title: Protein kinase A regulates C-terminally truncated CaV1.2 in Xenopus oocytes: roles of N- and C-termini of the α1Csubunit.
abstract: β-Adrenergic stimulation enhances Ca2+entry via L-type CaV1.2 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 CaV1.2 remain controversial despite extensive research. We show that PKA regulation of CaV1.2 can be reconstituted in Xenopus oocytes when the distal C-terminus (dCT) of the main subunit, α1C, is truncated. The PKA upregulation of CaV1.2 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 CaV1.2. The gating element within the initial segment of the N-terminus of the cardiac isoform of α1Cis 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 CaV1.2 in the heart.
  
  
  
  β-Adrenergic stimulation enhances Ca2+currents via L-type, voltage-gated CaV1.2 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 (α1C) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C. cAMP and the purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV1.2 currents by ∼40% (rabbit α1C) to ∼130% (mouse α1C). 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 CaVβ2bsubunit 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 α1Cas a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1Csubunit, is one of several mechanisms underlying the overall PKA action on CaV1.2 in the heart. We hypothesize that PKA is acting on CaV1.2, in part, by affecting a structural 'scaffold' comprising the interacting cytosolic N- and C-termini of α1C.
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 6997801, Israel.
authors.forename: Shimrit
authors.initials: S
authors.lastname: Oz
authors.affiliation: Max Delbrück Center for Molecular Medicine (MDC), D-13092, and the German Centre for Cardiovascular Research (DZHK) partner site, Berlin, Germany.
authors.forename: Ines
authors.initials: I
authors.lastname: Pankonien
authors.affiliation: Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421, Homburg, Germany.
authors.forename: Anouar
authors.initials: A
authors.lastname: Belkacemi
authors.affiliation: Experimentelle und Klinische Pharmakologie und Toxikologie, Universität des Saarlandes, 66421, Homburg, Germany.
authors.forename: Veit
authors.initials: V
authors.lastname: Flockerzi
authors.affiliation: Max Delbrück Center for Molecular Medicine (MDC), D-13092, and the German Centre for Cardiovascular Research (DZHK) partner site, Berlin, Germany.
authors.forename: Enno
authors.initials: E
authors.lastname: Klussmann
authors.affiliation: Max Delbrück Center for Molecular Medicine (MDC), D-13092, and the German Centre for Cardiovascular Research (DZHK) partner site, Berlin, Germany.
authors.forename: Hannelore
authors.initials: H
authors.lastname: Haase
authors.affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, 6997801, Israel.|Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, 6997801, Israel.
authors.forename: Nathan
authors.identifier: 0000-0002-5397-4146
authors.initials: N
authors.lastname: Dascal
mesh_terms: D000818:Animals; D020746:Calcium Channels, L-Type / Q000502:physiology*; D000242:Cyclic AMP / Q000502:physiology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D009865:Oocytes / Q000502:physiology*; D021122:Protein Subunits / Q000502:physiology*; D014982:Xenopus laevis
chemical_list: D020746:Calcium Channels, L-Type; C419385:L-type calcium channel alpha(1C); D021122:Protein Subunits; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: calcium channel; heterologous expression; protein kinase A; regulation