MOLECULAR BASIS OF SODIUM CHANNEL INACTIVATION

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The goal of this proposal is to elucidate the molecular basis of Na channel inactivation. Na channels are integral proteins that render membranes excitable by supporting rapid voltage-induced changes in permeability to Na ions. Inactivation plays a critical role in modulating the availability of Na channels to open and therefore directly influences excitability and drug binding in nerve, skeletal muscle and heart. The patch-clamp technique allows recording of Na currents from whole cells or from single Na channels. Inactivation in whole-cell records is evident as the decay of the Na current during maintained membrane depolarization. In single-channel recordings, inactivation is seen as a discrete transition from a current-conducting conformation of the channel protein to a long-lasting nonconducting conformation. The kinetics and voltage-dependence of transitions to the inactivated state can be explicitly described using analytic methods based on Markov chain theory or Eyring rate theory. Structural models of the transmembrane topology of the Na channel have led to hypotheses regarding the function of several highly conserved segments of the channel. These hypotheses can now be rigorously tested through the alliance of techniques in biophysics and molecular biology. This proposal targets the putative "inactivation gate" which resides near the inner vestibule of the Na channel and appears to swing or slide to a position which blocks the pore. Specific aims are: (1) to characterize Na channel inactivation in single-channel recordings; (2) to investigate links between the putative inactivation gate and Na channel inactivation; and (3) to assess the relationship of the inactivation gate to the overall behavior of Na channels. Inactivation will first be characterized and compared in rat skeletal muscle cells and in Xenopus oocytes which express functional Na channels from mRNA encoding the Na channel a subunit. Subsequently, site-directed mutagenesis will be used to synthesize mutant Na channels with altered primary amino acid sequence in the region of the inactivation gate. Proposed mutations include: (1) nick mutations which divide the inactivation gate; (2) point mutations which change the net charge on the inactivation gate; and (3) point mutations which change the flexibility of the inactivation gate. These mutant channels, expressed in oocytes, provide a means to probe specific aspects of structure-function relationships. Overall, the proposed studies promise to enhance our understanding of fundamental electrophysiological and structural properties of the Na channel.