The slow-channel congenital myasthenic syndrome (SCCMS) is a dominantly inherited disorder of neuromuscular transmission characterized by delayed closure of the skeletal muscle acetylcholine receptor (AChR) ion channel and degeneration of the neuromuscular junction. The identification of a series of AChR subunit mutations in the SCCMS supports the hypothesis that the altered kinetics of the endplate currents in this disease are attributable to inherited abnormalities of the AChR. To investigate the role of these mutant AChR subunits in the development of the synaptic degeneration seen in the SCCMS, we have studied the properties of the AChR mutation, ⑀L269F, found in a family with SCCMS, using both in vitro and in vivo expression systems. The mutation causes a sixfold increase in the open time of AChRs expressed in vitro, similar to the phenotype of other reported mutants. Transgenic mice expressing this mutant develop a syndrome that is highly reminiscent of the SCCMS. Mice have fatigability of limb muscles, electrophysiological evidence of slow AChR ion channels, and defective neuromuscular transmission. Pathologically, the motor endplates show focal accumulation of calcium and striking ultrastructural changes, including enlargement and degeneration of the subsynaptic mitochondria and nuclei. These findings clearly demonstrate the role of this mutation in the spectrum of abnormalities associated with the SCCMS and point to the subsynaptic organelles as principal targets in this disease. These transgenic mice provide a useful model for the study of excitotoxic synaptic degeneration.

Site-directed mutagenesis was used to mutate alpha Cys418 and beta Cys447 in the M4 domain of Torpedo californica acetylcholine receptor expressed in Xenopus laevis oocytes. The M4 region is a transmembrane domain thought to be located at the lipid-protein interface. By whole-cell voltage clamp analysis, mutation of both alpha subunits to alpha Trp418 increased maximal channel activity approximately threefold, increased the desensitization rate compared with wild-type receptor, and shifted the EC50 for acetylcholine from 32 microM to 13 microM. Patch measurements of single-channel currents revealed that the alpha Trp418 increased channel open times approximately 28-fold at 13 degrees C with no effect on channel conductance. All of our measured functional changes in the alpha Trp418 mutant are consistent with a simple kinetic model of the acetylcholine receptor in which only the channel closing rate is altered by the mutation. Our results show that changes in protein structure at the putative lipid-protein interface can dramatically affect receptor function.

Point mutations in the genes encoding the acetylcholine receptor (AChR) subunits have been recognized in some patients with slow-channel congenital myasthenic syndromes (CMS). Clinical, electrophysiological, and pathological differences between these patients may be due to the distinct effects of individual mutations. We report that a spontaneous mutation of the beta subunit that interrupts the leucine ring of the AChR channel gate causes an eightfold increase in channel open time and a severe CMS characterized by severe endplate myopathy and extensive remodeling of the postsynaptic membrane. The pronounced abnormalities in neuromuscular synaptic architecture and function, muscle fiber damage and weakness, resulting from a single point mutation are a dramatic example of a mutation having a dominant gain of function and of hereditary excitotoxicity.

Our previous amino acid substitutions at the postulated lipid-exposed transmembrane segment M4 of the Torpedo californica acetylcholine receptor (AChR) focused on the alpha C418 position. A tryptophan substitution on the alpha C418 produced a 3-fold increase in normalized macroscopic response to acetylcholine in voltage-clamped Xenopus laevis oocytes (Lee et al., 1994). This result was explained by a 23-fold decrease in the closing rate constant measured from single-channel analysis (Ortiz-Miranda et al., 1996). In this study, we introduce more tryptophan substitutions at different positions of this postulated lipid-exposed segment M4 in order to examine functional consequences at the single-channel level. From a series of amino acid substitutions at alpha G421, only phenylalanine and tryptophan produced a substantial increase in the open time constant. The lack of response from a tyrosine substitution at the alpha G421 suggests that the side chain volume is not the main structural element responsible for the effect of tryptophan on the stabilization of the open state of the channel. Three multiple mutants, alpha C418W/G421A, alpha C418W/G421W, and alpha C418W/beta C447W, were constructed in order to establish the correlation between the number of lipid-exposed tryptophans and the channel open time constant. The alpha C418W/G421A double mutant demonstrated that when both previous mutations are combined the open time constant was increased 1.5-fold relative to the alpha C418W. When the two mutants (alpha C418W and alpha G421W) were combined in a single mutation, a functional receptor was expressed and the open time constant of the new double mutant increased to 33.4 ms, an 80-fold increase relative to wild type. Estimations of free energy changes calculated from the rate constant for the opening transition suggest that each tryptophan contributes to the stabilization of the open state of the channel by about 0.8 kcal/mol, and the effect of tryptophan substitutions on the free energy is additive. This result suggests that in the channel gating mechanism of the AChR, each subunit contributes independently to the energy barrier between the open and closed state. At selected positions within the postulated lipid surface of the AChR, tryptophan substitutions could establish hydrophobic and perhaps dipole interactions that may play a dramatic role in the channel gating mechanism.

We studied the functional effects of single amino acid substitutions in the postulated M4 transmembrane domains of Torpedo californica nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes at the single-channel level. At low ACh concentrations and cold temperatures, the replacement of wild-type alpha418Cys residues with the large, hydrophobic amino acids tryptophan or phenylalanine increased mean open times 26-fold and 3-fold, respectively. The mutation of a homologous cysteine in the beta subunit (beta447Trp) had similar but smaller effects on mean open time. Coexpression of alpha418Trp and beta447Trp had the largest effect on channel open time, increasing mean open time 58-fold. No changes in conductance or ion selectivity were detected for any of the single subunit amino acid substitutions tested. However, the coexpression of the alpha418Trp and beta447Trp mutated subunits also produced channels with at least two additional conductance levels. Block by acetylcholine was apparent in the current records from alpha418Trp mutants. Burst analysis of the alpha418Trp mutations showed an increase in the channel open probability, due to a decrease in the apparent channel closing rate and a probable increase in the effective opening rate. Our results show that modifications in the primary structure of the alpha- and beta subunit M4 domain, which are postulated to be at the lipid-protein interface, can significantly alter channel gating, and that mutations in multiple subunits act additively to increase channel open time.