Fig. 2
From: CaVβ-subunit dependence of forward and reverse trafficking of CaV1.2 calcium channels
CaV1.2 endocytosis is dynamin-dependent and CaVβ subtype-independent. a Representative confocal images of tsA-201 cells expressing CaV1.2-BBS and labelled with BTX-AF488 (top panels). CaV1.2-BBS was co-expressed with CaVα2δ-1 and CaVβ1b. Cells were incubated at 17 °C with BTX-AF488 for 30 min and then fixed at different time point after incubation at 37 °C, from zero (T0) to 20 min (T20). The cells were then permeabilized and stained with a rabbit anti-CaV1.2 Ab and a secondary Ab anti-rabbit AF594 (bottom panels). Scale bar 20 µm. b Time course of endocytosis of cell surface CaV1.2-BBS co-expressed with CaVα2δ-1 and either CaVβ1b (filled circle) or CaVβ2a (open circle). The results are shown as the mean ± SEM. The n numbers correspond to independent experiments (average fluorescence from at least 25 cells per time point). The data were fitted with single exponentials. The time constants of the fits were 5.6 ± 0.2 min for CaVβ1b and 7.1 ± 0.2 min for CaVβ2a, respectively. c and d Effect of dominant negative dynamin Dyn K44E on CaV1.2 endocytosis. Cells were transfected with CaV1.2-BBS, CaVα2δ-1 and either CaVβ1b (c) or CaVβ2a (d) together with either empty pcDNA3.1 vector (filled symbols) or Dyn K44E (DDN, open symbols). Cells were incubated at 17 °C with BTX-AF488 for 30 min and then fixed at time point T0 and T20 after incubation at 37 °C. Cells were subjected to immunocytochemistry as described in a. BTX-AF488 fluorescence was normalized to the mean fluorescence at T0 for each condition. The results are shown as the mean ± SEM. The n numbers correspond to independent experiments (average fluorescence from at least 25 cells per time point). $$p < 0.01 Control T10 vs Control T0; *p < 0.05 DDN T10 vs Control T10, unpaired t-test