The application of molecular biology to electrophysiology has led to rapid progress during the last several years. Several voltage-gated cation channels have been cloned, and are composed of either large proteins with four repeated homologous subunits (Na+ and Ca2 + channels) or of smaller proteins which aggregate to form tetramers (K+ channels). Amino acid sequences show considerable structural homology among these channels; specifically, an area of conserved positively charged amino acids (found in each channel subunit and known as the S4 region) is thought to interact with the transmembrane electric field and to act as the voltage sensor which controls channel activation. Recent genetically-engineered mutations of this region by several laboratories support this hypothesis; a complete analysis of the function of S4 region has yet to be performed, however. Interactions between the subunits of the channel may play an important role in the voltage-gating process. This grant proposal describes the use of site-directed mutagenesis of the 54 region of a cloned delayed rectifier K+ channel (RCK1) to further elucidate the mechanisms of channel gating and the relationships between the channel subunits: a) less drastic mutations of the S4 residues which have previously formed nonfunctional channels will be engineered b) mutations will be restricted to a fraction of the subunits, and c) mutations will be made in acidic amino acids which may form ionic pairs with and stabilize the positively charged amino acids of the S4 region. Control and mutant channels will be expressed in-vitro in frog oocytes; gating properties will be determined by voltage-clamp, and compared to existing models of channel kinetics. Voltage-gated ion channels play key roles in pathophysiological and clinical pharmacology. A better understanding of the mechanisms of channel function may have profound clinical applications for the future.
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