FIGURE 2.

Inhibition of native SOC by the charge-swap TRPC mutants and rescue of SOC by STIM1(K684E,K685E). A, alignment of a C-terminal region of TRPC channels encompassing the conserved negative charges that are highlighted in boldface colored letters. The two negative charges were mutated to the positively charged lysines. B and C, HEK cells were transfected with GFP (black traces) and the following TRPC channel mutants: TRPC3(D697K,D698K) (TRPC3(DD/KK); blue traces and bars), TRPC4(E648K,E649K) (TRPC4(EE/KK); green traces and bars), TRPC5(D651K,E652K) (TRPC5(DE/KK); red traces and bars), and TRPC6(E755K,E756K) (TRPC6(EE/KK); magenta traces and bars). The cells were also transfected with empty vector (B) or STIM1(K684E,K685E) (S1(KK/EE); C). The Fura-2-loaded cells were incubated in Ca²⁺-free medium, treated with 25 μm cyclopiazonic acid (CPA) for 7.5 min to deplete the stores, and then exposed to medium containing 2 mm Ca²⁺ to assay Ca²⁺ influx. D, mean ± S.E. of at least 15 cells from two separate experiments.