Voltage-gated cation channels are membrane proteins responsible for the generation &propagation of action potentials central to neurological signal transmission. They are comprised of either four identical subunits (Kchannels) or homologous domains (Na &Ca channels), arranged about the normal to the membrane plane. Each of the four subunits (or domains) is comprised of six transmembrane helices, S1-S4 forming the voltage-sensor domain (VSD) and S5-S6 forming the pore domain (PD). Positively charged residues predominately in the S4 helix are responsible for voltage sensing by the VSD. The structural response of the VSD domain in each of the four subunits (or domains) to depolarizing transmembrane electrochemical potentials responsible for "gating currents" appears to be sequential. The activation of all four VSD's is required for the subsequent concerted opening of the cation pore formed by the four PD's resulting in selective "cation currents". An "electromechanical" coupling is required between the VSD's and the PD's to control the opening of the ion channel pore, otherwise closed at the membrane resting potential. The x-ray crystal structures of two voltage-gated potassium channels have revealed a "modular" structure showing the VSD to be somewhat "loosely associated" with the PD. This modular aspect has complicated attempts to understand the mechanism of the electromechanical coupling, which remains unknown. Crucial direct evidence is missing, namely definitive structural information concerning the nature of structural changes within the VSD and PD as a function of the applied transmembrane electrochemical potential.
The Specific Aims concern the utilization of state-of-the-art techniques throughout to investigate the structures of the intact voltage-gated K-channel, as well as only its voltage-sensor domain, vectorially-oriented within single lipid bilayers as a function of the applied transmembrane electrochemical potential. These techniques include membrane protein semi-synthesis, x-ray &neutron reflectivity, enhanced by interferometry, and computer simulation constrained by the experimental results.
The approach is applicable to all voltage-gated and ligand-gated ion channels central to neurological signal transmission phenomena. It can thereby contribute substantially to our understanding of not only the normal neurological phenomena, but also channelopathy diseases arising from ion channel dysfunction and drug action targeting these channels to modulate these phenomena.
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