Mutant CaM overexpression does not affect CRAC channel inactivation. (top) A TET-inducible HEK293 cell line expressing mCh-STIM1 and myc-Orai1 (double-stable, DS) was transiently transfected with GFP-STIM1, CaM-IRES-GFP, and rSK2. (bottom) HEK293H cells were transiently transfected with Ca_v1.2, β1b, α2δ1, and CaM-IRES-GFP or with mCh-STIM1, Orai1-GFP, and CaM-IRES-GFP. (A) Effects on SK and CaV currents (positive controls). (top) Summary of rSK2 current elicited by 100-ms voltage ramps for transfections with WT CaM, CaM₁₂, or CaM₁₂₃₄. CaM₁₂ and CaM₁₂₃₄ significantly reduced rSK2 current as compared with WT CaM (P < 0.01 at −100, −80, and −60 mV). Each point represents the mean ± SEM for n = 5–9 cells. (bottom) Summary of Ca_v1.2 CDI in cells overexpressing WT CaM or CaM₃₄. CaM₃₄ significantly suppressed Ca_v1.2 CDI as compared with WT CaM (P < 0.01 at all voltages). Each point represents the mean ± SEM for n = 4–5 cells. Representative Ca_v1.2 current traces are shown in Fig. S1. (B) Effects on CRAC channel CDI. (top) Representative CRAC currents elicited by 200-ms hyperpolarizations in DS cells expressing WT CaM, CaM₁₂, or CaM₁₂₃₄. Currents were recorded in 20 mM Ca²⁺_o. Current decay is due to Ca²⁺-dependent inactivation (CDI). (bottom) Representative CRAC currents elicited by 200-ms hyperpolarizations for HEK293H cells expressing WT CaM or CaM₃₄. (bottom right) Extent of CRAC channel CDI for each condition, summarized as the fraction of current remaining at the end of the pulse (see Materials and methods). Each point represents the mean ± SEM for n = 5–6 cells. There was no significant difference between CDI measured with WT CaM and any of the mutant CaMs tested (P > 0.2 compared with appropriate WT control in DS or 293H cells at all voltages).