The final common pathway of electrical excitability in neurons is generation of conducted action potentials. Action potentials in nerve and muscle are initiated by activation of voltage-gated Na channels, and the threshold and frequency of firing which encode information in the nervous system are critically dependent on Na channel properties. [unreadable] [unreadable] The ion conductance activity of Na channels is controlled on the millisecond time scale by two distinct but coupled gating processes: activation and inactivation. Activation controls the voltage- and time-dependence of conductance increase in response to depolarization, and inactivation controls the voltage- and time-dependence of the subsequent return of the sodium conductance to the basal level within one millisecond. In addition, Na channel activity is reduced during periods of prolonged or repetitive depolarization by a separate slow inactivation process and by neurotransmitters acting through G protein coupled receptors and protein phosphorylation. Both fast and slow gating processes are essential for normal electrical excitability of nerve and muscle cells, and elucidation of their molecular basis is a major challenge for molecular neurobiology. In the current project period, we have made substantial progress toward understanding the molecular basis for Na channel activation and inactivation and its modulation by second messenger-activated protein phosphorylation. We have mapped the inner pore of the Na channel by mutagenesis and analysis of binding of pore-blocking drugs, further defined the receptor site for local anesthetic and anticonvulsant drugs, provided new insights into the molecular basis for convergent regulation of Na channels by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), and elucidated the molecular mechanism of action of polypeptide toxins that alter Na channel gating. In addition, we have initiated a promising new avenue of research aimed at understanding Na channel structure and function by probing a small bacterial Na channel. In the next project period, we propose to build on this information about the molecular basis for control of Na channel activity by slow inactivation and second messenger-activated protein phosphorylation using biochemical, molecular biological, and electrophysiological methods to probe rat brain sodium channel Nav1.2 and the ancestral bacterial Na channel NaChBac. Our Specific Aims are to map the molecular determinants of slow inactivation and determine its molecular mechanism; to define the signaling complexes that mediate Na channel regulation and determine the functional relationship between slow inactivation and Na channel regulation by protein phosphorylation; and to compare the effects of slow inactivation and protein phosphorylation on movement of the S4 voltage sensors using gating current measurements. These proposed studies will give new insight into the molecular mechanisms of sodium channel gating and its modification by second messenger-activated protein phosphorylation. [unreadable] [unreadable] [unreadable]