Calcium current modulation by the ₁ subunit depends on alternative splicing of Ca_v1.1

The skeletal muscle voltage-gated calcium channel (Ca(v)1.1) primarily functions as a voltage sensor for excitation-contraction coupling. Conversely, its ion-conducting function is modulated by multiple mechanisms within the pore-forming a is subunit and the auxiliary alpha(2)delta-1 and gamma(1) subunits. In particular, developmentally regulated alternative splicing of exon 29, which inserts 19 amino acids in the extracellular IVS3-S4 loop of Ca(v)1.1a, 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 alpha(2)delta-1, beta(3), and STAC3. When the adult (Ca(v)1.1a) and embryonic (Ca(v)1.1e) 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 Ca(v)1.1a. Only when we further coexpressed the gamma(1) subunit was the current density of Ca(v)1.1a, but not that of Ca(v)1.1e, reduced by >50%. In addition, gamma(1) caused a shift of the voltage dependence of inactivation to negative voltages in both variants. Thus, the current-reducing effect of gamma(1), unlike its effect on inactivation, is specifically dependent on the inclusion of exon 29 in Ca(v)1.1a. Molecular structure modeling revealed several direct ionic interactions between residues in the IVS3-S4 loop and the y i subunit. However, substitution of these residues by alanine, individually or in combination, did not abolish the gamma(1)-dependent reduction of current density, suggesting that structural rearrangements in Ca(v)1.1a induced by inclusion of exon 29 may allosterically empower the gamma(1) subunit to exert its inhibitory action on Ca-v1.1 calcium currents.