The slow channel congenital myasthenic syndrome (SCCMS) is a hereditary, progressive muscle disease characterized by weakness, atrophy, degenerative changes confined to the neuromuscular junction (NMJ) and electrophysiological evidence of abnormal acetylcholine receptor (AChR) ion channel function. It may be the first degenerative disease recognized to result from an inherited abnormality of synaptic receptor function. The goal of this project is to demonstrate that the SCCMS is due to a mutation in the protein-coding region of one of four AChR subunit genes. This will be accomplished by first screening for mutations in these four candidate genes using a two-armed analysis, followed by functional confirmation of the phenotype of mutations: 1) The investigator will systematically screen for coding-region mutations along the entire length of each subunit in patients with SCCMS in parallel using the technique of single-stranded conformation polymorphism (SSCP) analysis. 2) They will determine the nucleotide sequence of any regions that show single-strand polymorphisms, as well as the sequences of the four transmembrane domains of each AChR subunit in cases where no polymorphism is found. 3) In order to be able to screen each exon completely, they will determine intron sequences and design intron-specific primers for each subunit. 4) Finally, the phenotype of any mutation identified in the SCCMS will be tested by generating the corresponding mouse AChR subunit cDNA mutation for co-expression along with the other mouse wild- type subunits in vitro. In this way they will directly correlate the clinical AChR phenotype with an AChR mutation and a molecular phenotype in vitro. The overall goal of this research is to characterize the role that abnormal receptors in excitatory synapses might play in degenerative disease using the NMJ and the SCCMS as models. In a related project, a transgenic mouse model for the SCCMS has been generated using mutant AChR subunits. This model and additional transgenic mice expressing the mutations in the SCCMS will be used to investigate the electro- physiological conditions and biochemical pathways responsible for the degenerative changes in muscle. This approach may lead to identification of potential targets for therapeutic intervention relevant to excitotoxicity.
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