Figure 6. Modulation of Ca_V1.2 surface expression via its interaction with α‐actinin‐1.

1. HEK293 cells were transfected with WT α₁1.2, α₂δ‐1, and β_2A ± WT α‐actinin‐1 and cultured for 22–24 h before surface biotinylation. Shown are representative immunoblots of NeutrAvidin pull‐down samples (from lysate containing 600 μg protein) and total lysate samples containing 20 μg protein. α₁1.2 was detected with an antibody against its HA tag (top), which is present in all constructs used throughout this work. Pull‐down and lysate samples are from the same blot but different exposures because signals from lysate samples were much stronger than from pull‐down samples. Immunoblotting for vinculin (middle) and tubulin (bottom) indicated that comparable amounts of these intracellular control proteins were present in lysate samples. Their absence in pull‐down samples as seen on the same blots showed that these prominent intracellular proteins did not undergo biotinylation as control for membrane integrity during surface biotinylation. Bar graph shows means ± SEM of the pull‐down immunosignals in mutants relative to surface labeling of control α₁1.2 samples lacking α‐actinin‐1 co‐expression (mean set to 100%; see Materials and Methods; **P < 0.01 two‐tailed t‐test; n = 7).
2. HEK293 cells were transfected with WT or K1647E mutant α₁1.2, α₂δ‐1, and β_2A, and WT or E847K/E851K mutant α‐actinin‐1, or CFP alone as negative control and cultured for 22–24 h before surface biotinylation. Shown are representative immunoblots of pull‐down and lysate samples as in (A). Bar graph shows means ± SEM of the pull‐down immunosignals in mutants relative to surface labeling in the WT/WT control (mean set to 100%; see Materials and Methods). **P < 0.01, ***P < 0.001; one‐way ANOVA with Tukey post hoc test; n = 4–5).