Mansour, N., Adler, M., Daly, J. W., Brown, G. B., Burger- The difference between the present results and those of King meister, W. & Witkop, B. (1977) Proc. Nati. Acad. Sci. USA 74, 2172- and Aronstam (19) is that measured the inhibition 2176. they only 7. Elliott, J. & Raftery, M. A. (1977) Biochem. Biophys. Res. Commun. 77, of [3H]PCP binding, an assay which is not capable of detect- 1347-1353. ing the high-affinity site. a-BuTx can inhibit the carbamoyl- 8. Krodel, E. K., Beckman, R. A. & Cohen, J. B. (1979) Mol. Pharmacol. choline-associated increase in affinity for [3H]PCP and bind- 15, 294-312. of but cannot inhibit the 9. Kloog, Y., Gabrialevitz, A., Kalir, A., Balderman, D. & Sokolovsky, M. ing 125I-a-BuTx carbamoylcholine- (1979) Biochem. Pharmacol. 28, 1447-1450. associated decrease in affinity for (-)-[3H]ANMC. This 10. Heidmann, T., Oswald, R. E. & Changeux, J. P. (1983) Biochemistry 22, suggests that an a-BuTx-insensitive binding site(s) for carba- 3112-3127. moylcholine exists on the AcChoR. This site would neces- 11. Cohen, J. B., Boyd, N. D. & Shera, N. S. (1980) in Molecular Mecha- be distinct from the two a-BuTx-sensi- nisms ofAnesthesia, ed. Fink, B. R. (Raven, New York), pp. 165-174. sarily high-affinity, 12. Kaldany, R. R. J. & Karlin, A. (1983) J. Biol. Chem. 258, 6232-6242. tive sites for carbamoylcholine that reside on the a subunits. 13. Sine, S. M. & Taylor, P. (1982) J. Biol. Chem. 257, 8106-8114. Additional sites for cholinergic agonist and antagonists have 14. Changeux, J. P. (1981) Harvey Lect. 75, 85-254. been proposed (29, 30), although the comparison with the 15. Quirion, R., Hammer, R. P., Herkenham, M. & Pert, C. B. (1981) Proc. results is not clear. The interaction Natl. Acad. Sci. USA 78, 5881-5885. present noncompetitive 16. Zukin, R. S. & Zukin, S. R. (1981) Mol. Pharmacol. 20, 246-254. between the high-affinity (-)-ANMC site and the PCP site, 17. Bamberger, M. J. & Gero, A. (1980) Arch. Int. Pharmacodyn. Ther. 245, the differential regulation by cholinergic effectors, and the 303-310. differential affinity labeling results (see below) suggest that 18. Eldefrawi, A. T., Miller, E. R., Murphy, D. L. & Eldefrawi, M. E. the site for PCP and the site for (1982) Mol. Pharmacol. 22, 72-81. binding high-affinity-binding 19. King, C. T. & Aronstam, R. S. (1983) Eur. J. Pharmacol. 90, 419-422. (-)-ANMC are distinct. The competitive interaction be- 20. Oswald, R. E. & Changeux, J. P. (1981) Proc. Natl. Acad. Sci. USA 78, tween the low-affinity site for (-)-ANMC and for the site for 3925-3929. PCP suggests that these sites may be identical. The weak 21. Saitoh, T., Oswald, R. E., Wennogle, L. P. & Changeux, J. P. (1980) inhibition of the initial rate of a-BuTx FEBS Lett. 116, 30-36. binding by (-)-ANMC 22. Oswald, R. E., Bamberger, M. J. & McLaughlin, J. T. (1984) Mol. and the heat sensitivity ofboth (-)-ANMC sites indicate that Pharmacol. 25, 360-368. these sites are neither the high-affinity sites for AcCho and 23. Oswald, R. E., Heidmann, T. & Changeux, J. P. (1983) Biochemistry 22, a-BuTx nor the lipid-associated sites reported previously for 3128-3136. some blockers 24. Oswald, R. E. & Changeux, J. P. (1981) Biochemistry 20, 7166-7174. noncompetitive (10). 25. Wennogle, L. P., Oswald, R. E., Saitoh, T. & Changeux, J. P. (1981) The UV-catalyzed cross-linking of (-)-[3H]ANMC to the Biochemistry 20, 2492-2497. AcChoR results in the specific labeling of the a and 8 sub- 26. Wennogle, L. P. & Changeux, J. P. (1980) Eur. J. Biochem. 106, 381- units for T. marmorata and of the 8 subunit for T. nobiliana 393. and T. The is controlled in a manner 27. Blanchard, S. G., Elliott, J. & Raftery, M. A. (1979) Biochemistry 18, californica. labeling 5880-5885. identical to that of the reversible binding of (-)-[3H]ANMC. 28. Strand, N. P. & Cohen, J. B. Neurosci. Abstr. 9, 160. The site of labeling on the T. marmorata 8 chain may be on a 29. Takeyasu, K., Udgaonkar, J. B. & Hess, G. P. (1983) Biochemistry 22, 16,000-dalton COOH-terminal portion of the subunit that 5973-5978. also a 30. Dunn, S. M. J. & Raftery, M. A. (1982) Proc. Natl. Acad. Sci. USA 79, contains cysteine, which is thought to be involved in 6757-6761. disulfide-linked 8-8 dimers of the receptor (25). This portion 31. Anderson, D. J., Blobel, G., Tzartos, S., Gullick, W. & Lindstrom, J. of the subunit most likely includes the 12,000-dalton tryptic (1983) J. Neurosci. 3, 1773-1784. Downloaded by guest on September 28, 2021