VOLTAGE INDUCED S4 CONFORMATION CHANGES BY NMR

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The long term goal of thais project is to investigate the structure and functional relationship of S4 voltage sensor during ion channel activation and inactivation by nuclear magnetic resonance (NMR). Voltage-gated Na+, K+, and Ca2+ channels contain highly conserved and positively charged 54 segments, and its movement during voltage gating is believed to control the activation of ion channels. Two possible models --"sliding helix" (or helix screwii) and "propagating helix" are proposed to describe the translational movement of S4 and its conformation changes during membrane depolarization. However, no structural evidence is available to support those models. We propose to characterize the conformation changes and charge movement of s4 segment (and its mutants) of the Shaker potassium channel in the presence and absence of electric field by the multi- dimensional NMR methods such as COSY, NOSEY, and electrophoretic NMR. Three specific aims will be accomplished: (l) To understand S4 conformational changes during cell depolarization, three-dimensional structures of S4 peptides of the Shaker potassium channel protein will be determined in the presence and absence of electric field; (2) The electrophoretic mobilities of 54 peptide (and mutants) of the shaker potassium channel will be correlated with the gating currents measured in the patch clamp experiments; the diffusion coefficients of 54 (and mutants) measured in the presence and absence of electric field will be correlated with the conformation changes of the peptides; (3)The propagation of the 54 conformation changes to the 54-55 linker will be monitored by determining structures of peptide that contains 54 and 54-55 linker in the presence and absence of electric field; the leucine-heptad-repeat in this region will be examined for its role in mediating the signal transduction from 54 to the 54-55 linker in shaker potassium channel. The knowledge gained on the molecular mechanism of ion channel signal transduction may be useful to control diseases related to ion channel abnormalities in cardiac excitation-contraction, nerve conduction, muscle contraction.