Biomedical subjects

S M Sine

Publications and source records attributed to S M Sine.

At least 37 records · Page 2Linked to original sources

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

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.

Animals↗

PubMed1998

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.

Frameshift Mutation↗

PubMed1998

Residues at the subunit interfaces of the nicotinic acetylcholine receptor that contribute to alpha-conotoxin M1 binding.

The two binding sites in the pentameric nicotinic acetylcholine receptor of subunit composition alpha2 beta gamma delta are formed by nonequivalent alpha-gamma and alpha-delta subunit interfaces, which produce site selectivity in the binding of agonists and antagonists. We show by sedimentation analysis that 125I-alpha-conotoxin M1 binds with high affinity to the alpha-delta subunit dimers, but not to alpha-gamma dimers, nor to alpha, gamma, and delta monomers, a finding consistent with alpha-conotoxin M1 selectivity for the alpha delta interface in the intact receptor measured by competition against alpha-bungarotoxin binding. We also extend previous identification of alpha-conotoxin M1 determinants in the gamma and delta subunits to the alpha subunit interface by mutagenesis of conserved residues in the alpha subunit. Most mutations of the alpha subunit affect affinity similarly at the two sites, but Tyr93Phe, Val188Lys, Tyr190Thr, Tyr198Thr, and Asp152Asn affect affinity in a site-selective manner. Mutant cycle analysis reveals only weak or no interactions between mutant alpha and non-alpha subunits, indicating that side chains of the alpha subunit do not interact with those of the gamma or delta subunits in stabilizing alpha-conotoxin M1. The overall findings suggest different binding configurations of alpha-conotoxin M1 at the alpha-delta and alpha-gamma binding interfaces.

Amino Acid Sequence↗

PubMed1997

Identification of residues in the adult nicotinic acetylcholine receptor that confer selectivity for curariform antagonists.

We identify residues in the epsilon and delta subunits of the adult nicotinic acetylcholine receptor that give the alphaepsilon and alphadelta binding sites different affinities for the curariform antagonist dimethyl d-tubocurarine (DMT). By constructing epsilon-delta subunit chimeras, coexpressing them with complementary subunits, and measuring DMT binding, we identify two pairs of residues, Ileepsilon58/Hisdelta60 and Aspepsilon59/Aladelta61, responsible for DMT site selectivity in the adult receptor. The two determinants contribute approximately equally to the binding site and interact in contributing to the site. Exchange of these residues from one subunit to the other exchanges the affinities of the resulting binding sites. These determinants in the adult receptor are far from those that confer site selectivity in the fetal receptor; determinants in the fetal receptor are Ilegamma116/Valdelta118, Tyrgamma117/Thrdelta119, and Sergamma161/Lysdelta163. Thus, alternative residues confer DMT selectivity in fetal and adult acetylcholine receptors.

Adult↗

PubMed1997

Identification of equivalent residues in the gamma, delta, and epsilon subunits of the nicotinic receptor that contribute to alpha-bungarotoxin binding.

Cysteine was introduced from residues 116 to 121 of the gamma subunit of the fetal mouse acetylcholine receptor, and the mutant receptors were treated with methanethiosulfonate reagents and examined for changes in ligand binding properties. Of the 18 combinations of mutant and reagent, only receptors harboring gammaL119C treated with the quaternary ammonium reagent MTSET (trimethylammonium-ethyl methanethiosulfonate) show a decreased number of alpha-bungarotoxin (alpha-btx) sites. The decrease of 50% suggests that alpha-btx binding to the site harboring gammaL119C is blocked. Analysis of binding of the site-selective ligands dimethyl-d-tubocurarine (DMT) and alpha-conotoxin M1 (CTX) confirm specificity of modification for the site harboring gammaL119C. Cysteines placed at equivalent positions of the delta and epsilon subunits also lead to selective loss of alpha-btx binding following MTSET treatment. gammaL119C receptors treated with the primary amine reagent MTSEA (aminoethyl methanethiosulfonate) retain alpha-btx binding to both sites but show reduced affinity for DMT and CTX at the modified site. Lysine mutagenesis of Leugamma119, Leudelta121, and Leuepsilon119 mimics MTSEA treatment, whereas mutagenesis of Thralpha119 and Glnbeta119 is without effect, demonstrating subunit and residue specificity of MTSEA modification. MTSET modification of nearby gammaY117C does not block alpha-btx binding but markedly diminishes affinity for DMT and CTX. The overall findings indicate a localized point of interaction between alpha-btx and the modified gammaL119C, deltaL121C, and epsilonL119C.

Animals↗

PubMed1997

Slow-channel myasthenic syndrome caused by enhanced activation, desensitization, and agonist binding affinity attributable to mutation in the M2 domain of the acetylcholine receptor alpha subunit.

We describe a novel genetic and kinetic defect in a slow-channel congenital myasthenic syndrome. The severely disabled propositus has advanced endplate myopathy, prolonged and biexponentially decaying endplate currents, and prolonged acetylcholine receptor (AChR) channel openings. Genetic analysis reveals the heterozygous mutation alphaV249F in the propositus and mosaicism for alphaV249F in the asymptomatic father. Unlike mutations described previously in the M2 transmembrane domain, alphaV249F is located N-terminal to the conserved leucines and is not predicted to face the channel lumen. Expression of the alphaV249F AChR in HEK fibroblasts demonstrates increased channel openings in the absence of ACh, prolonged openings in its presence, enhanced steady-state desensitization, and nanomolar rather than micromolar affinity of one of the two binding sites in the resting activatable state. Thus, neuromuscular transmission is compromised because cationic overloading leads to degenerating junctional folds and loss of AChR, because an increased fraction of AChR is desensitized in the resting state, and because physiological rates of stimulation elicit additional desensitization and depolarization block of transmission.

Acetylcholine↗

PubMed1997

A model of the nicotinic receptor extracellular domain based on sequence identity and residue location.

We have modeled the extracellular domains of individual subunits (amino acids 31-200) in the nicotinic acetylcholine receptor using sequence homology with copper binding proteins of known crystal structure, plastocyanin and pseudoazurin, and data from recent site-specific mutagenesis, antibody mapping, and site-directed labelling studies. These data formed an initial model that was refined using molecular dynamics and mechanics as well as electrostatic and solvation energy calculations. The sequences between residues 31 and 164 in the alpha 1-subunit and corresponding residues in homologous receptor subunits show similarity with the core sequence of the cation binding site in plastocyanin and pseudoazurin, a region in the template proteins characterized by multiple hairpin loops. In addition to defining the subunit interfaces that comprise the site for agonist and competitive antagonist binding in more detail, the findings show that negatively charged residues cluster in domains arranged to diminish electrostatic free energy of the complex. Electrostatic factors also appear to distinguish the ligand binding interfaces, alpha gamma and alpha delta, from the other three interfaces on the pentameric receptor.

Amino Acid Sequence↗

PubMed1997

Mutation in the M1 domain of the acetylcholine receptor alpha subunit decreases the rate of agonist dissociation.

We describe the kinetic consequences of the mutation N217K in the M1 domain of the acetylcholine receptor (AChR) alpha subunit that causes a slow channel congenital myasthenic syndrome (SCCMS). We previously showed that receptors containing alpha N217K expressed in 293 HEK cells open in prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Here we use single channel kinetic analysis to show that the prolonged activation episodes result primarily from slowing of the rate of acetylcholine (ACh) dissociation from the binding site. Rate constants for channel opening and closing are also slowed but to much smaller extents. The rate constants derived from kinetic analysis also describe the concentration dependence of receptor activation, revealing a 20-fold shift in the EC50 to lower agonist concentrations for alpha N217K. The apparent affinity of ACh binding, measured by competition against the rate of 125I-alpha-bungarotoxin binding, is also enhanced 20-fold by alpha N217K. Both the slowing of ACh dissociation and enhanced apparent affinity are specific to the lysine substitution, as the glutamine and glutamate substitutions have no effect. Substituting lysine for the equivalent asparagine in the beta, epsilon, or delta subunits does not affect the kinetics of receptor activation or apparent agonist affinity. The results show that a mutation in the amino-terminal portion of the M1 domain produces a localized perturbation that stabilizes agonist bound to the resting state of the AChR.

Acetylcholine↗

PubMed1997

Congenital myasthenic syndromes due to heteroallelic nonsense/missense mutations in the acetylcholine receptor epsilon subunit gene: identification and functional characterization of six new mutations.

We describe and functionally characterize six mutations of the acetylcholine receptor (AChR) epsilon subunit gene in three congenital myasthenic syndrome patients. Endplate studies demonstrated severe endplate AChR deficiency, dispersed endplate regions and well preserved junctional folds in all three patients. Electrophysiologic studies were consistent with expression of the fetal gamma-AChR at the endplates in one patient, prolongation of some channel events in another and gamma-AChR expression as well as some shorter than normal channel events in still another. Genetic analysis revealed two recessive and heteroallelic epsilon subunit gene mutations in each patient. One mutation in each (epsilonC190T [epsilon R64X], epsilon 127ins5 and epsilon 553del 7) generates a nonsense codon that predicts truncation of the epsilon subunit in its N-terminal, extracellular domain; and one mutation in each generates a missense codon (epsilon R147L, epsilon P245L and epsilon R311W). None of the mutations was detected in 100 controls. Expression studies in HEK cells indicate that the three nonsense mutations are null mutations and that surface expression of AChRs harboring the missense mutations is significantly reduced. Kinetic analysis of AChRs harboring the missense mutations show that epsilon R147L is kinetically benign, epsilon P245L prolongs burst open duration 2-fold by slowing the rate of channel closing and epsilon R311W shortens burst duration 2-fold by slowing the rate of channel opening and speeding the rate of ACh dissociation. The modest changes in activation kinetics are probably overshadowed by reduced expression of the missense mutations. The consequences of the endplate AChR deficiency are mitigated by persistent expression of gamma-AChR, changes in the release of transmitter quanta and appearance of multiple endplate regions on the muscle fiber.

Acetylcholine↗

PubMed1996

Asymmetric contribution of the conserved disulfide loop to subunit oligomerization and assembly of the nicotinic acetylcholine receptor.

The acetylcholine receptor (AChR) at the motor synapse is a pentamer of homologous subunits with the composition alpha2betagammadelta. Owing to the circular arrangement of subunits that forms a central ion channel, each subunit interface contains contributions from opposite faces of each subunit, designated + and -. Common to all subunits of the AChR and members of its superfamily is a disulfide loop formed between cysteines 128 and 142 of the major extracellular domain. To gain insight into the structural contribution of the disulfide loop and its possible location, we mutated the invariant proline at position 136 to glycine (P136G) and examined subunit assembly. When introduced into any AChR subunit, P136G disrupted assembly by affecting the - face of the subunit, suggesting equivalent positioning of the loop in each subunit and localization to the - face. Also, the contribution of the loop in the overall assembly process differed for each subunit. In the beta and gamma subunits, P136G prevented assembly of higher order heteroligomers, whereas in the alpha and delta subunits, P136G prevented transport of assembled pentamers to the cell surface. The results demonstrate asymmetry in the contribution of the disulfide loop to formation of subunit interfaces, and that the loop in each subunit contributes at different stages of assembly.

Amino Acid Sequence↗

PubMed1996

Molecular dissection of subunit interfaces in the acetylcholine receptor. Identification of residues that determine agonist selectivity.

Agonists and antagonists select between the alphagamma and ns31744adelta binding sites of the fetal muscle acetylcholine receptor owing to different contributions by the gamma and delta subunits. To identify determinants of selectivity for agonists, we constructed a panel of gamma-delta subunit chimeras, co-expressed them with the alpha subunit in 293 HEK cells, and measured carbamylcholine binding affinity of intracellular complexes. Wild-type alphadelta complexes bind carbamylcholine about 30-fold more tightly than alphagamma complexes. This degree of selectivity is similar to that of the resting state of the receptor determined by kinetic analysis of single-channel events. We identify a primary set of determinants of selectivity, Lysgamma34/Serdelta36 and Phegamma172/Iledelta178, and a secondary set, Glugamma57/Aspdelta59 and Cysgamma115/Tyrdelta117. The contributions of all four determinants are subunit-dependent and are modified by interaction with one another. Coexpression of point mutant subunits with complementary wild-type subunits to form cell surface pentamers shows that Lysgamma34/Serdelta36 and Phegamma172/Iledelta178 contribute in a manner consistent with affecting selectivity of the resting state of the receptor, while Glugamma57 appears to contribute to the affinity of the desensitized state. The four determinants either coincide with or are close to residues known to contribute to the acetylcholine binding site. These results suggest that a minimum of four loops in the gamma and delta subunits contribute to the agonist binding site.

Amino Acid Sequence↗

PubMed1996

End-plate acetylcholine receptor deficiency due to nonsense mutations in the epsilon subunit.

We describe a congenital myasthenic syndrome associated with severe end-plate (EP) acetylcholine receptor (AChR) deficiency not associated with an EP myopathy, and with evidence of immature AChR, containing the gamma instead of the epsilon subunit (gamma-AChR) at the EPs. Molecular genetic analysis of AChR-subunit genes revealed two mutations in the epsilon-subunit gene: insertion of a thymine after epsilon nucleotide 1101 (epsilon 11O1insT) that generates a nonsense codon directly, and insertion of a guanine after epsilon nucleotide 1293 (epsilon 1293insG) that generates three missense codons followed by a nonsense codon. Each mutation predicts truncation of the epsilon subunit at the level of the long cytoplasmic loop, between the third (M3) and fourth (M4) membrane spanning domains. The propositus' asymptomatic son carries epsilon 1293G, indicating that the two mutations are heteroallelic. Expression of AChR harboring either mutation in human embryonic kidney (HEK) fibroblasts was markedly reduced. Single-channel activity recorded from HEK cells expressing epsilon 11O1insT-AChR was infrequent but resembled activity of wild-type AChR channels in amplitude and open duration. No channel activity could be recorded from HEK cells expressing epsilon 1293insG-AChR. Expression of gamma-AChR at the EPs may serve as the means of phenotypic rescue from potentially fatal nonsense mutations in the epsilon-subunit gene.

Amino Acid Sequence↗

PubMed1996

Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor epsilon subunit.

We describe the genetic and kinetic defects for a low-affinity fast channel disease of the acetylcholine receptor (AChR) that causes a myasthenic syndrome. In two unrelated patients with very small miniature end plate (EP) potentials, but with normal EP AChR density and normal EP ultrastructure, patch-clamp studies demonstrated infrequent AChR channel events, diminished channel reopenings during ACh occupancy, and resistance to desensitization by ACh. Each patient had two heteroallelic AChR epsilon subunit gene mutations: a common epsilon P121L mutation, a signal peptide mutation (epsilon G-8R) (patient 1), and a glycosylation consensus site mutation (epsilon S143L) (patient 2). AChR expression in HEK fibroblasts was normal with epsilon P121L but was markedly reduced with the other mutations. Therefore, epsilon P121L defines the clinical phenotype. Studies of the engineered epsilon P121L AChR revealed a markedly decreased rate of channel opening, little change in affinity of the resting state for ACh, but reduced affinity of the open channel and desensitized states.

Acetylcholine↗

PubMed1996

New mutations in acetylcholine receptor subunit genes reveal heterogeneity in the slow-channel congenital myasthenic syndrome.

Mutations in genes encoding the epsilon, delta, beta and alpha subunits of the end plate acetylcholine (ACh) receptor (AChR) are described and functionally characterized in three slow-channel congenital myasthenic syndrome patients. All three had prolonged end plate currents and AChR channel opening episodes and an end plate myopathy with loss of AChR from degenerating junctional folds. Genetic analysis revealed heterozygous mutations: epsilon L269F and delta Q267E in Patient 1, beta V266M in Patient 2, and alpha N217K in Patient 3 that were not detected in 100 normal controls. Patients 1 and 2 have no similarly affected relatives; in Patient 3, the mutation cosegregates with the disease in three generations. epsilon L269F, delta Q267E and beta V266M occur in the second and alpha N217K in the first transmembrane domain of AChR subunits; all have been postulated to contribute to the lining of the upper half of the channel lumen and all but delta Q267E are positioned toward the channel lumen, and introduce an enlarged side chain. Expression studies in HEK cells indicate that all of the mutations express normal amounts of AChR. epsilon L269F, beta V266M, and alpha N217K slow the rate of channel closure in the presence of ACh and increase apparent affinity for ACh; epsilon L269F and alpha N217K enhance desensitization, and epsilon L269F and beta V266M cause pathologic channel openings in the absence of ACh, rendering the channel leaky, delta Q267E has none of these effects and is therefore a rare polymorphism or a benign mutation. The end plate myopathy stems from cationic overloading of the postsynaptic region. The safety margin of neuromuscular transmission is compromised by AChR loss from the junctional folds and by a depolarization block owing to temporal summation of prolonged end plate potentials at physiologic rates of stimulation.

Adolescent↗

PubMed1995

Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit.

In a congenital myasthenic syndrome with a severe endplate myopathy, patch-clamp studies revealed markedly prolonged acetylcholine receptor (AChR) channel openings. Molecular genetic analysis of AChR subunit genes demonstrated a heterozygous adenosine-to-cytosine transversion at nucleotide 790 in exon 8 of the epsilon-subunit gene, predicting substitution of proline for threonine at codon 264 and no other mutations in the entire coding sequences of genes encoding the alpha, beta, delta, and epsilon subunits. Genetically engineered mutant AChR expressed in a human embryonic kidney fibroblast cell line also exhibited markedly prolonged openings in the presence of agonist and even opened in its absence. The Thr-264-->Pro mutation in the epsilon subunit involves a highly conserved residue in the M2 domain lining the channel pore and is likely to disrupt the putative M2 alpha-helix. Our findings indicate that a single mutation at a critical site can greatly alter AChR channel kinetics, leading to a congenital myasthenic syndrome. This observation raises the possibility that mutations involving subunits of other ligand-gated channels may also exist and be the basis of various other neurologic or psychiatric disorders.

Acetylcholine↗

PubMed1995

Molecular dissection of subunit interfaces in the acetylcholine receptor: identification of determinants of alpha-conotoxin M1 selectivity.

The acetylcholine receptor from vertebrate skeletal muscle is a pentamer of homologous subunits with composition alpha 2 beta gamma delta. Its two ligand binding sites, formed at alpha-gamma and alpha-delta interfaces, differ in their affinities for agonists and competitive antagonists, owing to different contributions of the gamma and delta subunits. To identify portions of the gamma and delta subunits that contribute to the binding sites, the experiments described here use gamma-delta subunit chimeras and site-specific mutants to determine the basis of the 10,000-fold selectivity of conotoxin M1 for the sites. Three distinct regions of the extracellular domain were found to contribute to conotoxin M1 selectivity, each containing a single residue responsible for the contribution of that region. Residues K34, S111, and F172 of the gamma subunit confer low affinity to the alpha-gamma binding site, whereas the corresponding residues of the delta subunit, S36, Y113, and I178, confer high affinity to the alpha-delta site. Identification of three separate determinants of ligand selectivity suggests a limited model of the folding pattern of the extracellular domain of the subunits.

Amino Acid Sequence↗

PubMed1995

Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity.

In five members of a family and another unrelated person affected by a slow-channel congenital myasthenic syndrome (SCCMS), molecular genetic analysis of acetylcholine receptor (AChR) subunit genes revealed a heterozygous G to A mutation at nucleotide 457 of the alpha subunit, converting codon 153 from glycine to serine (alpha G153S). Electrophysiologic analysis of SCCMS end plates revealed prolonged decay of miniature end plate currents and prolonged activation episodes of single AChR channels. Engineered mutant AChR expressed in HEK fibroblasts exhibited prolonged activation episodes strikingly similar to those observed at the SCCMS end plates. Single-channel kinetic analysis of engineered alpha G153S AChR revealed a markedly decreased rate of ACh dissociation, which causes the mutant AChR to open repeatedly during ACh occupancy. In addition, ACh binding measurements combined with the kinetic analysis indicated increased desensitization of the mutant AChR. Thus, ACh binding affinity can dictate the time course of the synaptic response, and alpha G153 contributes to the low binding affinity for ACh needed to speed the decay of the synaptic response.

Acetylcholine↗