Fundamental gating mechanism of nicotinic receptor channel revealed by mutation causing a congenital myasthenic syndrome

We describe the genetic and kinetic defects in a congenital myasthenic synd rome due to the mutation epsilon A411P in the amphipathic helix of the acet ylcholine receptor (AChR) epsilon subunit. Myasthenic patients from three u nrelated families are either homozygous for epsilon A411P or are heterozygo us and harbor a null mutation in the second epsilon allele, indicating that epsilon A411P 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 individua l channels using hidden Markov modeling. For individual wild-type and mutan t 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 epsilon A411P. Prolines engineered into positions fl anking residue 411 of the epsilon subunit greatly increase the range of act ivation kinetics similar to epsilon A411P, whereas prolines engineered into positions equivalent to epsilon A411 in beta and delta subunits are withou t effect. Thus, the amphipathic helix of the epsilon subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to in dividual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in gene ral, and that they evolved to maintain uniformity of activation episodes. T he findings further suggest that the fundamental gating mechanism of the AC hR channel can be explained by a corrugated energy landscape superimposed o n a steeply sloped energy well.