FIGURE 2.
CaVβ stimulated CaV1.2 membrane expression in HEKT cells. A, HA-tagged CaV1.2 wt was co-expressed transiently either in the stable CaVβ3 or the stable CaVα2bδ cell line. Cell surface expression of CaV1.2 wt was determined in intact cells by flow cytometry using the anti-HA FITC conjugate antibody (Ab). The histogram shows the number of fluorescent cells as a function of the experimental conditions. Cell autofluorescence (HEKT no Ab) was <1% throughout, and the addition of the FITC did not significantly increase the level of fluorescence in HEKT cells not transfected with the HA-tagged CaV1.2 (HEKT with Ab). As seen, only co-expression of CaV1.2 with CaVβ3 significantly promoted membrane expression of CaV1.2 (p < 0.001). Co-expression of CaV1.2 with CaVα2bδ did not alter the number of CaV1.2 channels at the membrane (p > 0.1). Co-expression with both auxiliary subunits did not further improve the membrane expression of CaV1.2. The CaV1.2 + CaVβ3 + CaVα2bδ condition (either CaV1.2 + CaVβ3 expressed transiently in the stable CaVα2bδ cell line or CaV1.2 + CaVα2bδ expressed transiently in the CaVβ3 cell line) was not significantly different from the CaV1.2 + CaVβ3 condition (p > 0.1). Similar results were obtained with transient expression systems. The numerical values can be found in Table 1. B, Western blot analyses of HEKT cells transiently or stably transfected with CaVα2bδ or CaVβ3, using CaVα2δ-1 (1:200) and CaVβ3 (1:500) antibodies. Each lane was loaded with 50 μg of protein. Panel a, HEKT cells were transiently or stably transfected with CaVβ3. Lane 1, control nontransfected cells. Lane 2, transient transfection of CaVβ3. Lane 3, control nontransfected cells. Lane 4, stable CaVβ3 cell line. Lane 5, transient co-transfection of CaVβ3 and CaVα2bδ. Panel b, HEKT cells were transiently or stably transfected with CaVα2bδ. Lane 1, control nontransfected cells. Lane 2, transient transfection of CaVα2bδ. Lane 3, control nontransfected cells. Lane 4, stable CaVα2bδ cell line.