The overall purpose of this research proposal is to determine the properties and underlying kinetic mechanisms of two different ionic channels in cell membranes: the calcium-activated potassium channel and a voltage dependent chloride channel of large conductance. Currents that flow through single ionic channels in the sarcolemma of cultured rat skeletal muscle cells will be recorded under voltage clamp using the patch clamp technique. With this electrophysiological technique it is possible to study the kinetics of single channels by observing (through step changes in the current) when single channels open and close. The effects of Na, K, and Mg ions, step changes in [Ca] ion, and possible phosphorylation on the activity of the Ca-activated K channel will be characterized. The mechanism underlying the observation that openings of the Ca-activated K channel tend to occur in bursts will also be examined. The effect of membrane potential on activation, inactivation, and removal of inactivation for the voltage dependent C1 channel of large conduictance will also be characteized, as well as the permeability of this channelto various ions. The above data will be used to obtain information about the number of open and closed states, the mean lifetimes of the open and closed states, and the rate constants for transitions between the various states for each channel. These findings will then be used to develop a kinetic scheme for each channel. The Ca-activated K channel modulates repetitive firing in neurons and affects membrane potential and excitability in muscle. The C1 channel of large conductance would also affect membrane excitability. To understand the properties and function of nerve and muscle membranes, it will be necessary to understand the properties and mechanisms of these two channels. Understanding these channels may also help in understanding and treating nerve and muscle disease, as therapeutic agents and toxins often exert their specific effects on ionic channels in cell membranes.
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