Role of putative voltage-sensor countercharge D4 in regulating gating properties of Ca(V)1.2 and Ca(V)1.3 calcium channels

Voltage-dependent calcium channels (Ca(V)) activate over a wide range of membrane potentials, and the voltage-dependence of activation of specific channel isoforms is exquisitely tuned to their diverse functions in excitable cells. Alternative splicing further adds to the stunning diversity of gating properties. For example, developmentally regulated insertion of an alternatively spliced exon 29 in the fourth voltage-sensing domain (VSD IV) of Ca(V)1.1 right-shifts voltage-dependence of activation by 30 mV and decreases the current amplitude several-fold. Previously we demonstrated that this regulation of gating properties depends on interactions between positive gating charges (R1, R2) and a negative countercharge (D4) in VSD IV of Ca(V)1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of Ca(V)1.3 and in VSDs II and IV of Ca(V)1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of Ca(V)1.3 and in two splice variants of Ca(V)1.2. In both channels the D4N (VSD IV) mutation resulted in a ̴5 mV right-shift of the voltage-dependence of activation and in a reduction of current density to about half of that in controls. However in Ca(V)1.2 the effects were independent of alternative splicing, indicating that the two modulatory processes operate by distinct mechanisms. Together with our previous findings these results suggest that molecular interactions engaging D4 in VSD IV contribute to voltage-sensing in all examined Ca(V)1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle Ca(V)1.1 channel.