Ion channels mediate normal and abnormal cardiac excitation. Sodium (Na) and calcium (Ca) channels are particularly important in the pathogenesis of sudden cardiac death because they support propagation of the cardiac impulse and because they function as the receptors for a number of clinically useful antiarrhythmic drugs. Project 1 examines the fundamental structure-function correlation of Na and Ca channels. Using molecular genetic methods to mutate and express ion channels, we will investigate the structural basis of two fundamental channel processes, permeation and inactivation. Permeation deals with ion translocation through the channel pore, including the properties of ionic selectivity, conductance and block. We will characterize the phenotypes of Na and Ca channel point mutants in the loops between the fifth and sixth transmembrane segments of each of the four channel domains. Wild-type residues will be replaced by cysteine, producing an enhanced susceptibility to block by group IIb divalent cations (cadmium and zinc). The voltage dependence of block will be analyzed to determine fractional electrical distances to the binding site created by the altered residue, thereby enabling us to map the pore. Inactivation is the gating process whereby channels enter a nonconducting state during maintained depolarization. We will explore the sites involved in Na and Ca channel inactivation and determine, by single-channel analysis, how selectively and completely microscopic inactivation mutants. Having examined permeation and gating individually, we will then carefully analyze pore mutants for evidence of changes in inactivation, and inactivation mutants for changes in permeation with a view to defining the structural overlap between these two processes. Finally, we will determine how inactivation mutants and selected permeation mutants alter the interaction of pore- blocking drugs with Na and Ca channels.
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