FLUCTUATIONS IN IONIC CURRENT THROUGH MEMBRANE CHANNELS

Yale University, New Haven CT

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Abstract

Voltage-gated ion channels are responsible for the conduction of action potentials in nerve and muscle, and are critical in the timing of rhythmic electrical activity in the heart, smooth muscle and secretory cells, among others. The mechanism by which changes in membrane potential lead to the opening and closing of these channels is partly understood: it involves a series of conformational changes within the channel protein which result in a large changes in the disposition of charged residues. The result is a gating charge movement which couples membrane potential changes to channel opening. The most-studied voltage-gated channel is the Shaker potassium channel. The focus of our work will be to obtain physical and structural insights into the "voltage sensor" in each Shaker subunit and the nature of its voltage-dependent conformational change. Toward this goal we will first combine patch-clamp recordings of ionic and gating currents with computer solutions to the Poisson-Boltzmann equation to infer features of the geometry of solution access to the gating charges, and to search, through the use of chimaeric channels, for regions of the channel sequence that influence the extent of voltage-dependent conformational changes. We will search for conditions under which the channel protein can be locked into its "closed" and "open" states. Using cryo-electronmicroscopy, we will then obtain images of the purified, reconstituted channel protein in these states from vesicles rapidly frozen in vitreous ice. The three- dimensional structure of the channel at low resolution will be reconstructed by single-particle image-processing techniques. in support of this effort we will seek to develop new techniques for the reconstruction of structures of proteins in vesicle membranes.