The long-range goal of this research is to understand the pathophysiology and molecular mechanisms involved in the impairment of neuromuscular transmission in the slow-channel congenital myasthenic syndrome (SCS). Up to 14 distinct missense mutations in the genes coding for the four subunits of the muscle acetylcholine receptor (nAChR) are responsible for a dominantly-inherited syndrome of varied clinical severity and pathological findings. Although the common effect of each mutation identified in the SCS to date is to prolong the duration of the acetylcholine-induced channel bursts and endplate currents, differences in gating kinetics, ion permeability, desensitization rate, activation of cell death pathways, and sensitivity to choline and ethanol may affect pathogenesis. In this project we propose to explore specific, novel aspects of the molecular and cellular phenotype of each SCS mutation and to assess the relative contribution of each to neuromuscular weakness. Specifically, we propose to: 1) Determine whether pathogenicity in SCS correlates with activation of cell death pathways in vitro. This will be accomplished with cultured mammalian cells expressing SCS mutant AChRs using assays of cell lysis and of activation of cellular proteases. 2) Compare the capacity of ethanol as a model """"""""environmental agent"""""""" to modulate activity of mutant AChRs in SCS. This will be accomplished by studying the effect of ethanol on channel activity of AChRs bearing SCS mutations. 3) Determine whether pathogenicity in SCS correlates with differential response to serum choline or spontaneous activity. This will be accomplished by direct comparison of transgenic mice expressing choline-sensitive mutations with varied channel properties. 4) Determine whether reduced AChR channel opening rate (() alone can lead to significant weakness and impairment of synaptic responses. This will be accomplished using an AChR mutation that selectively alters rate of channel opening in transgenic mice. These studies extend the focus of the project by exploring newly recognized SCS mutant channel phenotypes in vitro and by using prototypic AChR subunit mutations to study pathogenic mechanisms in vivo. In addition, to extend the study of genetic and molecular mechanisms, we begin to investigate the influence of the environment on disease phenotype, and whether this influence may vary according to SCS mutation.
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