Beta-Adrenergic Stimulation of CAV1.2 Channels is Transduced via the IS6-Aid Linker

Cardiac CaV1.2 channels are prominently up-regulated by β-adrenergic agonists via activation of protein kinase A as part of the fundamental flight or fight response, yet detailed mechanisms of PKA activation of CaV1.2 remain unknown despite several decades of investigation. Recently, we reported that β-subunit binding to the α-interaction domain (AID) of the α1-subunit I-II loop is required for β-adrenergic regulation of CaV1.2 channels. Thereupon, we hypothesized that the β subunit-dependent stimulation of CaV1.2 gating induced by β-adrenergic agonists is transduced to pore-opening via the α1C IS6-AID linker. To test this hypothesis, we created transgenic mice with cardiac-specific expression of CaV1.2 α1C subunits with insertion of three glycine residues in the I-II loop (GGG-α1C), which increases the flexibility of the rigid α-helix connecting the IS6 domain and the AID. Introduction of these glycine residues markedly reduced basal open probability, similar to the effects observed by introducing mutations to the AID that disrupted α1-β binding. Despite binding to the β subunit, GGG-α1C CaV1.2 channels failed to be stimulated by β-adrenergic agonists, also similar to AID-mutant channels. Introduction of the 25 amino acid exon 9∗ in the I-II loop C-terminal to the AID did not attenuate PKA-mediated regulation of CaV1.2 and in contrast to GGG insertion, increased basal open probability. Taken together, these findings show that the response to adrenergic-stimulation is correlated with the presence of a bimodal Po distribution with channels transitioning between low and high activity.