L-type Ca²⁺channels play a critical role in regulating Ca²⁺-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 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_v1.2). Immunogold electron microscopy revealed that these proteins colocalize in caveolae. Immunoprecipitation from ventricular myocytes using anti-Ca_v1.2 or anti-caveolin-3 followed by Western blot analysis showed that caveolin-3, Ca_v1.2, β₂-AR (not β₁-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 β₂-AR/Ca_v1.2 signaling complex, β₂-AR stimulation (salbutamol plus atenolol) of I_Ca,Lwas examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I_Ca,Lin 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 I_Ca,Lwas 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.

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