Evidence of cysteine cross-bridging between R362C of KCa3.1 and K75C of CaM catalyzed by Cu(II)Phe. (A) Inside-out current recording illustrating the effect of 1 µM Cu(II)Phe on the F248W-R362C(KCa3.1) channel mutant coexpressed with K75C(CaM) in Xenopus oocytes. Exposure to Cu(II)Phe resulted in a total inhibition of channel activity that was irreversible after Cu(II)Phe washout. Channel activity could be recovered after exposure to 10 mM DDT, confirming disulfide bond formation between K75C of CaM and R362C of KCa3.1. (B) Inside-out single-channel current recording illustrating the action of 1 µM Cu(II)Phe on the K75C(CaM) mutant coexpressed with F248W(KCa3.1). A 1-min application of 1 µM Cu(II)Phe failed under these conditions to affect channel activity. (C) Single-channel recording performed with F248W-R362C(KCa3.1) channel mutant illustrating the absence of the effect of 1 µM Cu(II)Phe when applied internally. This mutant channel was also insensitive to the application of 10 mM DTT. Experiments were performed in symmetrical 200-mM K₂SO₄ conditions at a pipette potential of 60 mV. Label “c” refers to the channel closed state.