Ionic interactions between gating charges and countercharges in voltage-sensing domain I independently regulate kinetics and voltage-dependence of Cav1.1 gating

Voltage-gated calcium channels control a variety of processes in excitable cells. However, the exact mechanisms regulating kinetics and voltage-dependence of channel activation are not fully understood. Voltage-gated activation is determined by four distinct voltage-sensing domains (VSD I-IV) coupled to a common pore. Each VSD consists of four transmembrane helices (S1-S4) with S4 containing four to five gating charges. Upon membrane depolarization, consecutive interactions of these gating charges with negatively charged countercharges are believed to facilitate an upward movement of the S4 helices, leading to the opening of the pore and activation of the channel. Previous studies linked naturally occurring mutations of the innermost gating charge R4 (R174W) and its negative countercharge (E100) in CaV1.1 to muscle disease. To investigate the contribution of VSD I and the roles of its gating- and countercharges in channel gating, we combined structure-guided site-directed mutagenesis with patch-clamp analysis in dysgenic myotubes (CaV1.1-null). As E100 in helix S2 together with D126 in helix S3 form the highly conserved charge-transfer center of VSDs, we included this second negative countercharge (D126) in our analysis. While mutation of R174A resulted in a strong right-shift of voltage dependence, charge neutralizing mutations of the countercharges E100 and D126 both lead to a left-shift of voltage-dependence and to a slowing of channel activation. The double mutant of both countercharges resulted in an additive effect on activation kinetics, but left the voltage dependence of activation unaffected compared to the single mutants. Our findings indicate that VSD I substantially contributes to the regulation of both kinetics and voltage-dependence of activation; that the same ionic interactions are critical for both properties; but that the molecular mechanisms governing the two gating properties are functionally separate.