FIGURE 1.

Timing of ADP-induced endocytosis in DRG neurons measured by membrane capacitance (Cm). (A) A 200-ms depolarizing pulse (V_m) induced a whole-cell Ca²⁺ current (I_m) and a rapid Cm increase of 280 fF, which represented a normal depolarization-secretion coupling (17,28,29). Subsequently, the cell was stimulated with ADP (0.1 mM) for 5 s, which induced a Cm decrease of −260 fF, which represented a ligand-endocytosis coupling. The changes in Gs and Gm were negligible. Cm recovered partially after washout of ADP. (B) In another neuron, application of either ATP (0.1 mM, left panel) or ADP (0.1 mM, middle panel) induced comparable decreases in Cm (right panel). On average, the 7-s puffs of 0.1 mM ATP and ADP induced Cm signals of −125 ± 16 fF and −118 ± 17 fF, respectively (n = 8). Activation of P2X channels by ATP induced a transient inward current (I_m), which caused a transient Cm artifact (the overshooting part of the artifact is clipped). (C) Dose-dependent Cm signals induced by 1 μM–10 mM ADP. The Hill coefficient was 1.4. ADP-induced Cm signals saturated for ADP > 0.1 mM (Kd = 10 μM). The maximum Cm signal was −125 ± 16 fF (n = 8). (D) Puff time-dependent Cm signals induced by 0.1 mM ADP. The ADP-induced Cm signal saturated at t > 3 s (n = 6). (E) Determination of the kinetics of ADP-induced endocytosis. The time constant of the solution exchange was 55 ms (see Methods). After replacing the solution of this puffer channel with 0.1 mM ATP, the ADP-induced endocytosis (total −210 fF) with a time constant of 1.8 s was measured by Cm recording in a DRG neuron. On average, the time constants were 65 ± 7 ms (n = 9) and 1.7 ± 0.2 s (n = 22) for puffer solution exchange and cell endocytosis, respectively (inset). Times of puff solutions are indicated by the application bars, except panel (E), where the application continued after the onset arrow.