Dependence of P2X2aR desensitization kinetics on bath and intrapipette Ca²⁺ concentration. All experiments were performed in HEK293 cells. (A–C) Effects of variable bath Ca²⁺ concentrations on P2X2aR desensitization. Cells were bathed in Ca²⁺-deficient KR buffer containing 0.5 mM EGTA (A), Ca²⁺-deficient KR buffer (B), and 2 mM of Ca²⁺-containing buffer (C). In all these experiments, the intrapipette buffer did not contain EGTA. I_max: 4.6 (1), 4.5 (2), 4.5 (3), and 4.1 (4) nA (A); 3.5 (1), 3.5 (2), and 3.2 (3) nA (B); and 5.5 (1), 2.6 (2), 2.2 (3), and 2.2 (4) nA (C). (D and E) Dependence of the rate of P2X2aR desensitization on the mode of recording. Cells were bathed in KR buffer and stimulated with 100 µM ATP. (D) Whole cell (second agonist application, I_peak = 4.5 nA) versus outside-out macropatch (third agonist application, I_peak = 0.4 nA) recording. (E) Perforated cell recording. (Main panel) Cells were bathed in 2 mM of Ca²⁺-containing KR. I_peak = 4.7 (1), 2.6 (2), and 2.3 (3) nA. (Inset) Cells were bathed in Ca²⁺-deficient KR medium.