Calcium current modulation by the γ1 subunit depends on alternative splicing of CaV1.1

Abstract

The skeletal muscle voltage-gated calcium channel (CaV1.1) primarily functions as a voltage sensor for excitation–contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming α1S subunit and the auxiliary α2δ-1 and γ1 subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of CaV1.1a, greatly reduces the current density and shifts the voltage dependence of activation to positive potentials outside the physiological range. We generated new HEK293 cell lines stably expressing α2δ-1, β3, and STAC3. When the adult (CaV1.1a) and embryonic (CaV1.1e) splice variants were expressed in these cells, the difference in the voltage dependence of activation observed in muscle cells was reproduced, but not the reduced current density of CaV1.1a. Only when we further coexpressed the γ1 subunit was the current density of CaV1.1a, but not that of CaV1.1e, reduced by >50%. In addition, γ1 caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of γ1, unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in CaV1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the γ1 subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the γ1-dependent reduction of current density, suggesting that structural rearrangements in CaV1.1a induced by inclusion of exon 29 may allosterically empower the γ1 subunit to exert its inhibitory action on CaV1.1 calcium currents.

Advances in Ca V 1.1 gating: New insights into permeation and voltage-sensing mechanisms

“…The association of γ 1 with α 1S was shown to be supported by hydrophobic residues in RIVS3 and RIVS4, RIVS4-S5 linker, and III–IV loop with the carboxyl-terminus and transmembrane segment 2 of γ 1 . Recent functional work suggests that RIVS3-4 linker interacts with γ 1 and thus provides a plausible explanation as to why the γ 1 subunit differentially modulates Ca V 1.1e and Ca V 1.1a [ 64 ]; a 19 amino acid stretch in the domain IV S3–S4 linker of α1s is absent in the embryonic splice variant Ca V 1.1e and present in adult Ca V 1.1a [ 26 ] ( Figure 3a ).…”

“…The elegant study by Savalli and colleagues was carried out in a heterologous system which lacked α2δ-1 and γ1. While the channel was functional under these conditions, it is well established that α 2 δ-1 and γ 1 modulate the amplitude, kinetics and voltage-dependence of both L-type current and charge movement [ 64 , 94 , 95 ]. Each subunit likely modulates each VSD differently since each subunit interacts with a specific domain within α 1S [ 87 ].…”

From α1s splicing to γ1 function: A new twist in subunit modulation of the skeletal muscle L-type Ca2+ channel

“…Highlighted in red is the minimal set of molecular components that allowed functional reconstitution of voltage-dependent Ca 2+ release after heterologous expression in non-muscle cells ( Perni et al, 2017 ). (B) Proteins expressed in the study by El Ghaleb et al (2022 ) in HEK293 cells to investigate the impact of the γ 1 subunit and a 19 amino acid stretch in the domain IV S3–S4 linker of α 1s that is absent in the embryonic splice variant Ca V 1.1e and present in adult Ca V 1.1a (both structures indicated in yellow). (C) Alterations of functional characteristics of Ca V 1.1a and Ca V 1.1e caused by co-expressing γ 1 .…”

“…(C) Alterations of functional characteristics of Ca V 1.1a and Ca V 1.1e caused by co-expressing γ 1 . Inactivation (VDI) and surface expression are comparably enhanced, but L-type Ca 2+ current density is only reduced from a relatively high level in combination with the adult splice variant Ca V 1.1a ( El Ghaleb et al, 2022 ).…”

“…The auxiliary subunit γ 1 is highly specific for this channel even though it is not required for voltage-activated Ca 2+ release. A recent study by El Ghaleb et al (2022) in the Journal of General Physiology presents new evidence for a functional interaction of γ 1 with the channel molecule that is influenced by alternative splicing.…”