Reversible cysteine cross-linking is observed between Q52C-Kir6.2 and E203C-SUR1. Representative traces of inside-out patch voltage-clamp recordings from COSm6 cell transfected with Q52C-Kir6.2 and E203C-SUR1. Patches were exposed to oxidizing agent (0.2% H₂O₂, gray lines), 1 mM ATP (black lines), reducing agent (5 mM DTT, open lines), or 5 µM PIP₂ (thick dashed lines). Thin dashed lines represent zero current. (A) Current from Q52C-Kir6.2//E203C-SUR1 channels rapidly decreases to a plateau level in the presence of H₂O₂ and can be subsequently restored when DTT is applied. This pattern of activity suggests that the proximity of Q52C-Kir6.2 and E203C-SUR1 is close enough to allow intersubunit disulfide bond formation. (B) Cross-linking between Q52C-Kir6.2 and E203C-SUR1 can also occur from the ATP-bound, closed state as indicated by current decline with H₂O₂ in the presence of saturating ATP concentrations. This trace also shows that plateau current remains sensitive to ATP inhibition (inset; see also Fig. S3). (C) Cross-linking between Q52C-Kir6.2 and E203C-SUR1 locks channels in a PIP₂-insensitive closed state. However, once cross-linking is reversed with reducing agent, PIP₂-induced stimulation is also restored.