Figure 5.

The kinetics of channel blockade is significantly faster in 2-APB–gated channels compared to STIM1-gated channels. (A) STIM1- and 2-APB–activated Na⁺ currents in the presence of 20 µM Ca²⁺. Currents were elicited using a step pulse to −100 mV as described in Fig. 4. (B) Time constants of Ca²⁺ blockade of STIM1- and 2-APB–activated Orai3 currents as a function of voltage. Cells were stepped from the holding potential (+50 mV for STIM1-gated channels and −30 mV for 2-APB–gated channels) to the indicated test pulse. (C) Plot of the reciprocal of the time constant obtained at −100 mV against the blocking Ca²⁺ concentration. The slope and y-axis intercepts of the straight-line fits to the data are 4 × 10⁶ M⁻¹s⁻¹ and 33 s⁻¹ (STIM1-gated channels) and 3 × 10⁷ M⁻¹s⁻¹ and 246 s⁻¹ (2-APB–gated channels). (D) Relief from Ca²⁺ block at +100 mV. Ca²⁺ block was induced at 20 µM Ca²⁺_o through a 200-ms step to −100 mV, and the membrane voltage was stepped to +100 mV to induce relief of blockade. The normalized traces (normalized to the steady-state current at +100 mV) show the recovery from blockade after the step to +100 mV. (E) Summary of the dwell time of Ca²⁺ occupancy at −100 and +100 mV. Dwell times were calculated as 1/k_off. The values at −100 mV were derived from k_off estimates from the fit of the mean data shown in C; hence there are no error bars for these values. Data at +100 mV were determined from decay constants in experiments shown in D. Error bars represent SEM.