Sodium channels conduct the electrical impulse in excitable tissues including the heart, brain and skeletal muscle and serve as the receptors for clinically important antiarrhythmic and anticonvulant local anesthetic compounds. The essential features of Na channel function are selective yet rapid conduction of Na+ (permeation) and opening and closing in response to transmembrane voltage (gating). The long-term goals of this proposal are to understand the structural basis of ion permeation and selectivity in the Na channel. In the previous period of support we have demonstrated: 1. The importance of the P-segments in the formation of the outer pore. 2. Amino acids outside the selectivity filter are important determinants of ion selectivity. 3. There is considerable movement of the P segments that may be important both in permeation and gating. Despite considerable progress, the mechanism of Na+-selective permeation remains elusive, the cytoplasmic mouth of the pore structurally undefined, determinants of local anesthetic binding are incompletely characterized and the relationship of the channel pore to gating is incompletely understood. In this competitive renewal we focus on these central questions. The specific hypotheses of this proposal are: 1. The pore of the Na channel differs fundamentally from that of the recently crystallized KcsA potassium channel. 2. The P segment of domain IV is a crucial isoform-specific determinant of ion selectivity. 3. Motion in the external pore is important in gating of the channel. 4. The P segments significantly contribute to the formation of the local anesthetic binding site. We will use contemporary protein chemical, molecular biological, electrophysiological, fluorescence methods to study pore motion and place physical distance constraints on parts of the channel that comprise the pore. This proposal will provide insight into the dynamic structure, function and drug binding site(s) in the voltage- dependent Na channel and an enhanced understanding of the mechanism of Na+-selective permeation and guide rational Na channel-specific drug design.
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