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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. [ABSTRACT FROM AUTHOR]

We identify 2 homozygous mutations in the ε-subunit of the muscle acetylcholine receptor (AChR) in 3 patients with severe congenital myasthenia: εR218W in the pre-M1 region in 2 patients and εE184K in the β8-β9 linker in 1 patient. Arg218 is conserved in all eukaryotic members of the Cys-loop receptor superfamily, while Glu184 is conserved in the α-, δ-, and ε-subunits of AChRs from all species. εR218W reduces channel gating efficiency 338-fold and AChR expression on the cell surface 5-fold, whereas εE184K 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 εR218 and εE184, and between εR218 and εE45 from the β1-β2 linker, as also observed for equivalent residues in the principal coupling pathway of the α-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 α-subunit, but also between corresponding residues in the ε-subunit.

We describe a highly disabling congenital myasthenic syndrome (CMS) associated with rapidly decaying, low-amplitude synaptic currents, and trace its cause to a valine to leucine mutation in the signature cystine loop (cys-loop) of the AChR alpha subunit. The recently solved crystal structure of an ACh-binding protein places the cys-loop at the junction between the extracellular ligand-binding and transmembrane domains where it may couple agonist binding to channel gating. We therefore analyzed the kinetics of ACh-induced single-channel currents to identify elementary steps in the receptor activation mechanism altered by the alphaV132L mutation. The analysis reveals that alphaV132L markedly impairs ACh binding to receptors in the resting closed state, decreasing binding affinity for the second binding step 30-fold, but attenuates gating efficiency only about twofold. By contrast, mutation of the equivalent valine residue in the delta subunit impairs channel gating approximately fourfold with little effect on ACh binding, while corresponding mutations in the beta and epsilon subunits are without effect. The unique functional contribution of the alpha subunit cys-loop likely owes to its direct connection via a beta strand to alphaW149 at the center of the ligand-binding domain. The overall findings reveal functional asymmetry between cys-loops of the different AChR subunits in contributing to ACh binding and channel gating.

Choline acetyltransferase (ChAT; EC ) 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, V506L, and R560H) have significantly impaired catalytic efficiencies.

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.

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.

Congenital end-plate acetylcholinesterase (AChE) deficiency (CEAD), the cause of a disabling myasthenic syndrome, arises from defects in the COLQ gene, which encodes the AChE triple-helical collagenlike-tail subunit that anchors catalytic subunits of AChE to the synaptic basal lamina. Here we describe a patient with CEAD with a nonsense mutation (R315X) and a splice-donor-site mutation at position +3 of intron 16 (IVS16+3A-->G) of COLQ. Because both A and G are consensus nucleotides at the +3 position of splice-donor sites, we constructed a minigene that spans exons 15-17 and harbors IVS16+3A-->G for expression in COS cells. We found that the mutation causes skipping of exon 16. The mutant splice-donor site of intron 16 harbors five discordant nucleotides (at -3, -2, +3, +4, and +6) that do not base-pair with U1 small-nuclear RNA (snRNA), the molecule responsible for splice-donor-site recognition. Versions of the minigene harboring, at either +4 or +6, nucleotides complementary to U1 snRNA restore normal splicing. Analysis of 1,801 native splice-donor sites reveals that presence of a G nucleotide at +3 is associated with preferential usage, at positions +4 to +6, of nucleotides concordant to U1 snRNA. Analysis of 11 disease-associated IVS+3A-->G mutations indicates that, on average, two of three nucleotides at positions +4 to +6 fail to base-pair, and that the nucleotide at +4 never base-pairs, with U1 snRNA. We conclude that, with G at +3, normal splicing generally depends on the concordance that residues at +4 to +6 have with U1 snRNA, but other cis-acting elements may also be important in assuring the fidelity of splicing.

We describe the genetic and kinetic defects in a congenital myasthenic syndrome caused by heteroallelic mutations of the acetylcholine receptor (AChR) epsilon subunit gene. The mutations are an in-frame duplication of six residues in the long cytoplasmic loop (epsilon1254ins18) and a cysteine-loop null mutation (epsilonC128S). The epsilon1254 ins18 mutation causes mode switching in the kinetics of receptor activation in which three modes activate slowly and inactivate rapidly. The epsilon1245ins18-AChR at the endplate shows abnormally brief activation episodes during steady state agonist application and appears electrically silent during the synaptic response to acetylcholine. The phenotypic consequences are endplate AChR deficiency, simplification of the postsynaptic region, and compensatory expression of fetal AChR that restores electrical activity at the endplate and rescues the phenotype.

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.

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.

A method was developed to predict the severity of cerebral ischemia before permanent occlusion of a common carotid artery in gerbils by observing the diameter and appearance of the artery after temporary occlusion and observing clinical signs after permanent occlusion. The severity of cerebral ischemia was confirmed by a sensitive immunohistochemical method and measurement of focal cerebral blood flow after 30 minutes' ischemia. All gerbils with greater than 40% reduction of the diameter and a white arterial margin distal to temporary occlusion developed severe neurologic signs following permanent occlusion, but no gerbils with reduction of less than 30% and a red arterial margin developed neurologic signs. With the cumulative neurologic score, gerbils could be divided into classes with no, mild, moderate, and severe symptoms, mostly after 10 minutes. Severely symptomatic gerbils were identified in 3 minutes. Extensive ischemic damage was observed in severely symptomatic gerbils, but no immunohistochemical lesion was detected in mildly symptomatic gerbils. Cerebral blood flow was markedly reduced in severely symptomatic gerbils but more selectively reduced in the cortical structures of moderately symptomatic gerbils. This prediction method is useful for investigating early cerebral ischemia and for evaluating the effectiveness of pharmacologic agents.

We investigated progression and recovery of neuronal damage during and after global cerebral ischemia in gerbils after bilateral occlusion of the common carotid arteries, using the immunohistochemical method (reaction for tubulin and creatine kinase BB-isoenzyme). The earliest, but reversible, ischemic lesions occurred after 3 minutes' ischemia in the subiculum-CA1 and CA2 regions of the hippocampus. The lesions became irreversible after 4 minutes' ischemia. The ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen were partially or completely reversible if the ischemic period was 5 minutes, whereas delayed degeneration occurred in the pyramidal cells of the medial CA1 region after reperfusion for 48 hours (delayed neuronal death). After 10 minutes' ischemia and subsequent reperfusion, delayed neuronal death extended from the medial to the lateral CA1 region; the ischemic and postischemic lesions in the cerebral cortex, thalamus, and caudoputamen also expanded during reperfusion. Our investigation demonstrates that selective vulnerability existed in global cerebral ischemia as in incomplete or regional ischemia and suggests that neurons in many areas of the brain possessed the potential for recovery, progressive deterioration, and even delayed neuronal death depending on the severity and duration of cerebral ischemia.