Abstract 1506: Targeting of CaV1.2 Channels to Caveolae Requires CaVβ Subunit Association with Caveolin-3 in the Cardiomyocytes
Cavβ subunits regulate the function and membrane trafficking of Cav1.2 channels. Previously we have shown that Cavβ subunit isoforms show different subcellular distribution in ventricular myocytes and that a subpopulation of the CaV1.2 channels is localized to caveolae. We hypothesize that specific Cavβ subunit isoforms determine caveolar targeting of Cav1.2 channels in the heart. Using biochemical techniques, confocal microscopy, and electron microscopy we determined the composition and abundance of caveolar L-Type Ca2+ channels. Saturation co-immunoprecipitation (co-IP) assays using anti Cav-3 antibody and [3H]PN200–110 labeled L-type Ca2+ channels demonstrated that 43±7% of the Cav1.2 channels are associated with Cav-3 in mouse ventricular myocytes. Anti Cav-3 co-IP analysis of myocyte lysates, detected protein bands for β2C, β2a but not β1a, β1b, β1d, β3, or β4. Furthermore, co-expression and co-IP studies showed that β2C subunit was required for Cav-3 association of Cav1.2 subunit in transfected HEK293 cells. GSTCav-3 pull down analysis in HEK293 cells expressing different Cavβ isoforms demonstrated a similar specificity of Cavβ subunit and Cav-3 association. To characterize the composition of the caveolar Cav1.2 channels in native cells, we expressed GFP or a dominant negative mutation of Cav-3 (P104L) in neonatal mouse cardiomyocytes. P104LCav-3 is associated with limb-girdle muscular dystrophy and results in retention of Cav-3 in Golgi, thus disrupting caveolae formation. Cells were immunostained with specific antibodies to Cavβ subunits, Cav-3 and a 58 kDa Golgi protein. P104LCav-3 expression in the myocytes inhibited trafficking of β2C to the plasma membrane by trapping the protein along with Cav-3 in the Golgi complex. However, P104LCav3 did not alter trafficking of β1a, β1b, β3 subunits. Further, using immunogold labeling and electron microscopy on mouse ventricular sections we show that β2C isoform was specifically colocalized with Cav-3 in caveolae. In contrast β3 isoform was localized to T-tubules and did not colocalize with Cav-3 in caveolae. We conclude that Cavβ subunit isoforms differentially associate with Cav-3 and are responsible for targeting a subpopulation of L-type Ca2+ channels to caveolae in cardiomyocytes.
This research has received full or partial funding support from the American Heart Association, AHA National Center.