The long-term goal of this project is to determine the three-dimensional structure and to understand the molecular mechanisms of ion channel gating in the Streptomyces lividans K+ channel (SKCI). This channel is the smallest potassium-selective channel reported so far (160 residues), and appears to be an ideal model protein for the mammalian members of the K+ channel family. K+ channel function has been associated with such basic cellular functions as the regulation of electrical activity, signal transduction and osmotic balance. In higher organisms, K+ channel dysfunction may lead to uncontrolled periods of electrical hyperexcitability, like epileptic episodes and cardiac arrhythmia. Consequently, efforts to understand K+ channel structure, function and dynamics relate directly to human health and disease. The approach we plan to pursue combines reporter-group spectroscopic techniques (spin labeling/EPR, Fluorescence) and electrophysiological methods with classical biochemical and molecular biological procedures. Thus, we aim to develop a molecular understanding of K+ channel function by systematically probing its structure. Through site-directed spin labeling, cysteine chemistry is used to introduce nitroxide radicals into specific sites within a protein sequence with high reactivity and specificity. EPR spectroscopy analysis of the spin labeled mutants yields two types of structural information: 1) mobility and solvent accessibility of the attached nitroxide through collisional relaxation methods and 2) distances between pairs of nitroxides through dipole- dipole interactions. Additionally, time-resolved EPR spectroscopy makes it possible to resolve the sequential mechanism of channel gating. Data derived from EPR experiments will be interpreted structurally using distance geometry methods and restrained molecular dynamics to generate a three-dimensional structure at the level of backbone fold. A three- dimensional structure of the Streptomyces lividans K+ channel will directly impact structure-function studies in most voltage-dependent channels, while even a medium resolution pore structure will benefit efforts to design specific K+ channel blockers, with obvious clinical implications. We feel that this proposal opens up a new experimental avenue that will contribute to the understanding of biologically important events such as electrical signaling, signal transduction and ion channel gating.
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