Signalling in the nervous system, rhythmic beating of the heart, muscle excitation, and secretion by endocrine and epithelial cells are some of the important processes that are largely accomplished by ion channels. These molecules are ion selective pores in cell membranes that open and close in response to a wide variety of stimuli, such as membrane voltage, neurotransmitter molecules and intracellular messengers. Ions flowing through these channels transmit information either by directly acting as intracellular messengers themselves or by changing the membrane voltage. An important goal of modern neurobiology and physiology is to understand the molecular mechanisms whereby channel molecules change their conformation in response to voltage. The sensitivity to voltage of channel molecules and the time course of their response to changes in voltage largely determine the electrical properties of a given cell. The elucidation of these voltage-dependent gating mechanisms is important to our understanding of basic cellular mechanisms and their dysfunction during such pathological states as cardiac arrhythmias, periodic paralysis, and epilepsy. The understanding of molecular mechanisms of channel gating will require a combination of electrophysiological, molecular, and structural techniques. This proposal focuses on the molecular mechanisms of potassium channel gating. Single-channel, macroscopic and gating current measurements will be made on Shaker and related potassium channels expressed in Xenopus oocytes or cultured cell lines. These measurements of channel gating properties will be performed on channels that have been altered in specific ways by site-directed mutagenesis. Functional alterations that result from defined structural changes will be characterized to understand the conformational changes involved in voltage- dependent channel opening and in channel inactivation. The interaction between permeant ions and the conformational changes involved in gating and the regulation of gating by protein phosphorylation will also be elucidated.
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