Localization of cardiac L-type Ca2+ channels to a caveolar macromolecular signaling complex is required for β2-adrenergic regulation

Ravi C. Balijepalli, Jason D. Foell, Duane D. Hall, Johannes W Hell, Timothy J. Kamp

Research output: Contribution to journalArticle

287 Scopus citations

Abstract

L-type Ca2+ channels play a critical role in regulating Ca 2+-dependent signaling in cardiac myocytes, including excitation-contraction coupling; however, the subcellular localization of cardiac L-type Ca2+ channels and their regulation are incompletely understood. Caveolae are specialized microdomains of the plasmalemma rich in signaling molecules and supported by the structural protein caveolin-3 in muscle. Here we demonstrate that a subpopulation of L-type Ca2+ channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for β2-adrenergic receptor (AR) regulation of ICa,L. Immunofluorescence studies of isolated ventricular myocytes using confocal microscopy detected extensive colocalization of caveolin-3 and the major pore-forming subunit of the L-type Ca channel (Cav1-2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Cav1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Cav1.2, β2-AR (not β1-AR), G protein αs, adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized β2-AR/Ca v1.2 signaling complex, β2-AR stimulation (salbutamol plus atenolol) of ICa,L was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of ICa,L in response to β2-AR activation was eliminated by disruption of caveolae with 10 mM methyl β-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas β1-AR stimulation (norepinephrine plus prazosin) of ICa,L was not altered. These findings demonstrate that subcellular localization of L-type Ca2+ channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.

Original languageEnglish (US)
Pages (from-to)7500-7505
Number of pages6
JournalProceedings of the National Academy of Sciences of the United States of America
Volume103
Issue number19
DOIs
StatePublished - May 9 2006
Externally publishedYes

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Keywords

  • Caveolae
  • Electrophysiology
  • Ventricular myocyte

ASJC Scopus subject areas

  • Genetics
  • General

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