Role of individual S4 segments in gating of Ca_v3.1 T-type calcium channel by voltage

Contributions of voltage sensing S4 segments in domains I - IV of Ca_V3.1 channel to channel activation were analyzed. Neutralization of the uppermost charge in individual S4 segments by exchange of arginine for cysteine was employed. Mutant channels with single exchange in domains I - IV, in two adjacent domains, and in all four domains were constructed and expressed in HEK 293 cells. Changes in maximal gating charge Q_max and the relation between Q_max and maximal conductance G_max were evaluated. Q_max was the most affected by single mutation in domain I and by double mutations in domains I + II and I + IV. The ratio G_max/Q_max proportional to opening probability of the channel was significantly decreased by the mutation in domain III and increased by mutations in domains I and II. In channels containing double mutations G_max/Q_max ratio increased significantly when the mutation in domain I was included. Mutations in domains II and III zeroed each other. Mutation in domain IV prevented the decrease caused by the mutation in domain III. Neither ion current nor gating current was observed when channels with quadruple mutations were expressed. Immunocytochemistry analysis did not reveal the presence of channel protein in the cell membrane. Likely, quadruple mutation results in a structural change that affects the channel's trafficking mechanism. Altogether, S4 segments in domains I-IV of the Ca_V3.1 channel unequally contribute to channel gating by voltage. We suggest the most important role of the voltage sensor in the domain I and lesser roles of voltage sensors in domains II and III.