Protein kinase A regulates C-terminally truncated Ca_V 1.2 in Xenopus oocytes: roles of N- and C-termini of the α_1C subunit

Key points: β-Adrenergic stimulation enhances Ca²⁺ entry via L-type Ca_V 1.2 channels, causing stronger contraction of cardiac muscle cells. The signalling pathway involves activation of protein kinase A (PKA), but the molecular details of PKA regulation of Ca_V 1.2 remain controversial despite extensive research. We show that PKA regulation of Ca_V 1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α_1C , is truncated. The PKA upregulation of Ca_V 1.2 does not require key factors previously implicated in this mechanism: the clipped dCT, the A kinase-anchoring protein 15 (AKAP15), the phosphorylation sites S1700, T1704 and S1928, or the β subunit of Ca_V 1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α_1C is essential for the PKA effect. We propose that the regulation described here is one of two or several mechanisms that jointly mediate the PKA regulation of Ca_V 1.2 in the heart.

Abstract: β-Adrenergic stimulation enhances Ca²⁺ currents via L-type, voltage-gated Ca_V 1.2 channels, strengthening cardiac contraction. The signalling via β-adrenergic receptors (β-ARs) involves elevation of cyclic AMP (cAMP) levels and activation of protein kinase A (PKA). However, how PKA affects the channel remains controversial. Recent studies in heterologous systems and genetically engineered mice stress the importance of the post-translational proteolytic truncation of the distal C-terminus (dCT) of the main (α_1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated Ca_V 1.2 in Xenopus oocytes, which previously failed with the non-truncated α_1C . cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced Ca_V 1.2 currents by ∼40% (rabbit α_1C ) to ∼130% (mouse α_1C ). PKA blockers were used to confirm specificity and the need for dissociation of the PKA holoenzyme. The regulation persisted in the absence of the clipped dCT (as a separate protein), the A kinase-anchoring protein AKAP15, and the phosphorylation sites S1700 and T1704, previously proposed as essential for the PKA effect. The Ca_V β_2b subunit was not involved, as suggested by extensive mutagenesis. Using deletion/chimeric mutagenesis, we have identified the initial segment of the cardiac long-N-terminal isoform of α_1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α_1C subunit, is one of several mechanisms underlying the overall PKA action on Ca_V 1.2 in the heart. We hypothesize that PKA is acting on Ca_V 1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α_1C .