Table 1.

Cell-surface expression of Ca_Vα2δ1

Stable HEKT Ca_Vβ3 cells were transiently transfected simultaneously with 4 μg of pCMV-Ca_V1.2 and 4 μg of pmCherry-Ca_Vα2δ1 HA WT or mutants. Flow cytometry was used to determine the proportion of Ca_Vα2δ1 proteins expressed at the cell surface as reported in the “intact cells” column, whereas the “permeabilized cells” column represent the total expression level. Fluorescence intensity was estimated using FlowJo software® as detailed elsewhere (39). The relative cell-surface expression of Ca_Vα2δ1-HA constructs was estimated by calculating the ratio of the ΔMedFI for the FITC fluorophore over ΔMedFI values quantified for the FITC fluorophore for the WT construct (ΔMedFI mutant/ΔMedFI WT) in intact nonpermeabilized cells for the experiments carried out the same day under the same conditions. The relative total expression of Ca_Vα2δ1 constructs was estimated by calculating the ratio of the ΔMedFI values quantified for the FITC fluorophore over ΔMedFI values quantified for the FITC fluorophore for the WT construct after cell permeabilization. These ratios are expressed herein as a percentage. The total number of experiments is provided in parentheses, with each experiment being the result of a different transfection in a separate cell dish (>10,000 cells). Statistical analysis was carried out against the ΔMedFI for FITC measured with pmCherry-Ca_Vα2δ1-HA WT. *, p < 0.05, and **, p < 0.001.

CaV1.2 + CaVβ3	Relative ΔMedFI (%) FITC-conjugated anti-HA
	Cell surface, intact cells	Total, permeabilized cells
+ mCherry-CaVα2δ1-HA WT	92 ± 3 (12)	97 ± 0.9 (12)
D259A/S261A/S263A	10 ± 0.05 (3)**	36 ± 0.3 (3)**
D259A/S261A/S263A/D363A	6 ± 0.09 (3)**	31 ± 0.3 (3)**
D259A/S261A/S263A/T331A	11 ± 0.1 (3)**	33 ± 0.5 (3)**
D259A	55 ± 4 (3)**	81 ± 3 (3)**
D259R	20 ± 0.8 (3)**	73 ± 1 (3)**
V260A	77 ± 7 (3)*	99 ± 1 (3)
V260R	48 ± 3 (3)**	97 ± 1 (3)
S261A	36 ± 2 (3)**	72 ± 2 (3)**
S261R	39 ± 0.1 (3)**	66 ± 1 (3)**
G262A	110 ± 8 (3)*	95 ± 4 (3)
G262R	92 ± 0.5 (3)	87 ± 2 (3)**
S263A	97 ± 4 (3)	94 ± 0.3 (3)
S263R	105 ± 2 (3)*	86 ± 0.7 (3)**
T331A	133 ± 5 (3)**	106 ± 2 (3)*
T331G	103 ± 7 (3)	98 ± 2 (3)
T331R	108 ± 8 (3)	88 ± 2 (3)**
D363A	113 ± 9 (3)	89 ± 1 (3)*
D363G	92 ± 5 (3)	85 ± 1 (3)**
D363R	43 ± 3 (3)**	66 ± 1 (3)**