Activation of Ano1 currents by Ca²⁺ in the absence of ATP or exogenous CaM. (A) Kinetics of Ano1 current activation in response to photolysis of caged Ca²⁺ in whole-cell recording. Cells were whole-cell patch-clamped at a holding potential of 100 mV with an internal solution lacking ATP and containing NP-EGTA loaded with Ca²⁺. Ca²⁺ was released by photolysis using a 4-mJ/mm² flash from a flash lamp at the arrow as indicated. For a period of 10 ms (5.8–15.8 ms) starting at the time of current onset, the activation of the mean of five currents (black line) was fitted (red line) to the equation I_t = I₀ + I₁ × (1 − e^−t/τ1) + I₂ × (1 − e^−t/τ2), where t is time in ms, I₀ = 0.15 nA, I₁ = 3.14 nA, I₂ = 1.11 nA, τ₁ = 1.18 ms, τ₂ = 9.8 ms; r² = 0.999. A fit to a single exponential for the period 5.8–11.3 ms was slightly less good, (τ = 1.4 ms) r² = 0.9955. (B) The onset of the current shown in A is shown on an expanded time scale with the double exponential fit. (C) Repeated activation of Ano1 current by Ca²⁺ in an inside-out excised patch in the absence of added ATP. An inside-out excised patch at a holding potential of 100 mV was switched between 0 and 20 µM Ca²⁺ as indicated. The solution was changed by moving a theta tubing perfusion pipe mounted on a Piezo bimorph. The solution change occurred in <5 ms (Perez-Cornejo et al., 2012). (D) The amplitude of the Ano1 current evoked by each Ca²⁺ pulse (I_n) was normalized to the amplitude of the first response (I₀; I_n/I₀). This graph shows the entire experiment of C. C shows pulses 21–25 (2.4–2.9 min).