Myasthenic syndrome AChRα C-loop mutant disrupts initiation of channel gating.
ABSTRACT: Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in ace-tylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR α subunit (AChRα) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRα C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in β-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in β-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChRα is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.
Project description:OBJECTIVE:To characterize the molecular and phenotypic basis of a severe slow-channel congenital myasthenic syndrome (SCCMS). METHODS:Intracellular and single-channel recordings from patient endplates; alpha-bungarotoxin binding studies; direct sequencing of AChR genes; microsatellite analysis; kinetic analysis of AChR activation; homology modeling of adult human AChR structure. RESULTS:Among 24 variants reported to cause SCCMS only two appear in the AChR ?-subunit. We here report a 16-year-old patient harboring a novel ?L273F mutation (?L294F in HGVS nomenclature) in the second transmembrane domain (M2) of the AChR ? subunit. Kinetic analyses with ACh and the weak agonist choline indicate that ?L273F prolongs the channel opening bursts 9.4-fold due to a 75-fold increase in channel gating efficiency, whereas a previously identified ?L269F mutation (?L289F in HGVS nomenclature) at an equivalent location in the AChR ?-subunit prolongs channel opening bursts 4.4-fold due to a 30-fold increase in gating efficiency. Structural modeling of AChR predicts that inter-helical hydrophobic interactions between the mutant residue in the ? and ? subunit and nearby M2 domain residues in neighboring ? subunits contribute to structural stability of the open relative to the closed channel states. INTERPRETATION:The greater increase in gating efficiency by ?L273F than by ?L269F explains why ?L273F has more severe clinical effects. Both ?L273F and ?L269F impair channel gating by disrupting hydrophobic interactions with neighboring ?-subunits. Differences in the extent of impairment of channel gating in ? and ? mutant receptors suggest unequal contributions of ?/? and ?/? subunit pairs to gating efficiency.
Project description:We identify 2 homozygous mutations in the ?-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: ?R218W in the pre-M1 region in 2 patients and ?E184K in the ?8-?9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the ?-, ?-, and ?-subunits of AChRs from all species. ?R218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas ?E184K reduces channel gating efficiency 11-fold but does not alter AChR cell surface expression. Determinations of the effective channel gating rate constants, combined with mutant cycle analyses, demonstrate strong energetic coupling between ?R218 and ?E184, and between ?R218 and ?E45 from the ?1-?2 linker, as also observed for equivalent residues in the principal coupling pathway of the ?-subunit. Thus, efficient and rapid gating of the AChR channel is achieved not only by coupling between conserved residues within the principal coupling pathway of the ?-subunit, but also between corresponding residues in the ?-subunit.
Project description:To characterize the molecular basis of a novel fast-channel congenital myasthenic syndrome.We used the candidate gene approach to identify the pathogenic mutation in the acetylcholine receptor (AChR) ε subunit, genetically engineered the mutant AChR into HEK cells, and evaluated the level of expression and kinetic properties of the mutant receptor.An 8-year-old boy born to consanguineous parents had severe myasthenic symptoms since birth. He is wheelchair bound and pyridostigmine therapy enables him to take only a few steps. Three similarly affected siblings died in infancy. He carries a homozygous p.W55R mutation at the α/ε subunit interface of the AChR agonist binding site. The mutant protein expresses well in HEK cells. Patch-clamp analysis of the mutant receptor expressed in HEK cells reveals 30-fold reduced apparent agonist affinity, 75-fold reduced apparent gating efficiency, and strikingly attenuated channel opening probability (P(open)) over a range agonist concentrations.Introduction of a cationic Arg into the anionic environment of α/ε AChR binding site hinders stabilization of cationic ACh by aromatic residues and accounts for the markedly perturbed kinetic properties of the receptor. The very low P(open) explains the poor response to pyridostigmine and the high fatality of the disease.
Project description:Charge selectivity forms the basis of cellular excitation or inhibition by Cys-loop ligand-gated ion channels (LGICs), and is essential for physiological receptor function. There are no reports of naturally occurring mutations in LGICs associated with the conversion of charge selectivity. Here, we report on a CHRNA1 mutation (?1Leu251Arg) in a patient with congenital myasthenic syndrome associated with transformation of the muscle acetylcholine receptor (AChR) into an inhibitory channel. Performing patch-clamp experiments, the AChR was found to be converted into chloride conductance at positive potentials, whereas whole-cell currents at negative potentials, although markedly reduced, were still carried by sodium. Umbrella sampling molecular dynamics simulations revealed constriction of the channel pore radius to 2.4 Å as a result of the mutation, which required partial desolvation of the ions in order to permeate the pore. Ion desolvation was associated with an energetic penalty that was compensated for by the favorable electrostatic interaction of the positively charged arginines with chloride. These findings reveal a mechanism for the transformation of the muscle AChR into an inhibitory channel in a clinical context.
Project description:The mechanisms underlying lipid-sensing by membrane proteins is of considerable biological importance. A unifying mechanistic question is how a change in structure at the lipid-protein interface is translated through the transmembrane domain to influence structures critical to protein function. Gating of the nicotinic acetylcholine receptor (nAChR) is sensitive to its lipid environment. To understand how changes at the lipid-protein interface influence gating, we examined how a mutation at position 418 on the lipid-facing surface of the outer most M4 transmembrane ?-helix alters the energetic couplings between M4 and the remainder of the transmembrane domain. Human muscle nAChR is sensitive to mutations at position 418, with the Cys-to-Trp mutation resulting in a 16-fold potentiation in function that leads to a congenital myasthenic syndrome. Energetic coupling between M4 and the Cys-loop, a key structure implicated in gating, do not change with C418W. Instead, Trp418 and an adjacent residue couple energetically with residues on the M1 transmembrane ?-helix, leading to a reorientation of M1 that stabilizes the open state. We thus identify an allosteric link connecting the lipid-protein interface of the nAChR to altered channel function.
Project description:The nicotinic acetylcholine receptor (AChR) transduces binding of nerve-released ACh into opening of an intrinsic ion channel, yet the intraprotein interactions behind transduction remain to be fully elucidated. Attention has focused on the region of the AChR in which the beta1-beta2 and Cys-loops from the extracellular domain project into a cavity framed by residues preceding the first transmembrane domain (pre-M1) and the linker spanning transmembrane domains M2 and M3. Previous studies identified a principal transduction pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the beta1-beta2 loop. Here we identify a parallel pathway in which the pre-M1 domain is coupled to the M2-M3 linker through the Cys-loop. Mutagenesis, single-channel kinetic analyses and thermodynamic mutant cycle analyses reveal energetic coupling among alphaLeu 210 from the pre-M1 domain, alphaPhe 135 and alphaPhe 137 from the Cys-loop, and alphaLeu 273 from the M2-M3 linker. Residues at equivalent positions of non-alpha-subunits show negligible coupling, indicating these interresidue couplings are specific to residues in the alpha-subunit. Thus, the extracellular beta1-beta2 and Cys-loops bridge the pre-M1 domain and M2-M3 linker to transduce agonist binding into channel gating.
Project description:Acetylcholine receptor channel gating is a brownian conformational cascade in which nanometer-sized domains ("Phi blocks") move in staggering sequence to link an affinity change at the transmitter binding sites with a conductance change in the pore. In the alpha-subunit, the first Phi-block to move during channel opening is comprised of residues near the transmitter binding site and the second is comprised of residues near the base of the extracellular domain. We used the rate constants estimated from single-channel currents to infer the gating dynamics of Y127 and K145, in the inner and outer sheet of the beta-core of the alpha-subunit. Y127 is at the boundary between the first and second Phi blocks, at a subunit interface. alphaY127 mutations cause large changes in the gating equilibrium constant and with a characteristic Phi-value (Phi = 0.77) that places this residue in the second Phi-block. We also examined the effect on gating of mutations in neighboring residues deltaI43 (Phi = 0.86), epsilonN39 (complex kinetics), alphaI49 (no effect) and in residues that are homologous to alphaY127 on the epsilon, beta, and delta subunits (no effect). The extent to which alphaY127 gating motions are coupled to its neighbors was estimated by measuring the kinetic and equilibrium constants of constructs having mutations in alphaY127 (in both alpha subunits) plus residues alphaD97 or deltaI43. The magnitude of the coupling between alphaD97 and alphaY127 depended on the alphaY127 side chain and was small for both H (0.53 kcal/mol) and C (-0.37 kcal/mol) substitutions. The coupling across the single alpha-delta subunit boundary was larger (0.84 kcal/mol). The Phi-value for K145 (0.96) indicates that its gating motion is correlated temporally with the motions of residues in the first Phi-block and is not synchronous with those of alphaY127. This suggests that the inner and outer sheets of the alpha-subunit beta-core do not rotate as a rigid body.
Project description:We identify two heteroallelic mutations in the acetylcholine receptor ?-subunit from a patient with severe myasthenic symptoms since birth: a novel ?D140N mutation in the signature Cys-loop and a mutation in intron 7 of the ?-subunit gene that disrupts splicing of exon 8. The mutated Asp residue, which determines the disease phenotype, is conserved in all eukaryotic members of the Cys-loop receptor superfamily. Studies of the mutant acetylcholine receptor expressed in HEK 293 cells reveal that ?D140N attenuates cell surface expression and apparent channel gating, predicting a reduced magnitude and an accelerated decay of the synaptic response, thus reducing the safety margin for neuromuscular transmission. Substituting Asn for Asp at equivalent positions in the ?-, ?-, and ?-subunits also suppresses apparent channel gating, but the suppression is much greater in the ?-subunit. Mutant cycle analysis applied to single and pairwise mutations reveals that ?Asp-138 is energetically coupled to ?Arg-209 in the neighboring pre-M1 domain. Our findings suggest that the conserved ?Asp-138 and ?Arg-209 contribute to a principal pathway that functionally links the ligand binding and pore domains.
Project description:The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist-receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy.
Project description:We identify two novel mutations in acetylcholine receptor (AChR) causing a slow-channel congenital myasthenia syndrome (CMS) in three unrelated patients (Pts). Pt 1 harbors a heterozygous ?V266A mutation (p.Val289Ala) in the second transmembrane domain (M2) of the AChR ? subunit (CHRNB1). Pts 2 and 3 carry the same mutation at an equivalent site in the ? subunit (CHRNE), ?V265A (p.Val285Ala). The mutant residues are conserved across all AChR subunits of all species and are components of a valine ring in the channel pore, which is positioned four residues above the leucine ring. Both ?V266A and ?V265A reduce the amino acid size and lengthen the channel opening bursts by fourfold by enhancing gating efficiency by approximately 30-fold. Substitution of alanine for valine at the corresponding position in the ? and ? subunit prolongs the burst duration four- and eightfold, respectively. Replacing valine at ? codon 265 either by a still smaller glycine or by a larger leucine also lengthens the burst duration. Our analysis reveals that each valine in the valine ring contributes to channel kinetics equally, and the valine ring has been optimized in the course of evolution to govern channel gating.