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

Abstract

Auxiliary Ca_Vβ subunits interact with the pore forming Ca_Vα₁ subunit to promote the plasma membrane expression of high voltage-activated calcium channels and to modulate the biophysical properties of Ca²⁺ currents. However, the effect of Ca_Vβ subunits on channel trafficking to and from the plasma membrane is still controversial. Here, we have investigated the impact of Ca_Vβ1b and Ca_Vβ2a subunits on plasma membrane trafficking of Ca_V1.2 using a live-labeling strategy. We show that the Ca_Vβ1b subunit is more potent in increasing Ca_V1.2 expression at the plasma membrane than the Ca_Vβ2a subunit and that this effect is not related to modification of intracellular trafficking of the channel (i.e. neither forward trafficking, nor recycling, nor endocytosis). We conclude that the differential effect of Ca_Vβ subunit subtypes on Ca_V1.2 surface expression is likely due to their differential ability to protect Ca_V1.2 from degradation.

Keywords: CaVβ auxiliary subunits; Calcium channel; Trafficking.

Fig. 1

Effect of Ca_Vβ subunits on Ca_V1.2 cell surface expression in tsA-201 cells. a Schematic of Ca_V1.2 channel tagged with bungarotoxin binding site (Ca_V1.2-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 Ca_V1.2 WT (top traces) or Ca_V1.2-BBS (bottom traces) together with auxiliary subunits Ca_Vα₂δ-1 and Ca_Vβ1b. Mean I/V curves (right panel) for Ca_V1.2 WT (filled circle, n = 9) and Ca_V1.2-BBS (open circle, n = 18) co-expressed with auxiliary subunits Ca_Vα₂δ-1 and Ca_Vβ1b. c Confocal images showing plasma membrane expression of Ca_V1.2-BBS in tsA cells stained with α-bungarotoxin (BTX)-AF488 (top panels). Ca_V1.2-BBS was co-expressed with Ca_Vα₂δ-1 (left, no β) and either Ca_Vβ1b (center) or Ca_Vβ2a (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-Ca_V1.2 Ab and secondary Ab anti-rabbit AF594 (bottom panels). Scale 20 µm. d Average Ca_V1.2-BBS surface expression co-transfected with Ca_Vα₂δ-1 and either Ca_Vβ1b (black bar), or Ca_Vβ2a (open bar), or empty vector (gray bar). Bars are mean (± SEM) normalized to Ca_Vβ1b mean. ***p < 0.001, n = 14; ^$$$p < 0.001, n = 5; paired t-test, n numbers correspond to independent experiments

Fig. 2

Ca_V1.2 endocytosis is dynamin-dependent and Ca_Vβ subtype-independent. a Representative confocal images of tsA-201 cells expressing Ca_V1.2-BBS and labelled with BTX-AF488 (top panels). Ca_V1.2-BBS was co-expressed with Ca_Vα₂δ-1 and Ca_Vβ1b. 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-Ca_V1.2 Ab and a secondary Ab anti-rabbit AF594 (bottom panels). Scale bar 20 µm. b Time course of endocytosis of cell surface Ca_V1.2-BBS co-expressed with Ca_Vα₂δ-1 and either Ca_Vβ1b (filled circle) or Ca_Vβ2a (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 Ca_Vβ1b and 7.1 ± 0.2 min for Ca_Vβ2a, respectively. c and d Effect of dominant negative dynamin Dyn K44E on Ca_V1.2 endocytosis. Cells were transfected with Ca_V1.2-BBS, Ca_Vα₂δ-1 and either Ca_Vβ1b (c) or Ca_Vβ2a (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

Fig. 3

Ca_V1.2 forward trafficking and recycling are not Ca_Vβ-subtype dependent. a Confocal images of tsA-201 cells expressing Ca_V1.2-BBS and labelled with BTX-AF488 (top panels). Ca_V1.2-BBS was co-expressed with Ca_Vα₂δ-1 and Ca_Vβ1b. 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-Ca_V1.2 Ab and a secondary Ab anti-rabbit AF594 (bottom panels). Scale bar 20 µm. b Time course of insertion of Ca_V1.2-BBS at the cell surface when co-expressed with Ca_Vα₂δ-1 and either Ca_Vβ1b (filled circle), Ca_Vβ2a (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 Ca_Vβ1b and Ca_Vβ2a (n = 7), respectively. c and d Effect of Brefeldin A (BFA) treatment on Ca_V1.2 forward trafficking. tsA-201 cells were co-transfected with Ca_V1.2-BBS, Ca_Vα₂δ-1 and either Ca_Vβ1b (c) or Ca_Vβ2a (d). Cells were treated with BFA for 4 h before undergoing the forward trafficking protocol described in a. BTX-AF488 fluorescence was normalized to the mean fluorescence at T40 for the control condition (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 compared using an unpaired t-test. The data were fitted with single exponentials. The time constants of the fits were 10.6 ± 6.6 min and 11.6 ± 8.1 min for Ca_Vβ1b and Ca_Vβ2a (n = 7), respectively