hSK2-CaM fusion proteins exhibit similar inhibitory effects to that of coexpressed hSK2 channels with CaM. (A) Diagram illustrating experimental paradigms using SK2 fusion proteins with flexible G_n linker fused to CaM_WT (upper panel) compared with mutant CaMs (lower panel). (B) Current recordings before (black trace) and after (red trace) apamin application (10 nM) for hSK2 expressed with CaM_WT versus hSK2-G4-CaM_WT versus SK2-G4-CaM_WT expressed with DN CaM_1,2,3,4. (C) Summary data at −120 mV and +60 mV from B. (D and E) Summary data for apamin-sensitive current density in HEK 293 cells coexpressing hSK2 with CaM_WT or mutant CaM constructs (left bars) compared with SK2-G4-CaM_WT or SK2-G4-CaM_MUT (hashed bars on the right), where CaM_MUT refers to CaM_N54I (red bars), CaM_F90L (green bars), CaM_F93L (purple bars), CaM_D96V (blue bars), CaM_N98S (cyan bars), CaM_D130G (magenta bars), and CaM_F142L (yellow bars) at −120 mV (D) and +60 mV (E; n = 10–13 cells). Data represent mean ± SEM. Statistical analysis was performed using two-way ANOVA combined with Tukey’s test. The colors of the asterisks denote comparisons with the corresponding bar graphs of the same colors. *, P < 0.05. At −120 mV, P = 0.00001 for all pairwise comparisons except P = 0.04 for CaM_N54I versus CaM_F93L and CaM_N54I versus CaM_D96V, and P= NS for CaM_WT versus CaM_F90L, CaM_F93L versus CaM_D96V, CaM_F93L versus CaM_D130G, CaM_D96V versus CaM_D130G, and CaM_N98S versus CaM_F142L. At +60 mV, P = 0.00001 for all pairwise comparisons except P= NS for CaM_D96V versus CaM_D130G and CaM_N98S versus CaM_F142L. There were no significant differences between hSK2 coexpression with CaM compared with hSK2-G4-CaM for WT or mutant CaMs (P = NS).