The long term goal of this research is to elucidate the physical mechanism of voltage-dependent activation in the Shaker K+ channel of Drosophila. The proposed research will test the hypothesis that putative transmembrane segments S2, S3, and S4 interact structurally and comprise part of a domain that senses and responds to changes in the transmembrane voltage during activation. This hypothesis is based on preliminary results suggesting that charged residues in segments S2, S3, and S4 experience strong, short-range electrostatic interactions, and that some of these same charged residues contribute to the charge movement that accompanies activation. This hypothesis will be tested by accomplishing the following aims: 1) The packing of transmembrane segments will be determined. Likely short range structural interactions will be identified using a strategy of directed intragenic suppression. A packing model will be developed, tested by a suppression strategy, and refined. 2) The contribution of charged residues to the single channel gating charge will be investigated. Whether reductions in the charge/channel are additive will be determined. 3) A voltage-independent chimera between a voltage-dependent K+ channel and a voltage-independent channel will be characterized.
This aim i nvestigates structural features that stabilize the open conformation of a voltage-dependent channel. 4) The feasibility of alternative methods for determining the physical proximity will be evaluated. Whether metal ion binding sites can be formed by histidine substitution mutagenesis at adjacent positions will be tested. Whether disulfide bonds can be formed after cysteine substitution mutagenesis will be explored. This proposal describes basic research aimed at understanding the mechanism of voltage-dependent activation. The research is likely to have significant health relevance because ion channels play essential roles in the physiology of the brain, heart, and skeletal muscle.
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