Oxidation renders CLC-1 channels insensitive to ATP. All experiments were performed at a pHi of 6.8 in inside-out patches acutely excised from tsA201 cells under various conditions. The CLC-1 current was continuously monitored using protocol B. Each circle represents the initial tail current. The application of 3 mM ATP for 10 s, as indicated by the short horizontal lines, is repeated at a frequency of ∼1/min. (A) Continuous monitoring of the ATP sensitivity in the normal intracellular solution for mammalian cells. The original recording traces before (black trace) and after (red trace) ATP applications were obtained from the indicated ATP trials. Notice that the initial ATP inhibition of the current is significant, but the inhibition quickly reduces or even disappears within 2–3 min. Insets show the averaged decay of the normalized ATP sensitivity for patches without a delay (left, τ = 1.9 min) or with a delay of 2 min (right, τ = 2.8 min). (B) Continuous monitoring of the ATP inhibition of CLC-1 in the presence of reducing agents. The experiment was the same as that in A except that the patch was excised into the intracellular solution containing 100 μM β-ME. (Inset) Time course of the normalized ATP sensitivity in β-ME. (C) ATP inhibition of CLC-1 channels after the membrane patch was exposed to 300 μM MTSES for 30 s right after the first ATP application. MTSES was then washed out, and the intracellular solution contained no reducing agent. (Insets) Current traces taken from the indicated ATP trials. (D) Effects of ATP on the steady-state Poc-V curve of CLC-1 channels in the presence of 100 μM β-ME.
