Ensemble variance analysis of Orai1 channels with charged substitutions at position Y80. (A) A thapsigargin-pretreated cell expressing Ch-STIM1_1-469 and WT Orai1-GFP was held continuously at −100 mV in 2 mM Ca²⁺_o, and as indicated by the bars, DVF solutions containing variable low [Ca²⁺]_o were applied transiently to reveal and partially block the Na⁺-CRAC current. Colored circles indicate peak currents corresponding to the 200-ms sweeps shown in (B), which were divided into 25-ms segments for analysis (see Materials and methods). (C) Ca²⁺ block of DVF Na⁺ currents for WT Orai1, Y80K, and Y80E. Na⁺ current was measured immediately after applying DVF solution containing the indicated [Ca²⁺]_o, and normalized to the value for the base DVF solution. Each point represents the mean ± SEM for n = 3–4 cells. Lines are fits of the Hill equation, block = 1/[1 + (K_1/2/[Ca])ⁿ_H]. (D) Normalized variance versus current plots for WT, Y80E, and Y80K were fitted by a parabolic function to determine open probability (see Materials and methods). (E) Variance/current versus block plots were fitted by a linear function to determine unitary current (see Materials and methods). Unitary channel properties are summarized in Table 1.