Dependence on extracellular Ca²⁺ concentration of BzATP-induced P2X7R current. (A) Calculated free BzATP concentration in medium containing 100 µM BzATP and variable concentrations of Ca²⁺ in the presence of 1 mM Mg²⁺. Horizontal dotted lines indicate solutions used for activation of P2X7R shown in B and C. (B) Typical patterns of P2X7R current in response to application of 100 µM BzATP in medium containing 0.5, 5, and 10 mM Ca²⁺. (C) Correlation between divalent cation concentrations ([DC]_e) and peak amplitude of current responses (left) and between free BzATP concentration and peak amplitude of current response (right). r, correlation coefficient. Data shown are means ± SEM (n = 4 per data point). (D–F) Variable current responses in cells stimulated with media containing identical free BzATP but variable Ca²⁺ concentrations. (D) Comparable free BzATP concentrations achieved with variable total BzATP, 1 mM Mg²⁺, and variable Ca²⁺ concentrations. (E) A decrease in the amplitude of currents in response to media containing identical free BzATP concentrations (1.6 µM) but increasing Ca²⁺ concentrations. Inset shows the current response to medium containing 10 mM Ca²⁺ on an enlarged scale. (F) Mean ± SEM values of peak current amplitude measured 40 s after application of BzATP. n = 4–6 per data point. (G) Effects of bath Ca²⁺ on the peak amplitude of current in cells expressing the P2X7R-H130A mutant; representative traces and mean ± SEM values from four experiments. (H and I) Concentration-dependent effects of bath Ca²⁺ on the activation time and peak amplitude of current; representative traces (H) and mean ± SEM values from four experiments (I). hp, holding potential.