Objective: To identify the genetic basis of a slow-channel myasthenic syndrome, characterize functional properties of the mutant receptor, and selectively silence the mutant allele. Methods: We performed mutation analysis, cloning, and patch-clamp analysis of the functional properties of the mutant receptor; screening for a small interfering RNA with check plasmid; and assessed of the efficacy of small interfering RNA at the messenger RNA, protein, and functional levels. Results: We traced the cause of a slow-channel myasthenic syndrome to a C418W mutation in the M4 domain of the acetylcholine receptor α subunit. The mutation is the first one to occur spontaneously in an M4 domain of the receptor, and it is positioned within a stripe of hydrophobic residues facing the lipid bilayer. Kinetic analysis shows that αC418W enhances the channel opening equilibrium constant 26-fold without altering agonist affinity. Using a check plasmid as a screening tool, we identified a small interfering RNA that markedly suppresses the mutant but not the wild-type allele at the messenger RNA, protein, and functional levels. Interpretation: αC418W occurring in humans causes a slow-channel syndrome by enhancing the relative stability of the channel open state. Efficient and selective knockdown of the mutant allele holds promise of therapeutic gene silencing.
ASJC Scopus subject areas
- Clinical Neurology