Localization of cardiac L-type Ca ²⁺ channels to a caveolar macromolecular signaling complex is required for β ₂ -adrenergic regulation

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 ␤2-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 (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, ␤2-AR (not ␤1-AR), G protein ␣s, adenylyl cyclase, protein kinase A, and protein phosphatase 2a are closely associated. To determine the functional impact of the caveolar-localized ␤2-AR͞Cav1.2 signaling complex, ␤2-AR stimulation (salbutamol plus atenolol) of ICa,L was examined in pertussis toxin-treated neonatal mouse ventricular myocytes. The stimulation of I Ca,L in response to ␤2-AR activation was eliminated by disruption of caveolae with 10 mM methyl ␤-cyclodextrin or by small interfering RNA directed against caveolin-3, whereas ␤1-AR stimulation (norepinephrine plus prazosin) of ICa,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.caveolae ͉ electrophysiology ͉ ventricular myocyte

Caveolin proteins and estrogen signaling in the brain

“…Previous studies have not only indicated CAV1 to localize with ERα, but also mGluR1a [88]. Similarly, L-type calcium channels cluster in CAV3-generated caveolae [89,90]. As such, we find several individual signaling molecules linked to caveolin proteins to which they were previously ascribed.…”

Myoscape controls cardiac calcium cycling and contractility via regulation of L-type calcium channel surface expression

“…In this context, the overexpression of Myoscape resulted in enhanced global calcium transients with higher calcium amplitudes and LTCC currents. These results were additive to the effects of isoproterenol, implying that Myoscape acts independently of the adrenergic control of LTCC activity via PKA-dependent phosphorylation91326. Conversely, Myoscape ablation reduced LTCC currents irrespective of beta-adrenergic costimulation, which is associated with reduced global calcium transients and impaired contractile performance of cardiomyocytes.…”

Endothelial nitric oxide synthase decreases β-adrenergic responsiveness via inhibition of the L-type Ca2+ current

“…Exogenous NO (i.e., NO donors) and nonspecific NOS inhibitors have also been shown to regulate ␤-AR-stimulated I Ca (33,48), and it was hypothesized that this effect was via NOS3. Within cardiac myocytes, NOS3 is localized with the L-type Ca 2ϩ channel and the ␤ 2 -AR receptor to the caveolae (3,17). However, studies investigating ␤-ARstimulated I Ca in NOS3 Ϫ/Ϫ myocytes observed no difference compared with WT myocytes (21, 46).…”

“…Exogenous NO (i.e., NO donors) has also been found to decrease ␤-AR-stimulated I Ca (48). NOS3 is localized to the caveolae, along with the L-type Ca 2ϩ channel and the ␤ 2 -AR receptor (3,17). In addition, a study showed that myocytes from female mouse hearts have smaller ␤-AR -induced I Ca and a higher association between NOS3 and caveolin-3 compared with myocytes from male mouse hearts (43).…”