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

Background-Congenital long-QT syndrome (LQTS) is a primary arrhythmogenic syndrome stemming from perturbed cardiac repolarization. LQTS, which affects Ϸ1 in 3000 persons, is 1 of the most common causes of autopsy-negative sudden death in the young. Since the sentinel discovery of cardiac channel gene mutations in LQTS in 1995, hundreds of mutations in 8 LQTS susceptibility genes have been identified. All 8 LQTS genotypes represent primary cardiac channel defects (ie, ion channelopathy) except LQT4, which is a functional channelopathy because of mutations in ankyrin-B. Approximately 25% of LQTS remains unexplained pathogenetically. We have pursued a "final common pathway" hypothesis to elicit novel LQTS-susceptibility genes. With the recent observation that the LQT3-associated, SCN5A-encoded cardiac sodium channel localizes in caveolae, which are known membrane microdomains whose major component in the striated muscle is caveolin-3, we hypothesized that mutations in caveolin-3 may represent a novel pathogenetic mechanism for LQTS. Methods and Results-Using polymerase chain reaction, denaturing high-performance liquid chromatography, and direct DNA sequencing, we performed open reading frame/splice site mutational analysis on CAV3 in 905 unrelated patients referred for LQTS genetic testing. CAV3 mutations were engineered by site-directed mutagenesis and the molecular phenotype determined by transient heterologous expression into cell lines that stably express the cardiac sodium channel hNa v 1.5. We identified 4 novel mutations in CAV3-encoded caveolin-3 that were absent in Ͼ1000 control alleles. Electrophysiological analysis of sodium current in HEK293 cells stably expressing hNa v 1.5 and transiently transfected with wild-type and mutant caveolin-3 demonstrated that mutant caveolin-3 results in a 2-to 3-fold increase in late sodium current compared with wild-type caveolin-3. Our observations are similar to the increased late sodium current associated with LQT3-associated SCN5A mutations. Conclusions-The present study reports the first CAV3 mutations in subjects with LQTS, and we provide functional data demonstrating a gain-of-function increase in late sodium current. (Circulation. 2006;114:2104-2112.)