Determinants of Sodium Channel Function: Ion Pair Interactions Across Domains

项目摘要/摘要

Project Summary / Abstract This project will investigate basic mechanisms of bioelectric information as produced by the actions of sodium channel proteins of excitable cell membranes. We will focus our attention on the voltage-sensing domain of the sodium channel found in human skeletal muscle fibers. Mutations in this domain are responsible for a number of inherited diseases, called sodium channelopathies, and include muscle myotonia and periodic paralysis. In this work we will investigate the molecular means by which the segments of this domain, S1 to S4, interact to control basic sodium channel functions of activation, or opening, and fast inactivation, during which the channel is unable to respond to changes in membrane potential. Our hypotheses target putative interactions of negatively charged amino acids in segments S1 to S3, so-called countercharges, with positively charged amino acids in the segment S4. We will use voltage clamp electrophysiology to test the effects of mutations that reverse the charge of negatively, or positively charged amino acids. These charge-reversing mutations will be compared for effects on activation and for two forms of fast inactivation. Our goal is to identify countercharge interaction with the S4 segment of a given sodium channel domain, that determines a specific function of this asymmetric channel. To do this we will quantify the effects of all significant mutations on activation parameters using the IFM / QQQ inactivation deficient background, and using gating currents to directly test voltage sensor movement. Comparison of charge immobilization and its remobilization will allow a similar quantifiable measure of S1-S3 interaction with S4 segments during two forms of fast inactivation, and during recovery. Finally, we will build models of the voltage sensor domains, insert our mutations in these models, and then run computer simulations of the models in response to the change in membrane potential that elicits their typical function in muscle fibers. Our studies will further our understanding of the molecular basis of voltage-sensitivity in sodium channels and provide a foundation for studies on dysfunction produced by channelopathy mutations of muscle fibers.