Design and synthesis of peptides that bind α-bungarotoxin with high affinity and mimic the three-dimensional structure of the binding-site of acetylcholine receptor

α-Bungarotoxin (α-BTX) is a highly toxic snake neurotoxin that binds to acetylcholine receptor (AChR) at the neuromuscular junction, and is a potent inhibitor of this receptor. In the following we review multi-phase research of the design, synthesis and structure analysis of peptides that bind α-BTX and inhibit its binding to AChR. Structure-based design concomitant with biological information of the α-BTX/AChR system yielded 13-mer peptides that bind to α-BTX with high affinity and are potent inhibitors of α-BTX binding to AChR (IC₅₀ of 2 nM). X-Ray and NMR spectroscopy reveal that the high-affinity peptides fold into an anti-parallel β-hairpin structure when bound to α-BTX. The structures of the bound peptides and the homologous loop of acetylcholine binding protein, a soluble analog of AChR, are remarkably similar. Their superposition indicates that the toxin wraps around the binding-site loop, and in addition, binds tightly at the interface of two of the receptor subunits and blocks access of acetylcholine to its binding site. The procedure described in this article may serve as a paradigm for obtaining high-affinity peptides in biochemical systems that contain a ligand and a receptor molecule.     

Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin

Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) alpha subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to bind alpha-bungarotoxin (alpha-BTX) and several alpha-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the alpha subunit, and bind to AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corresponded to the complete sequence of Torpedo AChR alpha subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to alpha 166-203, were synthesized. alpha-BTX bound to a peptide containing the sequence alpha 181-200 and also, albeit to a lesser extent, to a peptide containing the sequence alpha 55-74. WF6 bound to alpha 181-200 and to a lesser extent to alpha 55-74 and alpha 134-153. The two other mAbs predominantly bound to alpha 55-74, and to a lesser extent to alpha 181-200. Peptides alpha 181-200 and alpha 55-74 both inhibited binding of 125I-alpha-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunits bound alpha-BTX or WF6, or interfered with their binding. Therefore, the cholinergic binding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR alpha subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the "discontinuous epitopes" observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al. (1988)].     

Two-dimensional measurement of proton T1rho relaxation in unlabeled proteins: mobility changes in alpha-bungarotoxin upon binding of an acetylcholine receptor peptide

A method for the measurement of proton T(1)(rho) relaxation times in unlabeled proteins is described using a variable spin-lock pulse after the initial nonselective 90 degrees excitation in a HOHAHA pulse sequence. The experiment is applied to alpha-bungarotoxin (alpha-BTX) and its complex with a 25-residue peptide derived from the acetylcholine receptor (AChR) alpha-subunit. A good correlation between high T(1)(rho) values and increased local motion is revealed. In the free form, toxin residues associated with receptor binding according to the NMR structure of the alpha-BTX complex with an AChR peptide and the model for alpha-BTX with the AChR [Samson, A. O., et al. (2002) Neuron 35, 319-332] display high mobility. When the AChR peptide binds, a decrease in the relaxation times and the level of motion of residues involved in binding of the receptor alpha-subunit is exhibited, while residues implicated in binding gamma- and delta-subunits retain their mobility. In addition, the quantitative T(1)(rho) measurements enable us to corroborate the mapping of boundaries of the AChR determinant strongly interacting with the toxin [Samson, A. O., et al. (2001) Biochemistry 40, 5464-5473] and can similarly be applied to other protein complexes in which peptides represent one of the two interacting proteins. The presented method is advantageous because of its simplicity, generality, and time efficiency and paves the way for future investigation of proton relaxation rates in small unlabeled proteins.     

Structural determinants of alpha-bungarotoxin binding to the sequence segment 181-200 of the muscle nicotinic acetylcholine receptor alpha subunit: effects of cysteine/cystine modification and species-specific amino acid substitutions

The sequence segment 181-200 of the Torpedo nicotinic acetylcholine receptor (nAChR) alpha subunit forms a binding site for alpha-bungarotoxin (alpha-BTX) [e.g., see Conti-Tronconi, B. M., Tang, F., Diethelm, B. M., Spencer, S. R., Reinhardt-Maelicke, S., & Maelicke, A. (1990) Biochemistry 29, 6221-6230]. Synthetic peptides corresponding to the homologous sequences of human, calf, mouse, chicken, frog, and cobra muscle nAChR alpha 1 subunits were tested for their ability to bind 125I-alpha-BTX, and differences in alpha-BTX affinity were determined by using solution (IC50S) and solid-phase (KdS) assays. Panels of overlapping peptides corresponding to the complete alpha 1 subunit of mouse and human were also tested for alpha-BTX binding, but other sequence segments forming the alpha-BTX site were not consistently detectable. The Torpedo alpha 1(181-200) and the homologous frog and chicken peptides bound alpha-BTX with higher affinity (KdS approximately 1-2 microM, IC50s approximately 1-2 microM) than the human and calf peptides (Kds approximately 3-5 microM, IC50s approximately 15 microM). The mouse peptide bound alpha-BTX weakly when attached to a solid support (Kd approximately 8 microM) but was effective in competing for 125I-alpha-BTX in solution (IC50 approximately 1 microM). The cobra nAChR alpha 1-subunit peptide did not detectably bind alpha-BTX in either assay. Amino acid substitutions were correlated with alpha-BTX binding activity peptides from different species. The role of a putative vicinal disulfide bound between Cys-192 and -193, relative to the Torpedo sequence, was determined by modifying the peptides with sulfhydryl reagents. Reduction and alkylation of the peptides decreased alpha-BTX binding, whereas oxidation of the peptides had little effect. Modifications of the cysteine/cystine residues of the cobra peptide failed to induce alpha-BTX binding activity. These results indicate that while the adjacent cysteines are likely to be involved in forming the toxin/alpha 1-subunit interface a vicinal disulfide bound was not required for alpha-BTX binding.     

Quantitation of junctional and extrajunctional acetylcholine receptors by electron microscope autoradiography after (125)I-α-bungarotoxin binding at mouse neuromuscular junctions

The distribution and quantitation of 125I-alpha-bungarotoxin (alpha-BTX) binding sites and thus acetylcholine receptor (AChR) were determined in mouse sternomastoid muscle by electron microscope autoradiography. We found that a valid criterion for receptor saturation at the neuromuscular junction was the complete elimination of neurally evoked tetanic muscle contractions, since, when such a criterion was used for the endpoint of toxin incubation, alpha-BTX was bound to approximately 90% of total available endplate sites. When, without implying localization, the presynaptic axonal membrane was used as a convenient reference structure, the concentration of alpha-BTX relative to this membrane was determined to be 46,000 +/- 27% sites/mum2.     

α-Bungarotoxin Binds to Human Acetylcholine Receptor α-Subunit Peptide 185–199 in Solution and Solid Phase but Not to Peptides 125–147 and 389–409

The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its alpha-subunits. By testing a panel of overlapping synthetic peptides encompassing the alpha-subunit segment 177-208 (containing cysteines 192 and 193) we found that specific binding of 125I-labelled alpha-bungarotoxin (alpha-BTx) was maximal in the region 185-199. Binding was inhibited by unlabelled alpha-BTx greater than d-tubocurarine greater than atropine greater than carbamylcholine. Peptide 193-208 did not bind alpha-BTx, whereas 177-192 retained 40% binding activity. Peptides corresponding to regions 125-147 (containing cysteines 128 and 142) and 389-409, or peptides unrelated to sequences of the AChR failed to bind alpha-BTx. No peptide bound 125I-alpha-labelled parathyroid hormone. The apparent affinity (KD) of alpha-BTx binding to immobilized peptides 181-199 and 185-199 was approximately 25 microM and 80 microM, respectively, in comparison with alpha-BTx binding to native Torpedo ACh receptor (apparent KD approximately 0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of alpha-BTx, and peptide 185-199 showed little evidence of dissociation after 24 h. Peptides that bound alpha-BTx did so when sulfhydryls were reduced. Cysteine modification, by N-ethylmaleimide or acetamidomethylation, abolished alpha-BTx-binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor.     

The main immunogenic region (MIR) of the nicotinic acetylcholine receptor and the anti-MIR antibodies

Myasthenia gravis (MG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) of the neuromuscular junction. The anti-AChR antibodies are heterogeneous. However, a small region on the extracellular part of the AChR alpha subunit, called the main immunogenic region (MIR), seems to be the major target of the anti-AChR antibodies, but not of the specific T-cells, in experimental animals and possibly in MG patients. The major loop of the overlapping epitopes for all testable anti-MIR monoclonal antibodies (MAbs) was localized within residues 67-76 (WNPADYGGIK for Torpedo and WNPDDYGGVK for human AChR) of the alpha subunit. The N-terminal half of alpha 67-76 is the most critical, Asn68 and Asp71 being indispensable for binding. Yet anti-MIR antibodies are functionally and structurally quite heterogeneous. Anti-MIR MAbs do not affect channel gating, but they are very potent in mediating acceleration of AChR degradation (antigenic modulation) in cell cultures and in transferring experimental MG in animals. Fab fragments of anti-MIR MAbs bound to the AChR prevent the majority of the MG patients' antibodies from binding to and causing loss of the AChR. Whether this inhibition means that most MG antibodies bind on the same small region or is a result of broad steric/allosteric effects is under current investigation.     

High acetylcholine concentrations cause rapid inactivation before fast desensitization in nicotinic acetylcholine receptors from Torpedo.

By using both a 3 to 4 ms quenched-86Rb+ flux assay and native acetylcholine receptor (AChR) rich electroplaque vesicles on which 50-60% of acetylcholine activation sites were blocked with alpha-BTX, we determined apparent rates of agonist-induced inactivation in AChR from Torpedo under conditions where measured flux response was directly proportional to initial 86Rb+ influx rate. Inactivation kinetics with acetylcholine in both the activating range (10 microM-10 mM) and the self-inhibiting range (15-100 mM) were measured at 4 degrees C. In the presence of 10 microM-1 mM acetylcholine, inactivation is characterized by a single exponential rate constant, kd (fast desensitization). Plots of kd vs. acetylcholine concentration display maximum kds [kd(max)] of 6.6-8.0 s-1, half-maximal kd at 102 +/- 16 microM, and a Hill coefficient of 1.6 +/- 0.3, closely paralleling the initial ion flux response of AChR. Thus, fast desensitization probably occurs from a doubly-liganded preopen state or the open channel state. In the self-inhibiting acetylcholine concentration range, inactivation is biphasic. A "rapid inactivation" phase is complete within 30 ms, followed by fast desensitization at a rate close to kd(max). Both the rate and extent of rapid inactivation increase with acetylcholine concentration, indicating that acetylcholine binds to its self-inhibition site with apparent kon approximately equal to 10(3) M-1s-1 and koff approximately equal to 40 s-1. This slow kon suggests either hindered access to the inhibitory allosteric site or that a fast binding step is followed by a slower conformational change leading to channel inhibition. Overall, our data suggest that acetylcholine binds preferentially to its inhibitory site when the receptor is in the open-channel conformation and that fast desensitization can occur from all multiple-liganded states.     

Localization of agonist and competitive antagonist binding sites on nicotinic acetylcholine receptors

Identification of all residues involved in the recognition and binding of cholinergic ligands (e.g. agonists, competitive antagonists, and noncompetitive agonists) is a primary objective to understand which structural components are related to the physiological function of the nicotinic acetylcholine receptor (AChR). The picture for the localization of the agonist/competitive antagonist binding sites is now clearer in the light of newer and better experimental evidence. These sites are located mainly on both alpha subunits in a pocket approximately 30-35 A above the surface membrane. Since both alpha subunits are identical, the observed high and low affinity for different ligands on the receptor is conditioned by the interaction of the alpha subunit with other non-alpha subunits. This molecular interaction takes place at the interface formed by the different subunits. For example, the high-affinity acetylcholine (ACh) binding site of the muscle-type AChR is located on the alphadelta subunit interface, whereas the low-affinity ACh binding site is located on the alphagamma subunit interface. Regarding homomeric AChRs (e.g. alpha7, alpha8, and alpha9), up to five binding sites may be located on the alphaalpha subunit interfaces. From the point of view of subunit arrangement, the gamma subunit is in between both alpha subunits and the delta subunit follows the alpha aligned in a clockwise manner from the gamma. Although some competitive antagonists such as lophotoxin and alpha-bungarotoxin bind to the same high- and low-affinity sites as ACh, other cholinergic drugs may bind with opposite specificity. For instance, the location of the high- and the low-affinity binding site for curare-related drugs as well as for agonists such as the alkaloid nicotine and the potent analgesic epibatidine (only when the AChR is in the desensitized state) is determined by the alphagamma and the alphadelta subunit interface, respectively. The case of alpha-conotoxins (alpha-CoTxs) is unique since each alpha-CoTx from different species is recognized by a specific AChR type. In addition, the specificity of alpha-CoTxs for each subunit interface is species-dependent.In general terms we may state that both alpha subunits carry the principal component for the agonist/competitive antagonist binding sites, whereas the non-alpha subunits bear the complementary component. Concerning homomeric AChRs, both the principal and the complementary component exist on the alpha subunit. The principal component on the muscle-type AChR involves three loops-forming binding domains (loops A-C). Loop A (from mouse sequence) is mainly formed by residue Y(93), loop B is molded by amino acids W(149), Y(152), and probably G(153), while loop C is shaped by residues Y(190), C(192), C(193), and Y(198). The complementary component corresponding to each non-alpha subunit probably contributes with at least four loops. More specifically, the loops at the gamma subunit are: loop D which is formed by residue K(34), loop E that is designed by W(55) and E(57), loop F which is built by a stretch of amino acids comprising L(109), S(111), C(115), I(116), and Y(117), and finally loop G that is shaped by F(172) and by the negatively-charged amino acids D(174) and E(183). The complementary component on the delta subunit, which corresponds to the high-affinity ACh binding site, is formed by homologous loops. Regarding alpha-neurotoxins, several snake and alpha-CoTxs bear specific residues that are energetically coupled with their corresponding pairs on the AChR binding site. The principal component for snake alpha-neurotoxins is located on the residue sequence alpha1W(184)-D(200), which includes loop C. In addition, amino acid sequence 55-74 from the alpha1 subunit (which includes loop E), and residues gammaL(119) (close to loop F) and gammaE(176) (close to loop G) at the low-affinity binding site, or deltaL(121) (close to the homologous region of loop G) at the high-affinity binding site, are i     

Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor alpha subunit. Dissecting a potentially myasthenogenic epitope.

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) alpha subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross-reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4-10-residues long were identified. One mAb could bind to either arm on both sides of a beta-turn structure. Five mAb bound to a very-immunogenic cytoplasmic epitope on alpha 373-380 (VICE-alpha). Three of the mAb against VICE-alpha were earlier found to cross-react with non-AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non-myasthenics. Since VICE-alpha has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE-alpha bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE-alpha-like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti-(AChR) mAb in AChR studies.     

The binding site of acetylcholine receptor as visualized in the X-Ray structure of a complex between alpha-bungarotoxin and a mimotope peptide.

We have determined the crystal structure at 1.8 A resolution of a complex of alpha-bungarotoxin with a high affinity 13-residue peptide that is homologous to the binding region of the alpha subunit of acetylcholine receptor. The peptide fits snugly to the toxin and adopts a beta hairpin conformation. The structures of the bound peptide and the homologous loop of acetylcholine binding protein, a soluble analog of the extracellular domain of acetylcholine receptor, are remarkably similar. Their superposition indicates that the toxin wraps around the receptor binding site loop, and in addition, binds tightly at the interface of two of the receptor subunits where it inserts a finger into the ligand binding site, thus blocking access to the acetylcholine binding site and explaining its strong antagonistic activity.     

Synthesis of an antigenic site of native acetylcholine receptor peptide 159-169 of Torpedo acetylcholine receptor alpha-chain.

A region of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of the Torpedo electric organ, containing residues 161-166, has been proposed to be a major antigenic site in the native AChR protein. We report the synthesis of a peptide corresponding to residues 159-169, which contains the proposed antigenic region. In quantitative radiometric titrations, radiolabelled anti-(native AChR) antibodies from three different species, rabbit, rat and dog, exhibited considerable binding (approx. 15% relative to native AChR) to Sepharose-immobilized peptide 159-169, but did not bind significantly to Sepharose-immobilized unrelated proteins or peptides. Specificity was further confirmed by the finding that no rabbit anti-AChR antibodies bound to the peptide after absorption with native AChR. These data indicate that the region 159-169 contains an antigenic site that is readily accessible in solubilized native Torpedo AChR.     

High affinity binding of alpha-bungarotoxin to the purified alpha-subunit and to its 27,000-dalton proteolytic peptide from Torpedo marmorata acetylcholine receptor. Requirement for sodium dodecyl sulfate

Intact nicotinic acetylcholine receptor (AChR) tightly binds alpha-bungarotoxin. The two toxin-binding sites are presumed to be on the two alpha-subunits, either on or near the ACh-binding sites. Isolated alpha-subunits have been found to maintain weak binding to alpha-bungarotoxin (KD approximately 0.2 microM). We describe here conditions under which the alpha-subunit and a 27,000-dalton proteolytic peptide bound alpha-bungarotoxin with high affinity. The four subunits of Torpedo marmorata AChR, as well as several proteolytic peptides of the alpha-subunit, were first purified by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. We found that the purified alpha-subunit (but not the beta-, gamma- or delta-subunits) and its 27,000-dalton peptide specifically bound 125I-labeled alpha-bungarotoxin with KD approximately 3 and 6 nM, i.e., about two orders of magnitude lower than the intact AChR. Nearly 100% of the sites were recovered. The recovery of this high affinity binding required the presence of SDS (approximately 0.02%) but non-denaturing detergents had a strongly inhibitory effect. Unlabeled alpha-toxins competed with labeled alpha-bungarotoxin, alpha-bungarotoxin being more effective than all the other toxins tested. Decamethonium and hexamethonium competed efficiently with alpha-bungarotoxin binding but carbamylcholine had only a weak effect. The main immunogenic region of the AChR was only partially preserved since conformation-dependent monoclonal antibodies to this region bound the alpha subunit-toxin complexes, but much less efficiently than the intact AChR. We conclude that SDS can be advantageous to the recovery of high toxin binding to the alpha subunit which still has not completely recovered its native conformation.     

Solution conformation of the antibody-bound tyrosine phosphorylation site of the nicotinic acetylcholine receptor beta-subunit in its phosphorylated and nonphosphorylated states

Phosphorylation of the acetylcholine receptor (AChR) seems to be responsible for triggering several effects including its desensitization and aggregation at the postsynaptic membrane and probably initiates a signal transduction pathway at the postsynaptic membrane. To study the structural and functional role of the tyrosine phosphorylation site of the AChR beta-subunit and contribute to the in-depth understanding of the structural basis of the ion channel function, we synthesized four peptides containing the phosphorylated and nonphosphorylated sequences (380-391) of the human and Torpedo AChR beta-subunits and studied their interaction with a monoclonal antibody (mAb 148) that is known to bind to this region and that is capable of blocking ion channel function. All four peptides were efficient inhibitors of mAb 148 binding to AChR, although the nonphosphorylated human peptide was considerably less effective than the three others. We then investigated the conformation acquired by all four peptides in their antibody-bound state, which possibly illustrates the local conformation of the corresponding sites on the intact AChR molecule. The phosphorylated human and Torpedo peptides adopted a distorted 3(10) helix conformation. The nonphosphorylated Torpedo peptide, which is also an efficient inhibitor, was also folded. In contrast, the nonphosphorylated human peptide (a less efficient inhibitor) presented an extended structure. It is concluded that the phosphorylation of the AChR at its beta-subunit Tyr site leads to a significant change in its conformation, which may affect several functions of the AChR.     

The tyrosine phosphorylation site of the acetylcholine receptor beta subunit is located in a highly immunogenic epitope implicated in channel function: antibody probes for beta subunit phosphorylation and function.

Tyrosine phosphorylation of the nicotinic acetylcholine receptor (AChR) seems to be involved in AChR desensitization and localization on the postsynaptic membrane. This study reveals a probable function of the single known beta subunit phosphorylation site (beta Tyr355) and provides suitable tools for its study. The epitopes for 15 monoclonal antibodies (mAbs) against the cytoplasmic side of the AChR beta subunit were precisely mapped using > 100 synthetic peptides attached on polyethylene rods. Eleven mAbs bound to a very immunogenic cytoplasmic epitope (VICE-beta) on Torpedo beta 352-359, which contains the beta Tyr355, and to the corresponding sequence of human AChR. The contribution of each VICE-beta residue to mAb binding was then studied by peptide analogues having single residue substitutions. Overall, each of the residues beta 354-359, including beta Tyr355, proved critical for mAb binding. Two of our four mAbs known to block the ion channel were found to bind at (mAb148) or close (mAb10) to VICE-beta. Tyrosine phosphorylation of Torpedo AChR by endogenous kinase(s) selectively reduced binding of some VICE-beta mAbs, including the channel blocking mAb148. We conclude that VICE-beta probably plays a key role in AChR function. Elucidation of this role should be facilitated by the identified mAb tools.     

Monoclonal antibodies to Torpedo acetylcholine receptor. Characterisation of antigenic determinants within the cholinergic binding site

Thirteen monoclonal antibodies (mAb) to the acetylcholine receptor (AChR) from Torpedo marmorata showed high avidity for the receptor but none exhibited binding to muscle AChR solubilised from seven other animal species. Five mAb and Fab monomer fragments prepared from two of them, inhibited alpha-bungarotoxin (alpha BuTx) binding to receptor by a maximum of 50%. In the presence of excess mAb the 125I-alpha BuTx bound could be precipitated by anti-IgG indicating that the mAb bound to only one of the two alpha BuTx binding sites on each AChR monomer. This site appeared to have a lower affinity for d-tubocurarine and decamethonium than the non-mAb site. Binding of five anti-site mAb was mutually competitive and four of them (AS2-AS5) were inhibited by other cholinergic ligands and influenced by four non-toxin binding site antibodies. One (AS1) bound within the toxin binding site yet outside the main neurotransmitter binding region. It is concluded that these five mAb distinguish between the two alpha BuTx binding sites on the Torpedo AChR, and bind only to the site which displays lower affinity for d-tubocurarine and other competitive ligands.     

Procaine rapidly inactivates acetylcholine receptors from Torpedo and competes with agonist for inhibition sites

The relationship between the high-affinity procaine channel inhibition site (apparent dissociation constant Kp congruent to 200 microM) and the agonist self-inhibition site on acetylcholine receptors (AChRs) from Torpedo electroplaque was investigated by using rapid 86Rb+ quenched-flux assays at 4 degrees C in native AChR-rich vesicles on which 50-60% of ACh activation sites were blocked with alpha-bungarotoxin (alpha-BTX). In the presence of channel-activating acetylcholine (ACh) concentrations (10 microM-10 mM) alone, AChR undergoes one phase of inactivation (fast desensitization, rate = kd) in under a second. Addition of procaine produces two-phase inactivation similar to that seen with self-inhibiting (greater than 10 mM) ACh concentrations [Forman & Miller (1988) Biophys. J. 54, 149-158]--rapid inactivation (rate = kr) complete in 30-75 ms is followed by fast desensitization at the same kd observed without procaine. The dependence of kr on [procaine] is consistent with a bimolecular association between procaine and its AChR site with kon = 2.5 X 10(5) M-1 s-1, koff = 36 s-1, and Kp = 145 +/- 36 microM). Inhibition of AChR function by mixtures of procaine (up to 12Kp) plus self-inhibiting concentrations of ACh or suberyldicholine ([SubCh] up to 13 X the 50% self-inhibiting agonist concentration, KB) was studied by reducing the level of alpha-BTX block in vesicles. The apparent KB increased in the presence of procaine, and the apparent KP increased linearly with [SubCh], indicating mutually exclusive actions at a common AChR site. Our data support a mechanism where procaine binds preferentially to the open-channel AChR state, since no procaine-induced inactivation is observed without agonist and kr's dependence on [ACh] in the channel-activating range closely parallels that of 86Rb+ flux response to ACh.     

Identification of a cytoplasmic region of the Torpedo nicotinic acetylcholine receptor alpha-subunit by epitope mapping.

Analysis of the binding of monoclonal antibodies (mAbs) by Torpedo nicotinic acetylcholine receptor (AChR) has demonstrated that a region of the alpha-subunit between alpha-156 and alpha-179 is exposed on the cytoplasmic surface of the nicotinic post-synaptic membrane. A panel of mAbs was produced that recognized sodium dodecyl sulfate-denatured subunits of the Torpedo AChR. Antibodies recognizing alpha-subunit were distinguished in terms of their ability to bind alpha-subunit fragments generated by Staphylococcus aureus V8 protease: an 18-kDa fragment beginning at Val-46, a 20-kDa fragment beginning at Ser-173/Ser-162, and a 10 kDa fragment beginning at Asn-339. Three mAbs, selected for binding to each of the V8-protease alpha-subunit fragments, respectively, were characterized in detail. The location of epitopes recognized by both anti-V8-18 and anti-V8-20 mAbs was determined to be within alpha-156 to alpha-179 by isolation of small immunoreactive peptides from proteolytic digests of the alpha-subunit, while the mAb reactive to V8-10 was bound to an epitope within alpha-339 to alpha-386. Quantitative evaluation of binding of the anti-V8-18 and anti-V8-20 mAbs to overlapping synthetic peptides corresponding to alpha-147 to alpha-179 localized the epitopes to distinct portions of this region. Further screening of the panel of mAbs using these synthetic peptides revealed three additional mAbs that bind in this region. The mAbs that bound the three distinct V8-protease alpha-subunit fragments were shown to bind to native AChR by indirect immunofluorescence on frozen sections of Torpedo electric organ. Binding to the native AChR was to the cytoplasmic surface of the AChR since the mAbs could bind to AChR in native vesicles, in which the AChR is oriented right-side-out, only after permeabilization of the vesicles by alkaline treatment or after scrambling of the orientation of the AChR by solubilization and reconstitution into liposomes. The location of the mAb-binding sites at the cytoplasmic surface of the AChR was visualized directly by freeze-etch immunoelectron microscopy. The identification of alpha-156 and alpha-179 as containing a cytoplasmic exposed sequence implies the existence of two non-hydrophobic transmembrane sequences between the site of N-glycosylation (Asn-141) and Cys-192, a site alkylated by the cholinergic affinity labels.