Effects of bath calcium on P2X7R current. (A–D) Concentration dependence on ATP (A and B) and BzATP (C and D) of P2X7R current in single HEK293 cells bathed in 2-mM Ca²⁺–containing (A and C) and Ca²⁺-deficient (B and D) medium with 1 mM Mg²⁺. In this and following figures, the whole-cell current recording was performed using Ca²⁺-deficient pipette medium containing 10 mM EGTA, and cells were held at −60 mV, if not otherwise specified. Horizontal bars above traces indicate the duration of agonist application. The current response was monophasic at low agonist concentrations and biphasic at higher concentrations. The ratio between rapid (I₁) and sustained (I₂) currents also changed with the increase in agonist concentration, as described previously (Yan et al., 2010). All traces shown were obtained during the initial agonist application and are representative of at least five records per dose. Mean ± SEM values are shown in Fig. 2 A. Numbers without parentheses indicate total agonist concentrations used in experiments, and numbers in parentheses indicate free agonist concentration calculated using the Maxc program for two chelators and two metals. The responses to ATP (A vs. B) and BzATP (C vs. D) were similar when the free concentration of each agonist was similar. However, there was a Ca²⁺-dependent shift in the dose–response of ∼0.5 log units of total agonist concentration. Dotted lines separate I₁ and I₂ currents.