FLUORESCENCE STUDIES OF THE SHAKER POTASSIUM CHANNEL

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Shaker, a model for the subfamily of voltage-dependent potassium (Kv) channels, catalyzes the transmembranous flow of potassium ions upon membrane depolarization. Kv channels are involved in a myriad of physiological processes including cardiac cell repolarization and frequency-encoding in neurons. Although this integral membrane protein has been subject to extensive biophysical characterization, details concerning the channel's activation mechanism are scant. In order to elucidate the manner in which the transmembrane electric field affects voltage sensor movement, a combination of optical and electrophysiological techniques will be utilized. The use of site-directed mutagenesis and thiol-specific fluorophores, allows specific structural modifications to be assayed via fluorescence techniques and correlated to gating charge displacement. Therefore, this research proposal will focus on two aspects of Shaker channel voltage-sensor dynamics: i.) determining equilibrium positions of S4 residues with the use of Lanthanide- Based Fluorescence Resonance Energy Transfer (LRET), and ii.) characterizing the kinetics of local conformational changes with the use of site-specific fluorescence The insight gained from this study will augment the contemporary view of ion channel biophysics and may result in a better understanding of normal physiology, in addition to the pathophysiology of numerous channelopathies.