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Introduction Congenital myasthenic syndromes (CMSs) are heterogeneous disorders in which the safety margin of neuromuscular transmission is impaired by one or more defects in motor endplate-associated proteins (1). Most defects [...], Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in acetylcholine 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 (AChR α) 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 AChR α is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.

An study of the acetylcholine receptor (AChR) subunit genes through molecular genetic analysis indicates the prolonged openings in the AChR channel to be due to disruption of the putative M2 alpha-helix, caused by a Thr-264 to Pro mutation. This mutation is present in the nucleotide 790 in exon 8 of the epsilon subunit gene. This variation in the kinetics of the AChR channels leads to congenital myasthenic syndrome, as suggested by patch-clamp studies.

Choline acetyltransferase (ChAT; EC 2.3.1.6) catalyzes the reversible synthesis of acetylcholine (ACh) from acetyl CoA and choline at cholinergic synapses. Mutations in genes encoding ChAT affecting motility exist in Caenorhabditis elegans and Drosophila, but no CHAT mutations have been observed in humans to date. Here we report that mutations in CHAT cause a congenital myasthenic syndrome associated with frequently fatal episodes of apnea (CMS-EA). Studies of the neuromuscular junction in this disease show a stimulation-dependent decrease of the amplitude of the miniature endplate potential and no deficiency of the ACh receptor. These findings point to a defect in ACh resynthesis or vesicular filling and to CHAT as one of the candidate genes. Direct sequencing of CHAT reveals 10 recessive mutations in five patients with CMS-EA. One mutation (523insCC) is a frameshifting null mutation. Three mutations (I305T, R420C, and E441K) markedly reduce ChAT expression in COS cells. Kinetic studies of nine bacterially expressed ChAT mutants demonstrate that one mutant (E441K) lacks catalytic activity, and eight mutants (L210P, P211A, I305T, R420C, R482G, S498L, VS06L, and R560H) have significantly impaired catalytic efficiencies.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation [Epsilon]A411P in the amphipathic helix of the acetylcholine receptor (AChR) [Epsilon] subunit. Myasthenic patients from three unrelated families are either homozygous for [Epsilon]A411P or are heterozygous and harbor a null mutation in the second [Epsilon] allele, indicating that [Epsilon]A411P 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 [Epsilon]A411P. Prolines engineered into positions flanking residue 411 of the [Epsilon] subunit greatly increase the range of activation kinetics similar to [Epsilon]A411P, whereas prolines engineered into positions equivalent to [Epsilon]A411 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. KEY WORDS: congenital myasthenic syndrome * single channel kinetics * hidden Markov modeling * channel gating * energy landscape