Modulation of cardiac Ca2+ channel by Gq-activating neurotransmitters reconstituted in Xenopus oocytes

J Biol Chem. 2004 Mar 26;279(13):12503-10. doi: 10.1074/jbc.M310196200. Epub 2004 Jan 13.

Abstract

L-type dihydropyridine-sensitive voltage dependent Ca(2+) channels (L-VDCCs; alpha(1C)) are crucial in cardiovascular physiology. Currents via L-VDCCs are enhanced by hormones and transmitters operating via G(q), such as angiotensin II (AngII) and acetylcholine (ACh). It has been proposed that these modulations are mediated by protein kinase C (PKC). However, reports on effects of PKC activators on L-type channels are contradictory; inhibitory and/or enhancing effects have been observed. Attempts to reproduce the enhancing effect of AngII in heterologous expression systems failed. We previously found that PKC modulation of the channel depends on alpha(1C) isoform used; only a long N-terminal (NT) isoform was up-regulated. Here we report the reconstitution of the AngII- and ACh-induced enhancement of the long-NT isoform of L-VDCC expressed in Xenopus oocytes. The current initially increased over several minutes but later declined to below baseline levels. Using different NT deletion mutants and human short- and long-NT isoforms of the channel, we found the initial segment of the NT to be crucial for the enhancing, but not for the inhibitory, effect. Using blockers of PKC and of phospholipase C (PLC) and a mutated AngII receptor lacking G(q) coupling, we demonstrate that the signaling pathway of the enhancing effect includes the activation of G(q), PLC, and PKC. The inhibitory modulation, present in both alpha(1C) isoforms, was G(q)- and PLC-independent and Ca(2+)-dependent, but not Ca(2+)-mediated, as only basal levels of Ca(2+) were essential. Reconstitution of AngII and ACh effects in Xenopus oocytes will advance the study of molecular mechanisms of these physiologically important modulations.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Acetylcholine / metabolism
  • Angiotensin II / chemistry
  • Animals
  • Calcium Channels, L-Type / chemistry*
  • Calcium Channels, L-Type / metabolism
  • Chelating Agents / pharmacology
  • DNA, Complementary / metabolism
  • Egtazic Acid / analogs & derivatives*
  • Egtazic Acid / pharmacology
  • Electrophysiology
  • Enzyme Inhibitors / pharmacology
  • GTP-Binding Protein alpha Subunits, Gq-G11 / chemistry*
  • Gene Deletion
  • Humans
  • Myocardium / metabolism*
  • Oocytes / metabolism*
  • Patch-Clamp Techniques
  • Protein Isoforms
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism
  • RNA, Messenger / metabolism
  • Rabbits
  • Signal Transduction
  • Time Factors
  • Type C Phospholipases / antagonists & inhibitors
  • Up-Regulation
  • Xenopus laevis

Substances

  • Calcium Channels, L-Type
  • Chelating Agents
  • DNA, Complementary
  • Enzyme Inhibitors
  • Protein Isoforms
  • RNA, Messenger
  • Angiotensin II
  • Egtazic Acid
  • Protein Kinase C
  • Type C Phospholipases
  • GTP-Binding Protein alpha Subunits, Gq-G11
  • 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
  • Acetylcholine