| Modeling the behavior of voltage-gated sodium channels requires gating schemes that consist of kinetics states in coupled, voltage-dependent equilibria. Each kinetic transition in such a scheme in presumed to correspond to a conformational change at the level of the current-conducting alpha-subunit of the channel protein. Because the kinetic models are underdetermined by the ionic currents, monitoring the movements of the protein directly can provide grounds for elimination of a subset of the possible gating schemes. The experiments in this thesis were organized around this principle.; Conformational markers for gating transitions were generated by engineering cysteine residues at specific sites in the cDNA of the rat adult skeletal muscle sodium channel alpha-subunit, and monitoring changes in reactivity of the substituted cysteines with methanethiosulfonate (MTS) reagents under voltage clamp in excised macropatches. Accessibility changes that correlated with gating transitions were used as conformational markers to probe movements of sodium channel gates that do not involve changes in the ionic currents.; In the experiments described in Chapter 3, a cysteine mutant was engineered into the sodium channel III-IV interdomain at a residue known to be important for fast inactivation gating, F1304. The voltage-dependence of the reaction rate of F1304C with an MTS reagent correlated with fast inactivation, and was used to monitor the position of the fast inactivation gate during slow inactivation. We found that both the fast and slow inactivation gates can be closed simultaneously, and that recovery from fast inactivation is not altered in slow-inactivated channels. The Appendix describes further experiments in which this conformational marker was used to probe the relationship between lidocaine binding and fast inactivation. We found, contrary to many models of lidocaine action, that use-dependent block does not involve accumulation of fast inactivated channels. Based on these results and previous work, a kinetic model of lidocaine action is proposed.; Chapter 4 discusses a series of cysteine mutants engineered into the voltage-sensing S4 segments. We found reactivity changes in three of these mutants (R663C in IIS4, R1128C in IIIS4, and R1447C in IVS4) consistent with outward movement of the S4 segments during depolarization. Although our experiments are preliminary, we found evidence of slow movement in IIS4, and found that IVS4, the voltage-sensor associated with fast inactivation, moves freely in slow-inactivated channels, consistent with the results of Chapter 3.; Chapter 5 presents a series of experiments designed to find conformational markers for activation gating in the putative inner vestibule of the sodium channel (segment IVS6). Though none of the four mutants we tested (L1580C, I1581C, V1582C, and V1583C) showed activation-associated movements, one of them, V1583C, revealed both a rapid conformational change coupled to fast inactivation and a slow movement closely associated with slow inactivation. Batrachotoxin, an inhibitor of both fast and slow inactivation, protected the site from modification, suggesting that the movements at site 1583 are important for gating, and that the toxin may work by preventing them. |
