The Ca²⁺-dependent inactivation of ANO 2 affects gating. (A) Ca²⁺-preloading experiments with ANO 1 and ANO 2 channels. During preloading with 7.5 µM free Ca²⁺ at 0 mV, channels were activated and conducted small, transient outward currents during equilibration of cytosol, pipette, and bath solution (left traces). Subsequent application of −70 mV induced current in ANO 1–expressing cells. In contrast ANO 2–expressing cells showed no Cl⁻ current as a consequence of complete ANO 2 inactivation during preloading (right traces). Arrows indicate whole cell breakthrough. (B) Nonstationary current recording at −70 mV from a cell expressing ANO 2 channels. After high-pass filtering of the data, two episodes of increased current noise are discernible (black trace; boxes). (C) Gating activity during the current transient. The peak of current variance (dots) during cell perfusion with 7.5 µM Ca²⁺ preceded the peak current amplitude (normalized current; red line). The maximal variance coincided with the half-maximal current in the activation phase (depicted as “A”). During the ensuing phase of Cl⁻ depletion (depicted as “D”), the variance remained unchanged at a very low value, reflecting the persistent low level of gating activity in the fully activated channels. About 10 s after the start of cell perfusion, the current variance increased again and reached a second peak within ∼8 s (inactivation). This second peak reflects channel inactivation, as the open probability returned from near unity at t = 8 s to near 0 at t = 32 s. The blue line results from a fit of the equation σ²(t) = NP_o(t) × [1 − P₀(t)] × i² to the normalized variance data, with σ²(t) for the normalized variance. n = 100, the number of channels; P_o(t), the open probability; i = 0.14 pA, the single-channel current. The fit demonstrates that the time course of the current variance can be described with P_o as the only variable, a characteristic feature of gating effects.