Activation mechanisms of voltage-gated K+ and Na+ channels

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Voltage-gated ion channels are critically important membrane proteins that are activated and deactivated by changes in electric potential across membranes. The activation mechanism of voltage-gated ion channels remains elusive, in part due to lack of high-resolution structural information on the channels in resting state in the presence of membrane potential. Knowledge of the structure of voltage-gated ion channels in resting state will allow understanding their activation mechanisms. Here, I propose to use a combination of membrane protein biochemistry, biophysics, and subtomogram averaging to obtain structural information on voltage-gated ion channels in closed lipid vesicles under a controlled membrane potential at high resolution. Specifically, we aim to determine the resting state structures of three different voltage-gated ion channels in the resting states: (1) a bacterial voltage-gated sodium channel NaChBac, (2) a conventional mammalian voltage-gated potassium channel Kv1.2 and (3) a non-conventional mammalian voltage-gated channel with non-swapped domains. Experimental approach involves heterologous expression of the the proteins, their purification, reconstitution into liposomes, biophysical characterization and structural analysis. In order to succeed in biological interpretation, it is critical to achieve high resolution with subtomogram averaging from cryo electron tomograms which is the method of choice for structural analysis in situ. For this method developments optimizing the image processing workflows will be performed. We will use modern approaches from the computer vision field in order to perform more precise registration of noisy 3D volumes with anisotropic resolution. We will further account for the geometrical and electron optical distortions typical to processing of subtomogram averaging data. Taken together the method developments will allow reaching alpha-helical or even near-atomic resolutions.The developed methods for subtomogram averaging will be of general use; they will be publically released and used by other researchers and by us for determining the structures of ion channels in closed lipid vesicles under the membrane potential. Knowledge of the structures of the voltage-gated ion channels in their resting states will significantly further our understanding of the molecular mechanisms of voltage gating which will serve as a basis for the development of more selective drugs against multiple cardiac and neurological disorders.