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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
Author(s) -
Oz Shimrit,
Pankonien Ines,
Belkacemi Anouar,
Flockerzi Veit,
Klussmann Enno,
Haase Hannelore,
Dascal Nathan
Publication year - 2017
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jp274015
Subject(s) - xenopus , protein kinase a , protein subunit , microbiology and biotechnology , phosphorylation , gene isoform , biology , gating , chemistry , biochemistry , biophysics , gene
Key points β‐Adrenergic stimulation enhances Ca 2+ 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 Ca 2+ 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 .