Molecular and Functional Mechanisms of PKA and PKC Phosphorylation of α1D L-type Calcium Channel.

Abstract

Introduction: Only recently that the L-type α1D calcium (Ca2+) channel has been shown to play an important role in the pacemaker activity of the heart. Knockout mice for α1D Ca2+ channel showed sinus bradycardia, AV block and were prone to atrial fibrillation. The SA node is heavily innervated by the sympathetic nervous system. Previously, our lab showed that α1D Ca2+ channel can be regulated by the adrenergic pathway [cAMP- dependent Protein Kinase A (PKA)/Protein Kinase C (PKC) pathways] but the molecular mechanisms are not known. Therefore, I hypothesized that the adrenergic regulation of α1D Ca2+ channel is mediated through the C-terminus of the α1 subunit of α1D Ca2+ channel for PKA and through the N-terminus of the α1 subunit of α1D Ca2+ channel for PKC. Methods and Results: GST fusion proteins of the N-terminus, intracellular loops, proximal and distal C-terminus of the α1 subunit of α1D Ca2+ channel were prepared. In vitro PKA kinase assay of GST fusion proteins of the N-terminus, intracellular loops and C-terminus of the α1 subunit of α1D Ca2+ channel was performed. The in vitro PKA kinase assay was followed by Western blotting using anti-PKA substrate and anti-phosphoserine antibodies. The proximal part of the C-terminus of the α1 subunit of α1D Ca2+ channel was phosphorylated. The distal part of the C-terminus of α1 subunit of α1D Ca2+ channel was minimally phosphorylated. Several PKA consensus sites were found phosphorylated of which: Serine 1703, serine 1743 and serine 1816 on the proximal C-terminus of α1D Ca2+ channel were found to be phosphorylated by Mass Spectrometry. Site directed mutagenesis and patch clamp studies showed that serine 1743 and serine 1816 were major phosphorylation sites. Also in vitro kinase assay of 35 amino acid peptides mimicking the N-terminus region of α1 subunit of α1D Ca2+ channel with PKC potential phosphorylation sites, showed that it can be regulated by βII- and ε-PKC isozymes. In vivo experiments on mice have shown that α1D Ca2+ channel was phosphorylated with adrenergic stimulation but not under basal conditions. The inhibition of ε-PKC in εV1low PKC antagonist mice has shown that α1D Ca2+ channel is not phosphorylated. Conclusion: The C-terminus of α1 subunit of α1D Ca2+ channel contains two functional sites that mediate the PKA regulation of α1D Ca2+ channel. The N-terminus of α1 subunit of α1D Ca2+ channel mediates the PKC regulation of α1D Ca2+ channel. α1D Ca2+ channel is phosphorylated in vivo with adrenergic stimulation but not with the inhibition of ε-PKC. These results provide novel insights into the PKA and PKC regulation of α1D Ca2+ channel in the heart especially in the setting of excessive adrenergic tone such as heart failure and to the pathogenesis of atrial arrhythmias such as atrial fibrillation.