BASIC MECHANISMS OF ION CHANNEL FUNCTION

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This proposal seeks to continue work on the fundamental mechanisms by which ion channel proteins work, and on how their ability to transport ions in a gated fashion is related to their underlying molecular structures. The focus of this research is on three different ion channels: the high- conductance Ca2+-activated K+ channel from mammalian muscle, the A-type K+ channel encoded by the Shaker gene of Drosophila, and the voltage-gated Cl- channel from the electric organ of Torpedo californica. We will probe the conduction pore of the Ca2+-activated K+ channel reconstituted into planar bilayer membranes by examining blocking by Ba2+ ion and by the interaction with the peptide charybdotoxin, and of site-directed mutants of this peptide. We will attempt to purify and reconstitute the Shaker K+ channel after high-level expression in the baculovirus-infected insect cell line, as a first attempt to carry out protein-level biochemical analysis of this channel. Finally, we will complete the purification of the Torpedo Cl- channel in order to launch a molecular analysis of a prominent member of the family of voltage-dependent anion channels. The general interest in this project lies in the central place of ion channel proteins in the process of electrical signalling. Researchers are now just beginning to glimpse the molecular architecture of these important proteins, and this project will contribute to that effort.