Localization of cardiac L-type Ca(2+) channels to a caveolar macromolecular signaling complex is required for beta(2)-adrenergic regulation.
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 beta(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, beta(2)-AR (not beta(1)-AR), G protein alpha(s), adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized beta(2)-AR/Ca(v)1.2 signaling complex, beta(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 beta(2)-AR activation was eliminated by disruption of caveolae with 10 mM methyl beta-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas beta(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.
Balijepalli
Ravi C
RC
Department of Medicine, University of Wisconsin, Madison, WI 53706, USA.
Foell
Jason D
JD
Hall
Duane D
DD
Hell
Johannes W
JW
Kamp
Timothy J
TJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D020746:Calcium Channels, L-Type / Q000235:genetics / Q000378:metabolism*; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism*; D051244:Caveolin 3 / Q000235:genetics / Q000378:metabolism; D003331:Coronary Vessels / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004285:Dogs; D004594:Electrophysiology; D006321:Heart / Q000187:drug effects; D007425:Intracellular Membranes / Q000378:metabolism; D051379:Mice; D046529:Microscopy, Electron, Transmission; D016253:Microscopy, Immunoelectron; D009206:Myocardium / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000235:genetics; D011943:Receptors, Adrenergic, beta / Q000378:metabolism*; D015398:Signal Transduction; D047392:beta-Cyclodextrins / Q000494:pharmacology
D020746:Calcium Channels, L-Type; D051244:Caveolin 3; D034741:RNA, Small Interfering; D011943:Receptors, Adrenergic, beta; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D017868:Cyclic AMP-Dependent Protein Kinases; D000262:Adenylyl Cyclases