Voltage-dependent K (Kv) channels control action potential duration and firing frequency in excitable membranes, and, therefore, m targets of drugs that regulate excitability. The availability of Kv clones makes possible a molecular approach to drug sites and mechanisms. The objective of this research is to elucidate the structural relationships between drug binding sites and other functional domains of the Kv polypeptide, such M the gating, ion conduction and selectivity regions that are critical regulators of drug action.
The specific aims are to: (1) map the solvent accessible residues in the external and internal mouths of the pore that are critical for ion conductance and blockade; (2) investigate the mechanisms and sites whereby highly conserved polar, aromatic and charged residues which line the pore, influence ion selectivity, gating and drug blockade; (3) determine the functional relationships between critical regions by identifying residues that couple voltage-dependent gating with the pore. The hypotheses to be tested are that: (1) the inner mouth is a mosaic structure formed by contributions from several transmembrane and linker segments, whereas the outer mouth is specified in its entirety by a single linker segment (S5-S6 linker) (Aim l); (2) the rectification, conduction, selectivity and drug blockade in the pore are determined by a combination of multiple K selective ion binding sites associated with specific side chains within the narrow tunnel region of the pore and long-range electrostatic forces contributed by charged residues in the wide mouths at either end of the pore (Aim 2); (3) critical residues associated with 4AP and ThA blockade at the inner mouth are coupled to the voltage.sensing transmembrane segment (s4) which controls activation gating, such that access to the binding sites is gating-dependent, where- residues in the pore region which specify ion selectivity and external TEA block are independent of the gating mechanism (Aim 3).
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