CaVβ-subunit dependence of forward and reverse trafficking of CaV1.2 calcium channels

Figures

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  Fig. 1. Effect of CaVβ subunits on CaV1.2 cell surface expression in tsA-201 cells. a Schematic of CaV1.2 channel tagged with bungarotoxin binding site (CaV1.2-BBS, BBS: red triangle) between S5 and the P-loop of domain II (DII). b Representative whole-cell current traces recorded in response to depolarizing steps from − 50 to + 40 mV from a holding potential of − 100 mV from tsA-201 cells expressing either CaV1.2 WT (top traces) or CaV1.2-BBS (bottom traces) together with auxiliary subunits CaVα2δ-1 and CaVβ1b. Mean I/V curves (right panel) for CaV1.2 WT (filled circle, n = 9) and CaV1.2-BBS (open circle, n = 18) co-expressed with auxiliary subunits CaVα2δ-1 and CaVβ1b. c Confocal images showing plasma membrane expression of CaV1.2-BBS in tsA cells stained with α-bungarotoxin (BTX)-AF488 (top panels). CaV1.2-BBS was co-expressed with CaVα2δ-1 (left, no β) and either CaVβ1b (center) or CaVβ2a (right). Cells were incubated at 17 °C with BTX-AF488 for 30 min and fixed. The cells were then permeabilized and stained with a rabbit anti-CaV1.2 Ab and secondary Ab anti-rabbit AF594 (bottom panels). Scale 20 µm. d Average CaV1.2-BBS surface expression co-transfected with CaVα2δ-1 and either…

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  Fig. 2. 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…

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  Fig. 3. CaV1.2 forward trafficking and recycling are not CaVβ-subtype dependent. a 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 untagged BTX for 30 min and then incubated at 37 °C with BTX-AF488. The cells were fixed at different time point after incubation at 37 °C, from zero (T0) to 40 min (T40). 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 insertion of CaV1.2-BBS at the cell surface when co-expressed with CaVα2δ-1 and either CaVβ1b (filled circle), CaVβ2a (open circle) or empty vector (open triangle). The results are shown as the mean ± SEM (n numbers correspond to independent experiments). Data were fitted with single exponentials. The time constants of the fits were 16.8 ± 12.6 min and 13.0 ± 14.2 min for CaVβ1b and CaVβ2a (n = 7), respectively. c and d Effect of Brefeldin A (BFA) treatment on CaV1.2 forward trafficking. tsA-201 cells were co-transfected with CaV1.2-BBS, CaVα2δ-1 and either CaVβ1b (c) or CaVβ2a (d). Cells were…