Simulations of use dependence of desensitization based on the kinetic model for P2X2aR. Currents correspond to stimulations with 100 µM ATP for 70 s, as indicated by the gray bar, in the presence of various [Ca²⁺]_e and were normalized by their maximum amplitudes (I_max is 3.5 nA in all of these simulations, and washout period is 250 s). (A–C) Repetitive stimulation at variable [Ca²⁺]_e modeled by varying the fraction of free Ca²⁺, f. Current simulations obtained by setting (A) f = 0 to represent Ca²⁺-deficient medium plus EGTA (the four traces overlie each other, indicating that desensitization did not increase because of the loss of Ca²⁺-dependent desensitization); (B) f = 0.005 to represent Ca²⁺-deficient medium but no EGTA; and (E) f = 0.05 to represent 2 mM [Ca²⁺]_e; panels correspond to Fig. 11 (A–C), respectively. (D) Receptor desensitization of P2X2aR changes from slow to fast during the course of the second ATP stimulation (gray line) because of activation of the toggle switch (see Fig. 2 and Results for details); β₁ increased to 130 s⁻² µM⁻¹. (E) Normalized current (left) and [Ca²⁺]_i (right) simulations in the whole cell (black) and outside-out (gray) configuration. The two configurations were established by setting f = 0 for the former and f = 0.3 for the latter. Calcium entry and accumulation inside the cell in the whole cell configuration are slower than in the outside-out configuration.