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

Abstract

The voltage-gated calcium channel Ca_V1.1a primarily functions as voltage-sensor in skeletal muscle excitation-contraction (EC) coupling. In embryonic muscle the splice variant Ca_V1.1e, which lacks exon 29, additionally function as a genuine L-type calcium channel. Because previous work in most laboratories used a Ca_V1.1 expression plasmid containing a single amino acid substitution (R165K) of a critical gating charge in the first voltage-sensing domain (VSD), we corrected this substitution and analyzed its effects on the gating properties of the L-type calcium currents in dysgenic myotubes. Reverting K165 to R right-shifted the voltage-dependence of activation by ~12 mV in both Ca_V1.1 splice variants without changing their current amplitudes or kinetics. This demonstrates the exquisite sensitivity of the voltage-sensor function to changes in the specific amino acid side chains independent of their charge. Our results further indicate the cooperativity of VSDs I and IV in determining the voltage-sensitivity of Ca_V1.1 channel gating.

Keywords: Ca1.1; Voltage-gated calcium channel; dysgenic myotubes; skeletal muscle; voltage-sensing.

Figure 1.

Structure of the first VSD of Ca_V1.1. (a) Amino acid sequence alignment of the S4 transmembrane helices of the first VSD of Ca_V1.1 from five vertebrate species; Oryctolagus cuniculus (UniProtKB, P07293), Homo sapiens (UniProtKB, Q13698), Mus musculus (UniProtKB, Q02789), Rattus norvegicus (UniProtKB, Q02485) and Danio rerio (UniProtKB, Q6RKB0). The conserved arginines in position R1 (R₁₆₅ in the rabbit clone) are marked in red, the other four positively charged residues are marked in blue. (b–e) Structure models of the first VSDs of Ca_V1.1a-K₁₆₅ and Ca_V1.1a-R₁₆₅ in the up-state, based on the cryo-EM structure of Ca_V1.1a [3,13]. (b, d) S1, S2, S3, and S4 denote the transmembrane helices; R/K1, R2, R3, and R4 the positively charged amino acids (blue) in every third position of S4 that transit the electric field across the membrane upon activation and deactivation. The frame indicates the area enlarged at right. (c, e) In the up-state the additional amino group of R165, compared to K165, enables the formation of an additional H-bond with the countercharge E87 in the S2 helix (arrows).

Figure 2.

Expression and localization of Ca_V1.1 constructs in dysgenic myotubes. (a) The conventional (K₁₆₅) and the corrected (R₁₆₅) constructs of the classical/adult Ca_V1.1a splice variant including exon 29; (b) the conventional (K₁₆₅) and the corrected (R₁₆₅) constructs of the embryonic Ca_V1.1e splice variant lacking exon 29. The GFP-tagged Ca_V1.1 constructs were localized with anti-GFP (green) and co-stained with anti-RyR1 (red). Co-clustering of all Ca_V1.1 constructs with RyR1 is indicative of their normal expression and targeting into the skeletal muscle triads. Scale bar, 10 µm.

Figure 3.

Correcting the R165K substitution in the VSD I of Ca_V1.1a right-shifts the voltage-dependence of activation. (a) Representative calcium currents of Ca_V1.1a-K₁₆₅ (black) and Ca_V1.1a-R₁₆₅ (red) at the maximally activating test pulses. (b,c) I/V curves and the fractional activation plot show an 11.4 mV right shift of the voltage-dependence of activation of the corrected Ca_V1.1a-R₁₆₅ (red) compared to Ca_V1.1a-K₁₆₅ (black), but little difference in current density. (d) The scatter-plot of V½ shows a significant increase of the mean voltage at half-activation of Ca_V1.1a-R₁₆₅ (red) compared to Ca_V1.1a-K₁₆₅ (black) (mean±SE, N = 6–8, *P < 0.05, student t-test). (e,f) Scatter-plots of time to peak and fractional inactivation at the end of the 500-ms test pulse (R₅₀₀) show that the K165R substitution did not affect the kinetics of the calcium currents.

Figure 4.

The correction of the R165K substitution in the VSD I of Ca_V1.1e right-shifts the voltage-dependence of activation. (a) Representative calcium currents of Ca_V1.1e-K₁₆₅ (black) and Ca_V1.1e-R₁₆₅ (red) at the maximally activating test pulses. (b,c) I/V curves and the fractional activation plot show a >12 mV right shift of the voltage-dependence of activation of the corrected Ca_V1.1e-R₁₆₅ (red) compared to Ca_V1.1e-K₁₆₅ (black), with only an insignificant increase in current density. (D) The scatter-plot of the V½ shows a significant increase of the mean voltage at half-activation of Ca_V1.1e-R₁₆₅ (red) compared to Ca_V1.1e-K₁₆₅ (black) (mean±SE, N = 8, ***P < 0.001, student t-test). (e,f) Scatter-plots of time to peak and fractional inactivation at the end of the 500-ms test pulse (R₅₀₀) show that the K165R substitution does not affect the kinetics of the calcium current.