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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

Authors

  • S. Oz
  • I. Pankonien
  • A. Belkacemi
  • V. Flockerzi
  • E. Klussmann
  • H. Haase
  • N. Dascal

Journal

  • Journal of Physiology

Citation

  • J Physiol 595 (10): 3181-3202

Abstract

  • β-adrenergic stimulation enhances Ca(2+) currents via L-type, voltage-gated CaV 1.2 channels, strengthening cardiac contraction. The signaling 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 posttranslational proteolytic truncation of distal C-terminus (dCT) of the main (α1C ) subunit. Here, we successfully reconstituted the cAMP/PKA regulation of the dCT-truncated CaV 1.2 in Xenopus oocytes, which previously failed with the non-truncated α1C . cAMP and purified catalytic subunit of PKA, PKA-CS, injected into intact oocytes, enhanced CaV 1.2 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 PKA effect. CaV β2b 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 α1C as a previously unrecognized essential element involved in PKA regulation. We propose that the observed regulation, that exclusively involves the α1C subunit, is one of several mechanisms underlying the overall PKA action on CaV 1.2 in the heart. We hypothesize that PKA is acting on CaV 1.2, in part, by affecting a structural "scaffold" comprising the interacting cytosolic N- and C-termini of α1C .


DOI

doi:10.1113/JP274015