Role of putative voltage-sensor countercharge D4 in regulating gating properties of Ca_V1.2 and Ca_V1.3 calcium channels

Abstract

Voltage-dependent calcium channels (CaV) 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 CaV1.1 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 CaV1.1. Here we investigated whether this molecular mechanism plays a similar role in the VSD IV of CaV1.3 and in VSDs II and IV of CaV1.2 by introducing charge-neutralizing mutations (D4N or E4Q) in the corresponding positions of CaV1.3 and in two splice variants of CaV1.2. 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 CaV1.2 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 CaV1 channels, however its striking role in regulating the gating properties by alternative splicing appears to be a unique property of the skeletal muscle CaV1.1 channel.

Correcting the R165K substitution in the first voltage-sensor of CaV1.1 right-shifts the voltage-dependence of skeletal muscle calcium channel activation

“…Paraformaldehyde-fixed cultures were immunolabeled as previously described [17,27] with rabbit polyclonal anti-GFP (1:10,000; Molecular Probes) and mouse monoclonal anti-RyR (34-C; 1:1000; Alexis Biochemicals) and fluorescently labeled with goat anti-rabbit Alexa-488 and secondary goat antimouse Alexa-594 (1:4000), respectively. Thus, the anti-GFP label and the intrinsic GFP signal were both recorded in the green channel.…”

“…EC coupling) [14,15]. This divergent voltage-dependence of EC-coupling and current activation depends on the developmentally regulated insertion of exon 29 and on a unique interaction of the outermost gating charges (R1 and R2) with countercharges in the fourth VSD [12,[16][17][18]. In contrast, the characteristic slow activation kinetics of skeletal muscle calcium currents are determined by sequences in the first VSD [19,20]; thus suggesting that the specific properties of first and fourth VSDs of Ca V 1.1 independently determine the kinetics and voltage-dependence of current activation, respectively [20].…”

Stabilization of negative activation voltages of Cav1.3 L-Type Ca2+-channels by alternative splicing

“…developmentally regulated insertion of exon 29 right-shifts voltage-dependence of activation by ≈30 mV [35]. This regulation depends on interactions between positive gating charges and a negative countercharge in the voltage-sensing domain of Ca V 1.1 35 . Neutralization of the corresponding negative countercharge in Cav1.3 lacking exon 32 and exon 11 (Cav1.3 8a-42 ) also shifts V 0.5,act to more positive voltages (although only slightly, ≈ 5 mV, 35 ).…”

“…the opposite effect as reported here, measured in 10 mM Ba 2+ ) upon inclusion of exon 32 [ 22 ]. Exclusion of the corresponding exon (exon 33) in Cav1.2 α1-subunits resulted in a hyperpolarizing shift in activation of about 5–10 mV [ 35 , 39 ], which has been associated with the development of ventricular arrhythmia and a higher risk for heart failure [ 39 ].…”

“…Alternative splicing at a homologous position in exon 32 of Cav1.1 channels, i.e. developmentally regulated insertion of exon 29 right-shifts voltage-dependence of activation by ≈30 mV [35]. This regulation depends on interactions between positive gating charges and a negative countercharge in the voltage-sensing domain of Ca V 1.1 35 .…”