Orientation of alpha-neurotoxin at the subunit interfaces of the nicotinic acetylcholine receptor.

The alpha-neurotoxins are three-fingered peptide toxins that bind selectively at interfaces formed by the alpha subunit and its associating subunit partner, gamma, delta, or epsilon of the nicotinic acetylcholine receptor. Because the alpha-neurotoxin from Naja mossambica mossambica I shows an unusual selectivity for the alpha gamma and alpha delta over the alpha epsilon subunit interface, residue replacement and mutant cycle analysis of paired residues enabled us to identify the determinants in the gamma and delta sequences governing alpha-toxin recognition. To complement this approach, we have similarly analyzed residues on the alpha subunit face of the binding site dictating specificity for alpha-toxin. Analysis of the alpha gamma interface shows unique pairwise interactions between the charged residues on the alpha-toxin and three regions on the alpha subunit located around residue Asp(99), between residues Trp(149) and Val(153), and between residues Trp(187) and Asp(200). Substitutions of cationic residues at positions between Trp(149) and Val(153) markedly reduce the rate of alpha-toxin binding, and these cationic residues appear to be determinants in preventing alpha-toxin binding to alpha 2, alpha 3, and alpha 4 subunit containing receptors. Replacement of selected residues in the alpha-toxin shows that Ser(8) on loop I and Arg(33) and Arg(36) on the face of loop II, in apposition to loop I, are critical to the alpha-toxin for association with the alpha subunit. Pairwise mutant cycle analysis has enabled us to position residues on the concave face of the three alpha-toxin loops with respect to alpha and gamma subunit residues in the alpha-toxin binding site. Binding of NmmI alpha-toxin to the alpha gamma interface appears to have dominant electrostatic interactions not seen at the alpha delta interface.

The extracellular linker of muscle acetylcholine receptor channels is a gating control element.

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2-M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the alpha subunit (alphaS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the alphaS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an approximately 92-fold increased gating equilibrium constant, which is consistent with an approximately 10-fold decreased EC(50) in the presence of ACh. With choline, this mutation accelerates channel opening approximately 28-fold, slows channel closing approximately 3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, alphaS269I acetylcholine receptors open at a rate of approximately 1.4 x 10(6) s(-1) and close at a rate of approximately 760 s(-1). These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of approximately 140 s(-1). Ile mutations at positions flanking alphaS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the alpha subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the alphaS269I mutation, Ile mutations at equivalent positions of the beta, straightepsilon, and delta subunits do not affect apparent open-channel lifetimes. However, in beta and straightepsilon, shifting the mutation one residue to the NH(2)-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2-M3L of the alpha subunit moves before the corresponding linkers of the beta and straightepsilon subunits.

Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation epsilonA411P in the amphipathic helix of the acetylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three unrelated families are either homozygous for epsilonA411P or are heterozygous and harbor a null mutation in the second epsilon allele, indicating that epsilonA411P is recessive. We expressed human AChRs containing wild-type or A411P epsilon subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by epsilonA411P. Prolines engineered into positions flanking residue 411 of the epsilon subunit greatly increase the range of activation kinetics similar to epsilonA411P, whereas prolines engineered into positions equivalent to epsilonA411 in beta and delta subunits are without effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

Hydrophobic pairwise interactions stabilize alpha-conotoxin MI in the muscle acetylcholine receptor binding site.

The present work delineates pairwise interactions underlying the nanomolar affinity of alpha-conotoxin MI (CTx MI) for the alpha-delta site of the muscle acetylcholine receptor (AChR). We mutated all non-cysteine residues in CTx MI, expressed the alpha(2)betadelta(2) pentameric form of the AChR in 293 human embryonic kidney cells, and measured binding of the mutant toxins by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The CTx MI mutations P6G, A7V, G9S, and Y12T all decrease affinity for alpha(2)betadelta(2) pentamers by 10,000-fold. Side chains at these four positions localize to a restricted region of the known three-dimensional structure of CTx MI. Mutations of the AChR reveal major contributions to CTx MI affinity by Tyr-198 in the alpha subunit and by the selectivity determinants Ser-36, Tyr-113, and Ile-178 in the delta subunit. By using double mutant cycles analysis, we find that Tyr-12 of CTx MI interacts strongly with all three selectivity determinants in the delta subunit and that deltaSer-36 and deltaIle-178 are interdependent in stabilizing Tyr-12. We find additional strong interactions between Gly-9 and Pro-6 in CTx MI and selectivity determinants in the delta subunit, and between Ala-7 and Pro-6 and Tyr-198 in the alpha subunit. The overall results reveal the orientation of CTx MI when bound to the alpha-delta interface and show that primarily hydrophobic interactions stabilize the complex.

Pairwise electrostatic interactions between alpha-neurotoxins and gamma, delta, and epsilon subunits of the nicotinic acetylcholine receptor.

alpha-Neurotoxins bind with high affinity to alpha-gamma and alpha-delta subunit interfaces of the nicotinic acetylcholine receptor. Since this high affinity complex likely involves a van der Waals surface area of approximately 1200 A(2) and 25-35 residues on the receptor surface, analysis of side chains should delineate major interactions and the orientation of bound alpha-neurotoxin. Three distinct regions on the gamma subunit, defined by Trp(55), Leu(119), Asp(174), and Glu(176), contribute to alpha-toxin affinity. Of six charge reversal mutations on the three loops of Naja mossambica mossambica alpha-toxin, Lys(27) --> Glu, Arg(33) --> Glu, and Arg(36) --> Glu in loop II reduce binding energy substantially, while mutations in loops I and III have little effect. Paired residues were analyzed by thermodynamic mutant cycles to delineate electrostatic linkages between the six alpha-toxin charge reversal mutations and three key residues on the gamma subunit. Large coupling energies were found between Arg(33) at the tip of loop II and gammaLeu(119) (-5.7 kcal/mol) and between Lys(27) and gammaGlu(176) (-5.9 kcal/mol). gammaTrp(55) couples strongly to both Arg(33) and Lys(27), whereas gammaAsp(174) couples minimally to charged alpha-toxin residues. Arg(36), despite strong energetic contributions, does not partner with any gamma subunit residues, perhaps indicating its proximity to the alpha subunit. By analyzing cationic, neutral and anionic residues in the mutant cycles, interactions at gamma176 and gamma119 can be distinguished from those at gamma55.

Pairwise interactions between neuronal alpha(7) acetylcholine receptors and alpha-conotoxin PnIB.

This work uses alpha-conotoxin PnIB to probe the agonist binding site of neuronal alpha(7) acetylcholine receptors. We mutated the 13 non-cysteine residues in CTx PnIB, expressed alpha(7)/5-hydroxytryptamine-3 homomeric receptors in 293 HEK cells, and measured binding of each mutant toxin to the expressed receptors by competition against the initial rate of (125)I-alpha-bungarotoxin binding. The results reveal that residues Ser-4, Leu-5, Pro-6, Pro-7, Ala-9, and Leu-10 endow CTx PnIB with affinity for alpha(7)/5-hydroxytryptamine-3 receptors; side chains of these residues cluster in a localized region within the three-dimensional structure of CTx PnIB. We next mutated key residues in the seven loops of alpha(7) that converge at subunit interfaces to form the agonist binding site. The results reveal predominant contributions by residues Trp-149 and Tyr-93 in alpha(7) and smaller contributions by Ser-34, Arg-186, Tyr-188, and Tyr-195. To identify pairwise interactions that stabilize the receptor-conotoxin complex, we measured binding of receptor and toxin mutations and analyzed the results by double mutant cycles. The results reveal a single dominant interaction between Leu-10 of CTx PnIB and Trp-149 of alpha(7) that anchors the toxin to the binding site. We also find weaker interactions between Pro-6 of CTx PnIB and Trp-149 and between both Pro-6 and Pro-7 and Tyr-93 of alpha(7). The overall results demonstrate that a localized hydrophobic region in CTx PnIB interacts with conserved aromatic residues on one of the two faces of the alpha(7) binding site.

Mutation causing congenital myasthenia reveals acetylcholine receptor beta/delta subunit interaction essential for assembly.

We describe a severe postsynaptic congenital myasthenic syndrome with marked endplate acetylcholine receptor (AChR) deficiency caused by 2 heteroallelic mutations in the beta subunit gene. One mutation causes skipping of exon 8, truncating the beta subunit before its M1 transmembrane domain, and abolishing surface expression of pentameric AChR. The other mutation, a 3-codon deletion (beta426delEQE) in the long cytoplasmic loop between the M3 and M4 domains, curtails but does not abolish expression. By coexpressing beta426delEQE with combinations of wild-type subunits in 293 HEK cells, we demonstrate that beta426delEQE impairs AChR assembly by disrupting a specific interaction between beta and delta subunits. Studies with related deletion and missense mutants indicate that secondary structure in this region of the beta subunit is crucial for interaction with the delta subunit. The findings imply that the mutated residues are positioned at the interface between beta and delta subunits and demonstrate contribution of this local region of the long cytoplasmic loop to AChR assembly.

Pairwise interactions between neuronal alpha7 acetylcholine receptors and alpha-conotoxin ImI.

The present work uses alpha-conotoxin ImI (CTx ImI) to probe the neurotransmitter binding site of neuronal alpha7 acetylcholine receptors. We identify key residues in alpha7 that contribute to CTx ImI affinity, and use mutant cycles analysis to identify pairs of residues that stabilize the receptor-conotoxin complex. We first mutated key residues in the seven known loops of alpha7 that converge at the subunit interface to form the ligand binding site. The mutant subunits were expressed in 293 HEK cells, and CTx ImI binding was measured by competition against the initial rate of 125I-alpha-bungarotoxin binding. The results reveal a predominant contribution by Tyr-195 in alpha7, accompanied by smaller contributions by Thr-77, Tyr-93, Asn-111, Gln-117, and Trp-149. Based upon our previous identification of bioactive residues in CTx ImI, we measured binding of receptor and toxin mutations and analyzed the results using thermodynamic mutant cycles. The results reveal a single dominant interaction between Arg-7 of CTx ImI and Tyr-195 of alpha7 that anchors the toxin to the binding site. We also find multiple weak interactions between Asp-5 of CTx ImI and Trp-149, Tyr-151, and Gly-153 of alpha7, and between Trp-10 of CTx ImI and Thr-77 and Asn-111 of alpha7. The overall results establish the orientation of CTx ImI as it bridges the subunit interface and demonstrate close approach of residues on opposing faces of the alpha7 binding site.

Acetylcholine and epibatidine binding to muscle acetylcholine receptors distinguish between concerted and uncoupled models.

The muscle acetylcholine receptor (AChR) has served as a prototype for understanding allosteric mechanisms of neurotransmitter-gated ion channels. The phenomenon of cooperative agonist binding is described by the model of Monod et al. (Monod, J., Wyman, J., and Changeux, J. P. (1965) J. Mol. Biol. 12, 88-118; MWC model), which requires concerted switching of the two binding sites between low and high affinity states. The present study examines binding of acetylcholine (ACh) and epibatidine, agonists with opposite selectivity for the two binding sites of mouse muscle AChRs. We expressed either fetal or adult AChRs in 293 HEK cells and measured agonist binding by competition against the initial rate of 125I-alpha-bungarotoxin binding. We fit predictions of the MWC model to epibatidine and ACh binding data simultaneously, taking as constants previously determined parameters for agonist binding and channel gating steps, and varying the agonist-independent parameters. We find that the MWC model describes the apparent dissociation constants for both agonists but predicts Hill coefficients that are far too steep. An Uncoupled model, which relaxes the requirement of concerted state transitions, accurately describes binding of both ACh and epibatidine and provides parameters for agonist-independent steps consistent with known aspects of AChR function.

Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating.

By defining the functional defect in a congenital myasthenic syndrome (CMS), we show that the third transmembrane domain (M3) of the muscle acetylcholine receptor governs the speed and efficiency of gating of its channel. The clinical phenotype of this CMS results from the mutation V285I in M3 of the alpha subunit, which attenuates endplate currents, accelerates their decay and causes abnormally brief acetylcholine-induced single-channel currents. Kinetic analysis of engineered alpha V285I receptors demonstrated a predominant effect on channel gating, with abnormally slow opening and rapid closing rates. Analysis of site-directed mutations revealed stereochemical and volume-dependent contributions of alpha V285 to channel gating. Thus, we demonstrate a functional role for the M3 domain as a key component of the nicotinic acetylcholine receptor channel-gating mechanism.

Subunit interface selectivity of the alpha-neurotoxins for the nicotinic acetylcholine receptor.

Peptide toxins selective for particular subunit interfaces of the nicotinic acetylcholine receptor have proven invaluable in assigning candidate residues located in the two binding sites and for determining probable orientations of the bound peptide. We report here on a short alpha-neurotoxin from Naja mossambica mossambica (NmmI) that, similar to other alpha-neurotoxins, binds with high affinity to alphagamma and alphadelta subunit interfaces (KD approximately 100 pM) but binds with markedly reduced affinity to the alphaepsilon interface (KD approximately 100 nM). By constructing chimeras composed of portions of the gamma and epsilon subunits and coexpressing them with wild type alpha, beta, and delta subunits in HEK 293 cells, we identify a region of the subunit sequence responsible for the difference in affinity. Within this region, gammaPro-175 and gammaGlu-176 confer high affinity, whereas Thr and Ala, found at homologous positions in epsilon, confer low affinity. To identify an interaction between gammaGlu-176 and residues in NmmI, we have examined cationic residues in the central loop of the toxin and measured binding of mutant toxin-receptor combinations. The data show strong pairwise interactions or coupling between gammaGlu-176 and Lys-27 of NmmI and progressively weaker interactions with Arg-33 and Arg-36 in loop II of this three-loop toxin. Thus, loop II of NmmI, and in particular the face of this loop closest to loop III, appears to come into close apposition with Glu-176 of the gamma subunit surface of the binding site interface.

Congenital myasthenic syndromes: recent advances.

Congenital myasthenic syndromes (CMS) can arise from presynaptic, synaptic, or postsynaptic defects. Mutations of the acetylcholine receptor (AChR) that increase or decrease the synaptic response to acetylcholine (ACh) are a common cause of the postsynaptic CMS. An increased response occurs in the slow-channel syndromes. Here, dominant mutations in different AChR subunits and in different domains of the subunits prolong the activation episodes of AChR by either delaying channel closure or increasing the affinity of AChR for ACh. A decreased synaptic response to ACh occurs with recessive, loss-of-function mutations. Missense mutations in the low-affinity, fast-channel syndrome and in a disorder associated with mode-switching kinetics of AChR result in brief activation episodes and reduce the probability of channel opening. Mutations causing premature termination of the translational chain or missense mutations preventing the assembly or glycosylation of AChR curtail the expression of AChR. These mutations are concentrated in the epsilon subunit, probably because substitution of the fetal gamma for the adult epsilon subunit can rescue humans from fatal null mutations in epsilon. Recent molecular genetic studies have also elucidated the pathogenesis of the CMS caused by absence of the asymmetric form of acetylcholinesterase from the synaptic basal lamina. Endplate acetylcholinesterase deficiency is now known to be caused by mutations in the collagenic tail subunit of the asymmetric enzyme that prevents the association of the collagenic tail subunit with the catalytic subunit or its insertion into the basal lamina.

Toxins selective for subunit interfaces as probes of nicotinic acetylcholine receptor structure.

The pentameric structure of the nicotinic acetylcholine receptor with two of the five subunit interfaces serving as ligand binding sites offers an opportunity to distinguish features on the surfaces of the subunits and their ligand specificity characteristics. This problem has been approached through the study of assembly of subunits and binding characteristics of selective peptide toxins. The receptor, with its circular order of homologous subunits (alpha gamma alpha delta beta), assembles in only one arrangement, and through mutagenesis, the residues governing assembly can be ascertained. Selectivity of certain toxins is sufficient to readily distinguish between sites at the alpha gamma and alpha delta interfaces. By interchanging residues on the gamma and delta subunits, and ascertaining how they interact with the alpha-subunit, determinants forming the binding sites can be delineated. The alpha-conotoxins, which contain two disulfide loops and 12-14 amino acids, show a 10,000-fold preference for the alpha delta over the alpha gamma subunit interface with alpha epsilon falling between the two. The waglerins, as 22-24 amino acid peptides with a single core disulfide loop, show a 2000-fold preference for alpha epsilon over the alpha gamma and alpha delta interfaces. Finally, the 6700 Da short alpha-neurotoxin from N. mossambica mossambica shows a 10,000-fold preference for the alpha gamma and alpha delta interfaces over alpha epsilon. Selective mutagenesis enables one to also distinguish alpha-neurotoxin binding at the alpha gamma and alpha delta subunits. This information, when coupled with homology modeling of domains and site-directed residue modification, reveals important elements of receptor structure and conformation.

Molecular dissection of subunit interfaces in the nicotinic acetylcholine receptor.

Ligand binding sites in the muscle nicotinic acetylcholine receptor are generated by pairs of alpha and non-alpha subunits. The non-alpha subunits, gamma, delta and epsilon, contribute significantly to overall affinity of agonists and antagonists, and confer selectivity of these ligands for the two binding sites. By constructing chimeras composed of segments of the various non-alpha subunits and determining ligand selectivity, we have identified four loops, well separated in the linear sequence, that contribute to the non-alpha portion of the binding site. Studies of point mutations in these loops and labeling of engineered cysteines show that the peptide backbones of each non-alpha subunit fold into similar basic scaffolds. Studies of mutations of the peptide antagonists alpha-conotoxin M1 and ImI reveal pairs of residues in the binding site and the toxin that stabilize the complex.

Congenital myasthenic syndromes: experiments of nature.

Congenital myasthenic syndromes (CMS) can arise from presynaptic, synaptic, or postsynaptic defects. Recent studies indicate that mutations in the acetylcholine receptor (AChR) subunit genes are a common cause of the postsynaptic CMS. The mutations, which increase or decrease the response to acetylcholine, are experiments of nature that highlight functionally significant domains of the AChR.

Epibatidine activates muscle acetylcholine receptors with unique site selectivity.

We recently showed that at desensitized muscle nicotinic receptors, epibatidine selects by 300-fold between the two agonist binding sites. To determine whether receptors in the resting, activatible state show similar site selectivity, we studied epibatidine-induced activation of mouse fetal and adult receptors expressed in 293 HEK cells. Kinetic analysis of single-channel currents reveals that (-)-epibatidine binds with 15-fold selectivity to sites of adult receptors and 75-fold selectivity to sites of fetal receptors. For each receptor subtype, site selectivity arises solely from different rates of epibatidine dissociation from the two sites. To determine the structural basis for epibatidine selectivity, we introduced mutations into either the gamma or the delta subunit and measured epibatidine binding and epibatidine-induced single-channel currents. Complexes formed by alpha and mutant gamma(K34S+F172I) subunits bind epibatidine with increased affinity compared to alphagamma complexes, whereas the kinetics of alpha2betadeltagamma(K34S+F172I) receptors reveal no change in affinity of the low-affinity site, but increased affinity of the high-affinity site. Conversely, complexes formed by alpha and mutant delta(S36K+I178F) subunits bind epibatidine with decreased affinity compared to alphadelta complexes, whereas the kinetics of alpha2betagammadelta(S36K+I178F) and alpha2betaepsilondelta(S36K+I178F) receptors show markedly reduced sensitivity to epibatidine. The overall data show that epibatidine activates muscle receptors by binding with high affinity to alphagamma and alphaepsilon sites, but with low affinity to the alphadelta site.

Identification of residues in the neuronal alpha7 acetylcholine receptor that confer selectivity for conotoxin ImI.

To identify residues in the neuronal alpha7 acetylcholine subunit that confer high affinity for the neuronal-specific toxin conotoxin ImI (CTx ImI), we constructed alpha7-alpha1 chimeras containing segments of the muscle alpha1 subunit inserted into equivalent positions of the neuronal alpha7 subunit. To achieve high expression in 293 human embryonic kidney cells and formation of homo-oligomers, we joined the extracellular domains of each chimera to the M1 junction of the 5-hydroxytryptamine-3 (5HT-3) subunit. Measurements of CTx ImI binding to the chimeric receptors reveal three pairs of residues in equivalent positions of the primary sequence that confer high affinity of CTx ImI for alpha7/5HT-3 over alpha1/5HT-3 homo-oligomers. Two of these pairs, alpha7Trp55/alpha1Arg55 and alpha7Ser59/alpha1Gln59, are within one of the four loops that contribute to the traditional non-alpha subunit face of the muscle receptor binding site. The third pair, alpha7Thr77/alpha1Lys77, is not within previously described loops of either the alpha or non-alpha faces and may represent a new loop or an allosterically coupled loop. Exchanging these residues between alpha1 and alpha7 subunits exchanges the affinities of the binding sites for CTx ImI, suggesting that the alpha7 and alpha1 subunits, despite sequence identity of only 38%, share similar protein scaffolds.

Structural elements in alpha-conotoxin ImI essential for binding to neuronal alpha7 receptors.

The neuronal-specific toxin alpha-conotoxin ImI (CTx ImI) has the sequence Gly-Cys-Cys-Ser-Asp-Pro-Arg-Cys-Ala-Trp-Arg-Cys-NH2, in which each cysteine forms a disulfide bridge to produce a constrained two-loop structure. To investigate the structural basis for bioactivity we mutated individual residues in CTx ImI and determined bioactivity. Bioactivity of the toxins was determined by their competition against 125I-labeled alpha-bungarotoxin binding to homomeric receptors containing alpha7 sequence in the major extracellular domain and 5HT-3 sequence elsewhere. The results reveal two regions in CTx ImI essential for binding to the alpha7/5HT-3 receptor. The first is the triad Asp-Pro-Arg in the first loop, where conservative mutations of each residue diminish affinity by 2-3 orders of magnitude. The second region is the lone Trp in the second loop, where an aromatic side chain is required. The overall results suggest that within the triad of the first loop, Pro positions the flanking Asp and Arg for optimal interaction with one portion of the binding site, while within the second loop, Trp stabilizes the complex through its aromatic ring.