Western blot analysis of KCa3.1 channels coexpressed with CaM mutants subjected to Cu(II)Phe treatment. (A) Membranes were prepared from oocytes expressing either the F248W-R362C(KCa3.1)–K75C(CaM), F248W-R362C(KCa3.1), or the F248W(KCa3.1)–K75C(CaM) mutants. Disulfide cross-linking catalyzed by incubating oocytes in 100 µM Cu(II)Phe and 10 µM ionomycin resulted in a band shift from ≈40 kD for KCa3.1 monomer to ≈57 kD for KCa3.1 linked to CaM (KCa3.1-CaM) (A, lane 2). Samples were subjected to SDS/PAGE (10% gel) under either reducing (+DTT) or nonreducing (−DTT) conditions. Exposing membranes to DTT caused the loss of the ≈57-kD band (A, lane 3). Cu(II)Phe was ineffective in initiating disulfide bond formation using the F248W-R362C(KCa3.1) (A, lane 5) or F248W(KCa3.1)–K75C(CaM) (A, lane 8) systems. (B) Western blotting obtained under the conditions described in A using an antibody directed against CaM. The same band at ≈57 kD could be detected after Cu(II)Phe treatment and disappeared after disulfide bond reduction with DDT (B, lane 3). Also shown is a band at 17 kD corresponding to CaM.