The goal of this project is to understand better the molecular mechanism of dynamic interactions between the voltage-gated Na channel and specifically bound ligands. Among these ligands are local anesthetic (L.A.) drugs which inhibit the Na currents and alpha- and beta- scorpion toxins which modify primarily the channel inactivation and activation gating process respectively. The Na channel is an extremely dynamic protein which exhibits rapid time- and voltage- dependent conformational changes upon depolarization. Channel-specific ligands interfere with these structural rearrangements and thus are potential probes for the ligand binding sites and their corresponding functions. In this proposal, we plan to examine the ion pathway and gating property of the ligand-bound Na channel in muscle and heart cells using planar lipid bilayer and/or patch-clamp techniques. The action of the L.A. drug appears to be strongly modulated by voltage, Na+ ions, and pH as outlined in Hille's modulated receptor hypothesis. In his model, the receptor conformation is changed upon depolarization and accordingly, the receptor-ligand binding is altered. At present, the physico-chemical properties of the L.A. receptor and the molecular basis of its modulation remain largely unknown. Toward this understanding, we will determine the number of L.A. receptors within the Na channel, the mechanism of receptor modulation, as well as the structure-activity relationship of L.A. drugs. Beside L.A. drugs, we also plan to use alpha- and beta-scorpion toxins to examine their separate """"""""external"""""""" binding sites. These peptide toxins are ideal tools for characterizing the activation and inactivation processes influenced by external Na channel structure. In turn, scorpion toxins may later be applied to relate the inactivation or activation gating process to the L.A. action. Together, these studies should provide us an integrated view of ligand-Na channel interactions.
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