Alternative Splicing at N Terminus and Domain I Modulates Ca_V1.2 Inactivation and Surface Expression

The Ca_V1.2 L-type calcium channel is a key conduit for Ca²⁺ influx to initiate excitation-contraction coupling for contraction of the heart and vasoconstriction of the arteries and for altering membrane excitability in neurons. Its α_1C pore-forming subunit is known to undergo extensive alternative splicing to produce many Ca_V1.2 isoforms that differ in their electrophysiological and pharmacological properties. Here, we examined the structure-function relationship of human Ca_V1.2 with respect to the inclusion or exclusion of mutually exclusive exons of the N-terminus exons 1/1a and IS6 segment exons 8/8a. These exons showed tissue selectivity in their expression patterns: heart variant 1a/8a, one smooth-muscle variant 1/8, and a brain isoform 1/8a. Overall, the 1/8a, when coexpressed with Ca_Vβ_2a, displayed a significant and distinct shift in voltage-dependent activation and inactivation and inactivation kinetics as compared to the other three splice variants. Further analysis showed a clear additive effect of the hyperpolarization shift in V_1/2inact of Ca_V1.2 channels containing exon 1 in combination with 8a. However, this additive effect was less distinct for V_1/2act. However, the measured effects were β-subunit-dependent when comparing Ca_Vβ_2a with Ca_Vβ₃ coexpression. Notably, calcium-dependent inactivation mediated by local Ca²⁺-sensing via the N-lobe of calmodulin was significantly enhanced in exon-1-containing Ca_V1.2 as compared to exon-1a-containing Ca_V1.2 channels. At the cellular level, the current densities of the 1/8a or 1/8 variants were significantly larger than the 1a/8a and 1a/8 variants when coexpressed either with Ca_Vβ_2a or Ca_Vβ₃ subunit. This finding correlated well with a higher channel surface expression for the exon 1-Ca_V1.2 isoform that we quantified by protein surface-expression levels or by gating currents. Our data also provided a deeper molecular understanding of the altered biophysical properties of alternatively spliced human Ca_V1.2 channels by directly comparing unitary single-channel events with macroscopic whole-cell currents.