SODIUM-CHANNEL ACTIVATION GATING IS AFFECTED BY SUBSTITUTIONS OF VOLTAGE SENSOR POSITIVE CHARGES IN ALL 4 DOMAINS

The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigat ed by mutating the second and fourth conserved positive charges in the S4 nts of all four homologous domains. Both charge-neutralizing (by g lutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel a subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III-IV link er (West, J.W., D.E, Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A . Catterall. 1992, Proc. Natl. Acad. Sci. USA. 89:10910-10914.). A tot al of 16 single and 2 double mutants were constructed and analyzed wit h respect to voltage dependence and kinetics of activation and deactiv ation. The most significant effects were observed with substitutions o f the fourth positive charge in each domain. Neutralization of the fou rth positive charge in domain I or II produced the largest shifts in t he voltage dependence of activation, both in tile positive direction. This change was accompanied by positive shifts in the voltage dependen ce of activation and deactivation kinetics. Combining the two mutation s resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger posi tive shifts in the voltage dependence of activation and deactivation k inetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal acti vation, while the charge-conserving mutation resulted in a positive sh ift. Neutralization of the fourth charge in domain IV did not shift th e half-maximal voltage of activation, but the conservative substitutio n produced a positive shift. These data support the idea that both cha rge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segmen ts contribute to voltage-dependent activation of the sodium channel.