In:
Proceedings of the National Academy of Sciences, Proceedings of the National Academy of Sciences, Vol. 103, No. 19 ( 2006-05-09), p. 7500-7505
Abstract:
L-type Ca 2+ 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 Ca 2+ 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 Ca 2+ channels is localized to caveolae in ventricular myocytes as part of a macromolecular signaling complex necessary for β 2 -adrenergic receptor (AR) regulation of I Ca,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 (Ca v 1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca v 1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca v 1.2, β 2 -AR (not β 1 -AR), G protein α s , adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized β 2 -AR/Ca v 1.2 signaling complex, β 2 -AR stimulation (salbutamol plus atenolol) of I Ca,L was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I Ca,L in response to β 2 -AR activation was eliminated by disruption of caveolae with 10 mM methyl β-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas β 1 -AR stimulation (norepinephrine plus prazosin) of I Ca,L was not altered. These findings demonstrate that subcellular localization of L-type Ca 2+ channels to caveolar macromolecular signaling complexes is essential for regulation of the channels by specific signaling pathways.
Type of Medium:
Online Resource
ISSN:
0027-8424
,
1091-6490
DOI:
10.1073/pnas.0503465103
Language:
English
Publisher:
Proceedings of the National Academy of Sciences
Publication Date:
2006
detail.hit.zdb_id:
209104-5
detail.hit.zdb_id:
1461794-8
SSG:
11
SSG:
12
