©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure of the Nicotinic Receptor Acetylcholine-binding Site
IDENTIFICATION OF ACIDIC RESIDUES IN THE SUBUNIT WITHIN 0.9 nm OF THE alpha SUBUNIT-BINDING SITE DISULFIDE (*)

(Received for publication, October 12, 1994)

Cynthia Czajkowski (§) Arthur Karlin (¶)

From the Center for Molecular Recognition, College of Physicians and Surgeons, Columbia University, New York, New York 10032

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

In the nicotinic receptor, the quaternary ammonium group of acetylcholine (ACh) binds to a negative subsite at most 1 nm from a readily reducible disulfide formed between alpha-subunit residues Cys and Cys. The cross-linker S-(2-[^3H]glycylamidoethyl)dithio-2-pyridine formed a disulfide bond with reduced alphaCys/Cys and an amide bond with an acidic residue in the subunit (Czajkowski, C., and Karlin, A. (1991) J. Biol. Chem. 266, 22603-22612). The fully extended cross-linking moiety -NHCH(2)CONHCH(2)CH(2)S- is 0.9 nm long. After the disulfide bond linking -NHCH(2)CONHCH(2)CH(2)S- to the alpha subunit was reduced, -NHCH(2)CONHCH(2)CH(2)SH remained linked to the subunit by an amide bond. The subunit was cleaved at Met residues, and the radioactive fragments were isolated and sequenced by automated Edman degradation. Additionally, the isolated radioactive fragments were further cleaved at Trp residues and sequenced. Peaks of release of radioactivity were obtained in the sequencing cycles corresponding to Asp, Asp, and Glu. The mutation of Asp to Asn decreased the affinity of the receptor for ACh 100-fold, whereas the mutation of either Asp, Glu, or 8 other acidic residues in the same region of decreased the affinity by 3-fold or less (Czajkowski, C., Kaufmann, C., and Karlin, A.(1993) Proc. Natl. Acad. Sci. U. S. A 90, 6285-6289). Because Asp both contributes to ACh binding and is suitably close to the binding site disulfide, it is likely to be part of the ACh-binding site formed in the interface between the alpha and the subunits.


INTRODUCTION

The binding of acetylcholine (ACh) (^1)by nicotinic receptors elicits the rapid opening of an ion-conducting channel and eventually desensitization (Katz and Thesleff, 1957). Even in the absence of high resolution structures of these receptors, we can begin to understand their function in terms of their structures through the identification of the amino acid residues forming the ACh-binding sites and the ion-conducting pathway (reviewed in Karlin, 1993; Galzi and Changeux, 1994; Akabas et al., 1994).

The effects of reduction and the different susceptibilities of the reduced receptor to affinity labels of different lengths led to the inferences that there is a readily reducible disulfide in the ACh-binding site and that this disulfide is within 1 nm of the negative subsite that binds the quaternary ammonium group of ACh (Karlin, 1969). The cysteines forming the binding site disulfide were first shown to be in the alpha subunit (Reiter et al., 1972) and subsequently identified as alphaCys and alphaCys (Kao et al., 1984; Kao and Karlin, 1986).

Four other alpha-subunit residues, all aromatic, have also been associated with the ACh-binding sites by affinity labeling. These are alphaTyr (Galzi et al., 1990; Cohen et al., 1991), alphaTrp (Dennis et al., 1988), alphaTyr (Dennis et al., 1988; Abramson et al., 1989), and alphaTyr (Middleton and Cohen, 1991). Their involvement in ACh binding was further supported by the functional consequences of site-directed mutagenesis (Mishina et al., 1985; Tomaselli et al., 1991; Galzi et al., 1991; O'Leary and White, 1992). The adjacent cysteines and the 4 aromatic residues are highly conserved among alpha subunit sequences.

Acidic residues also contribute to the binding of ACh. The cross-linker S-(2-glycylaminoethyl)dithio-2-pyridine (GCP) specifically forms a disulfide bond with a sulfhydryl (SH) and in the presence of a carbodiimide an amide bond with carboxyl groups; the fully extended cross-linking moiety -NHCH(2)CONHCH(2)CH(2)S- (GC) is 0.9 nm long. In torpedo ACh receptor, GC, attached by a disulfide bond to alphaCys or alphaCys, formed an amide bond with aspartate or glutamate residues on the subunit (Czajkowski and Karlin, 1991). The cross-linked aspartate or glutamate residues were located within a segment, 164-224, containing 6 aspartates and 5 glutamates (Fig. 1). To determine the functional importance of these Asp and Glu residues, we mutated, 1 at a time, each of the Asp and Glu residues in the homologous segment of the mouse-muscle subunit to Asn and Gln (Fig. 1). Each mutant subunit, together with wild-type alpha and beta subunits, was expressed in Xenopus oocytes, and the ACh concentration eliciting half-maximal current (K) and the K for ACh block of alpha-bungarotoxin binding were determined. By both measures, the mutation of Asp to Asn decreased the affinity for ACh 100-fold, and the mutation of Glu to Gln decreased the affinity for ACh 10-fold; by contrast, the neutralization by mutation of the other acidic residues decreased the ACh affinity 3-fold or less (Czajkowski and Karlin, 1993). Thus, we concluded that Asp and Glu contribute to the binding of ACh. We did not know, however, whether these residues specifically reacted with GC and thus how close they were to the binding site disulfide. In this paper, we demonstrate by peptide mapping and sequencing that in torpedo ACh receptor Asp, Asp, and Glu are cross-linked by GC to alphaCys or alphaCys and thus are within 0.9 nm of the binding site disulfide. Asp is both at an appropriate distance and functionally important and thus is likely to be part of the negative subsite.


Figure 1: Aligned sequences of torpedo and mouse subunits from residue 161 to residue 224. In torpedo, this sequence starts after Met and ends just before Pro, the postulated first residue of the first membrane-spanning segment. In torpedo , the sequence Asp to Lys includes the third CNBr cleavage fragment and the extracellular part of the fourth CNBr cleavage fragment. All the Asp and Glu in the mouse sequence were mutated individually to N and Q as shown below the sequence, and the mutations that strongly affected ACh binding are marked with an asterisk.




EXPERIMENTAL PROCEDURES

Materials

[^3H]GCP, specific activity 0.5-1 Ci/mmol, and non-radioactive GCP were synthesized as before (Czajkowski and Karlin, 1991). ACh receptor in Triton X-100 solution was purified by affinity chromatography on the adduct of bromoacetylcholine and Affi-Gel 401 (Chak and Karlin, 1992). [^3H]N-Ethylmaleimide ([^3H]NEM), 55 Ci/mmol, was purchased from DuPont NEN; 1-ethyl-3-(3`-dimethylaminopropyl)carbodiimide (EPCD) was from Sigma; 2-(2`-nitrophenysulfenyl)-3-methyl-3-bromoindolenine (BNPS-skatole) was from Pierce; and cyanogen bromide (CNBr) was from FLUKA. Reagent grade and HPLC grade solvents were from commercial sources and used as received.

Cross-linking the alpha and Subunits with GCP

As before (Czajkowski and Karlin, 1991), purified ACh receptor (50 nmol of binding sites) was alkylated with 20 mM NEM, dialyzed, reduced for 20 min with 0.2 mM dithiothreitol (DTT) at pH 8.1, dialyzed, reacted with 250 nmol of either [^3H]GCP (Fig. 2, variation A) or GCP (Fig. 2, variation B), dialyzed, reacted with EPCD, acetone precipitated, and finally reduced with 50 mM DTT in 2% SDS at 50 °C. In variation A, all free SH were alkylated with 110 mM NEM. In variation B, the DTT concentration was decreased by precipitation with 90% acetone. The dried protein was dissolved in 2% SDS, 20 mM Tris (pH 8.0), and reacted with 10 mM 5,5`-dithio-bis-(2-nitrobenzoate) (NBS)(2) to form mixed disulfides with the remaining protein-associated SH, including the free SH on GC linked to the subunit. The subunit linked via an amide bond to either [^3H]GC-NEM (variation A) or GC-NBS (variation B) was isolated by SDS-polyacrylamide gel electrophoresis (PAGE) on 7.5% slab gels (Laemmli, 1970). Following electrophoresis, the gel was briefly stained with 0.01% Coomassie Brilliant Blue in 25% isopropyl alcohol, 10% acetic acid, and destained in 10% acetic acid. The stained band corresponding to the subunit was excised, washed in 50 mM ammonium bicarbonate, 0.1% SDS (electroelution buffer), transferred to an electroelution cup (ISCO), and electroeluted at 50 V. We recovered approximately 0.04 nmol of [^3H]GC amide-coupled to /nmol of receptor input initially.


Figure 2: Cross-linking the receptor with GCP. The intact receptor complex is represented as a rectangle, within which are indicated the relevant reacting entities: COO is a carboxylate side chain on the subunit, alphaSS is the binding site disulfide between alphaCys and alphaCys on the alpha subunit. Isolated subunits or their derivatives are not enclosed by a rectangle. CNBr4 is the CNBr4 fragment of . Other abbreviations are DTT, dithiothreitol; H(3)NRSSP, S-(2-glycylaminoethyl)dithio-2-pyridine) (elsewhere, GCP); NEM, N-ethylmaleimide; EPCD, 1-ethyl-3-(3`-dimethylaminopropyl)carbodiimide; NBS, 5-thio-2-nitrobenzoate; (NBS)(2), 5,5`-dithio-(2-nitrobenzoate). In one variant of the procedure, A, H(3)NRSSP was synthesized with tritiated glycine. In the other variant, B, H(3)NRSSP was not radioactive, the thiol of CNBr4-COHNRSH was protected with NBS until the the fragment was isolated, after which the free SH was restored by reduction and reacted with [^3H]NEM.



CNBR Cleavage of

The electroeluted was dried under vacuum in a centrifuge (Savant Speed-Vac) and washed twice by acetone precipitation. The dried pellet was dissolved in 100 µl of 1 M CNBr in 80% trifluoroacetic acid and kept for 12 h in the dark at room temperature.

Reverse-phase HPLC

The CNBr cleavage products were directly injected onto a Vydac C4, 25 cm times 0.46-cm column and eluted with a mixture of 0.07% trifluoroacetic acid (buffer A) and 60% acetonitrile, 40% isopropyl alcohol, 0.03% trifluoroacetic acid (buffer B) as described previously (Czajkowski and Karlin, 1991). HPLC fractions containing 164-257 (CNBr 4) and 161-257 (CNBr 3 + 4) were pooled and dried down in a Savant Speed-Vac. The peptides were dissolved in 70% formic acid and sequenced on a polybrene-coated, glass-fiber filter.

Labeling GC Amide Bonded to the Subunit with ^3H-NEM (Fig. 1, Variation B)

The HPLC fractions containing CNBr 4 and CNBr (3 + 4) were combined, dried, and dissolved in 100 µl of 1 mM DTT in 1% SDS, 10 mM Tris, 1 mM EDTA pH 8.0, and incubated 1 h at 50 °C to remove the protecting NBS and restore the free SH of GC. The DTT was removed from the peptides by their precipitation with 90% acetone in the presence of carboxymethylated, succinylated lysozyme as a carrier. The precipitated peptides were washed with acetone, dried, dissolved in 100 µl of 1% SDS, 10 mM Tris, 1 mM EDTA, pH 7.0, and reacted with 25 µCi of [^3H]NEM for 1 h at room temperature. All SH not labeled with [^3H]NEM in 1 h were alkylated with non-radioactive NEM (10 mM). The peptides were acetone precipitated, washed with acetone, dissolved in Laemmli sample buffer, separated by Tricine SDS-PAGE, and electroblotted onto PVDF paper (as described below). Approximately 0.39 nmol of [^3H]NEM was incorporated/nmol initial input of subunit.

N-terminal Sequencing

Sequencing was performed by automated Edman degradation on an Applied Biosystem Inc. model 477A pulsed-liquid phase protein/peptide sequenator. Unless stated otherwise, the sequenator cycles were modified so the anilinothiazolinone (ATZ) amino acid conversion to phenylthiohydantoin (PTH) derivative was omitted and the freshly cleaved ATZ amino acids were directly transferred to a fraction collector with butylchloride. For each sequenator cycle, the radioactivity of the ATZ amino acids recovered was measured by evaporating the residual butyl chloride to dryness, extracting the contents of each tube two times with 100 µl of methanol, and counting the methanol extract, as well as the individual fraction tubes. Alternatively, peptides and blots were sequenced, and the PTH-amino acids produced by each cycle of Edman degradation were analyzed with an on-line 120A PTH analyzer. The repetitive yield of residues averaged 90%/sequencer cycle.

Tricine Gel Electrophoresis and Electroblotting

CNBr fragments of the subunit, isolated by HPLC, were further purified by Tricine-SDS-PAGE (Schagger and von Jagow, 1987; Czajkowski and Karlin, 1991). After electrophoresis, the peptides were electroblotted onto polyvinylidene difluoride (PVDF) paper as described by Matsudaira (1987).

Tryptophan Cleavage of CNBr 4 and CNBr (3 + 4)

The band of ^3H-labeled CNBr 4 and CNBr 3 + 4, blotted on PVDF paper, was excised, washed three times with water, and reacted with 150 µl of 1 µg/µl BNPS-skatole in 75% acetic acid for 1 h in the dark at 50 °C (Crimmens et al., 1990). The liquid was removed and concentrated to approximately 30 µl in a Savant Speed-Vac. The paper was washed five times with water and dried. The concentrated liquid and the paper were combined and sequenced together.


RESULTS

Isolated subunit, cross-linked via an amide bond to [^3H]GC was cleaved with CNBr, and the cleavage fragments were separated by HPLC (Fig. 3). There was a single major peak of radioactivity centered on the fraction eluting at 63 min (Fig. 3B), which corresponded to a major peak of peptide, as detected by absorbance at 205 nm (Fig. 3A). The fractions centered on fraction 63 were pooled and sequenced. The amino acids released corresponded to the predicted sequences for CNBr 4 (residues 164-257) and to CNBr 3 + 4 (residues 161-257) (data not shown). The latter arises because the cleavage of the Met-Thr bond by CNBr is incomplete (cf. Czajkowski and Karlin, 1991). Additionally, a minor sequence was present that corresponded to CNBr 1 ( 1-58). Because we showed previously (Czajkowski and Karlin, 1991) that all of the radioactivity in this peak is associated with CNBr 4 and CNBr 3 + 4, we did not need to consider CNBr 1 in analyzing the release of radioactivity in the sequencer cycles. The HPLC elution patterns after CNBr cleavage were the same for cross-linked with radioactive [^3H]GC, according to the reaction scheme in variation A of Fig. 1, and for cross-linked with non-radioactive GC, which was finally radioactively tagged with [^3H]NEM, according to the reaction scheme in variation B of Fig. 1.


Figure 3: The isolation by HPLC of CNBr cleavage fragments of covalently linked via an amide bond to radioactive GC. Starting with 50 nmol of torpedo receptor, the subunit was cross-linked to [^3H]GC as in Fig. 1, variation A. The subunit was isolated by electrophoresis and cleaved with CNBr, and the cleavage products were separated by HPLC. A, the elution of peptides monitored by A. The elution rate was 1 ml/min. B, the elution of tritiated peptide in 1-ml fractions as determined by scintillation counting of 50-µl aliquots. Input of tritium was 110,000 counts/min; recovery was 98,400 counts/min. The peak eluting around 63 min contained 36,000 counts/min.



The HPLC fractions containing CNBr 4 and CNBr 3 + 4 either were directly sequenced or were first subjected to Tricine-SDS-PAGE and then sequenced. In the latter case, CNBr 4 and CNBr 3 + 4 were separated from CNBr 1. The patterns of radioactive release were similar in all cases. Peaks of radioactive release were obtained in sequencer cycles 2, 5, 8, and 17 through 22 (Fig. 4).


Figure 4: Release of radioactive amino acids during automated Edman degradation of [^3H]GC-labeled fragments. The data are the means ± S.E. of nine independent sequencing experiments. In six experiments, the fractions eluting from the HPLC in the peak centered on 63 min were combined and sequenced (see ``Experimental Procedures''). In three experiments, the HPLC fractions were further purified by Tricine-SDS-PAGE and blotted onto PVDF paper, and the 15,700-dalton band was sequenced. In all cases, the radioactivity of the ATZ-derivatized amino acid was determined and is expressed as a fraction of the total radioactivity recovered. The radioactivity is that observed, not corrected for the repetitive yield of the cleavage cycles. Of the radioactivity loaded on the sequencer, about 30% was released in the sequencer cycles, about 50% remained on the filter and cartridge seal, and about 20% was lost in the washes.



CNBr 4 and CNBr 3 + 4 contain 2 Trp residues (Trp and Trp) (Fig. 1). We used BNPS-skatole to cleave the mixture of CNBr 4 and CNBr 3 + 4 at these Trp residues, and we sequenced the mixture of products to determine the success of the cleavage. The amino acid residues released corresponded to those of the four expected fragments: the N-terminal part of CNBr 4, starting at Thr (SKAT 1), the N-terminal part of CNBr 3 + 4, starting at Asp (SKAT 2), the fragment starting after Trp at Ile (SKAT 3), and the fragment starting after Trp at Glu (SKAT 4) ( Fig. 1and Table 1). Of the residues detected, residues in eight cycles were unique for SKAT 1, residues in five cycles were unique for SKAT 2, residues in six cycles were unique for SKAT 3, and residues in six cycles were unique for SKAT 4 (Table 1). Thus, each of the four expected sequences were present in the mixture. In parallel experiments, with CNBr 4 and CNBr 3 + 4 amide coupled to [^3H]GC, sequencing of the products generated by cleavage at Trp yielded peaks of radioactive release in sequencer cycles 2, 4, and 6 (Fig. 5).




Figure 5: Release of radioactive amino acids during automated Edman degradation of [^3H]GC-labeled CNBr cleavage fragments also cleaved at Trp residues. The fragments were separated by HPLC and then by Tricine-SDS-PAGE. The 15,700-dalton band blotted on PVDF paper was treated with BNPS-skatole (``Experimental Procedures''), and the resulting cleaved peptides on the paper were subjected to automated Edman degradation. The radioactivity released is expressed as the fraction of the total recovered and is not corrected for repetitive yield. The means ± S.E. of four independent experiments are shown. In a typical experiment, fragments from the HPLC containing 15,000 counts/min were separated by Tricine-SDS-PAGE, 90% of this radioactivity was electroblotted onto PVDF paper, and 8,700 counts/min were associated with the 15,700-dalton band (CNBr 4 and CNBr 3 + 4). After cleavage at Trp and sequencing, 2,900 counts/min were recovered in the sequencing cycles, and 1,000 counts/min were associated with the blot, filter, and cartridge seal.




DISCUSSION

The reaction whereby the subunit was cross-linked to GC uniquely formed an amide bond between a carboxylate on and the Gly-alpha-amino group of GC. Therefore, in sequential Edman degradation of labeled CNBr 4 and CNBr 3 + 4, or of their subfragments generated by cleavage at Trp, release of radioactivity should correspond to the release of either an Asp or a Glu residue. Sequential Edman degradation of the mixture of CNBr 4 and CNBr 3 + 4 gave peaks of radioactivity in cycle 2, uniquely specifying Asp, cycles 5 and 8, consistent with either Asp or Asp, in addition to Asp, cycles 17, 18, and 21, uniquely specifying Asp, and cycle 19 and 22, uniquely specifying Glu (Fig. 6). Sequential Edman degradation of the subfragments generated by cleavage at Trp gave peaks of radioactivity in cycle 2, specifying Asp, cycle 4, specifying Asp, and cycle 6, specifying Glu (Fig. 6).


Figure 6: The expected amino acid residues released in each sequencer cycle for the mixture of CNBr 4 and CNBr 3 + 4 and for the BNPS-skatole-cleavage fragments (SKAT1, 2, 3, and 4). The suffix a indicates the amino acids released without a lag at Pro, and the suffix b indicates the amino acids released with a lag at Pro. (Under normal sequencing conditions, cleavage at the Pro carboxyl is incomplete and significant further cleavage occurs in the next cycle; this causes a lag by one cycle in the release of residues subsequent to the Pro (Brandt et al., 1976).) The amino acids in the cycles yielding peaks of radioactivity are boxed. The Asp and Glu residues are numbered according to the torpedo sequence as in Fig. 1.



The results of the sequencing of the CNBr fragments and of their subfragments cleaved at Trp are consistent in implicating Asp, Asp, and Glu as residues that were cross-linked to GC (Fig. 6). The labeling of Asp or Asp, although suggested by the peaks in cycles 5 and 8 in the sequencing of the CNBr fragments, was not supported by the sequencing after cleavage at Trp. (The peak in cycle 5 was at least in part due to Asp.) Because of the incomplete cleavage and incomplete recovery of radioactivity from the sequencer, we can determine with greater certainty which residues were labeled than which residues were not.

Since the fully extended GC cross-link is 0.9 nm long, the side chain carboxylate group of the 3 labeled residues in the subunit, Asp, Asp, and Glu, are not more than 0.9 nm from alphaCys or alphaCys. Of the 3 residues, only the mutation of Asp had a large effect on the binding of ACh: the mutation to Asn caused an approximately 100-fold decrease in the affinity for ACh (Czajkowski et al., 1993). We conclude that Asp is likely to contribute directly to the binding of ACh. Asp and Glu are also within 0.9 nm of alphaCys/Cys, but mutation of these residues to Asn and Gln, respectively, caused only a 2.3- and 3.3-fold increase in the K for ACh.

The mutation of Glu to Gln caused a 10-fold decrease in the affinity of ACh (Czajkowski et al., 1993). The current experiments provide no evidence, however, that Glu is within the binding site. Glu was at best 13 cycles out in fragment SKAT 3; given a repetitive yield of about 90%, about 25% of input labeled Glu might have been released, and the labeling of this residue might have been undetected. On the other hand, the functional effect of mutating Glu to Gln could have been an indirect one mediated through non-local structural changes.

The cross-linking of Asp, Asp, and Glu to alphaCys/Cys by the hydrophilic GC is consistent with these residues facing alphaCys/Cys across a water-filled crevice between the alpha and subunits. We hypothesize that this crevice between subunits contains the ACh-binding site. The location of these negatively charged residues at the surface of this crevice is consistent with the negative electrostatic potential at alphaCys/Cys (Stauffer and Karlin, 1994). Only the neutralization by mutation of Asp, however, causes a large decrease in the affinity for ACh (Czajkowski et al., 1993). The mutation of Asp could either alter the structure of the binding site, or it could eliminate the electrostatic interaction between the carboxylate of Asp and the positively charged quaternary ammonium group of ACh. If the latter is the case, then Asp and Glu must not be as close to the quaternary ammonium group of bound ACh as is Asp, although they are also within 0.9 nm of alphaCys/Cys. For example, a molecular model of Asp-Pro-Glu shows that the side chain carboxylates of Asp and Glu could be 1 nm or more apart and still face alphaCys/Cys, 0.9 nm away. Thus, when ACh is bound, the acetyl group could bind next to alphaCys/Cys (Karlin, 1969) and the quaternary ammonium group could bind next to Asp but not next to either Glu or Asp.

The receptor has two, non-identical ACh-binding sites (Damle and Karlin, 1978; Neubig and Cohen, 1979; Dowding and Hall, 1987). In the native muscle-type ACh receptor, with composition alpha(2)beta (Reynolds and Karlin, 1978), one site is formed between the first alpha and the subunit, and the other site is formed between the second alpha and the subunit (Kurosaki et al., 1987; Blount and Merlie, 1989; Pedersen and Cohen, 1990; Sine and Claudio, 1991). Consistent with the intersubunit location of the binding sites, a number of uncharged residues in and contribute directly or indirectly to the binding of the competitive inhibitor d-tubocurarine (Chiara and Cohen, 1992; Sine, 1993; O'Leary et al., 1994).

In 164-224, only 3 carboxylate residues, Asp, Glu, and Glu, are identically conserved among all aligned sequences of the , , and subunits (Czajkowski et al., 1993). (The subunit substitutes for in adult muscle ACh receptor (Mishina et al., 1986).) The conservation of Asp is consistent with its role in the ACh-binding site formed between alpha and and with the similar role of the homologous residue in , , and . The mutation to Asn of mouse Asp, which aligns with Asp, also causes a two-order-of-magnitude decrease in the affinity of the receptor for ACh. (^2)The location of the ACh-binding sites in the interfaces between subunits is consistent with ACh binding inducing a sliding of one subunit past another (Unwin, 1989), a change that could be transmitted from the ACh-binding sites on the extracellular side of the membrane to the channel gate on the cytoplasmic side of the membrane (Czajkowski et al., 1993; Akabas et al., 1994).


FOOTNOTES

*
This research was supported in part by Research Grant NS07065 from the National Institutes of Health and by grants from the Muscular Dystrophy Association, Inc. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported in part by National Institutes of Health Training Grant NS07258. Present address: Dept. of Neurophysiology, University of Wisconsin, Madison, WI 53703.

To whom correspondence should be addressed. Tel.: 212-305-3973; Fax: 212-305-5594.

(^1)
The abbreviations used are: ACh, acetylcholine; ATZ, anilinothiazolinone; BNPS-skatole, 2-(2`-nitrophenysulfenyl)-3-methyl-3-bromoindolenine; CNBr, cyanogen bromide; DTT, dithiothreitol; GC, N-glycylcysteamine; GCP, S-(2-glycylamidoethyl)dithio-2-pyridine; EPCD, 1-ethyl-3-(3`-dimethylaminopropyl) carbodiimide; NBS, 5-thio-2-nitrobenzoate; (NBS)(2), 5,5`-dithio-bis-(2-nitrobenzoate); NEM, N-ethylmaleimide; PTH, phenylthiohydantoin; PVDF, polyvinylidene difluoride; SH, sulfhydryl; Tricine, N-tris(hydroxymethyl)methylglycine; HPLC, high performance liquid chromatography.

(^2)
M. Martin, C. Czajkowski, and A. Karlin, manuscript in preparation.


ACKNOWLEDGEMENTS

We thank M. Akabas, J. Javitch, and M. Martin for advice and G. Salazar-Jimenez for technical assistance.


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