(A) Representative basal ITPC2 step currents elicited by a voltage step protocol in the whole-endolysosome (EL) configuration. Voltage steps from −140 to 100 mV with a voltage increment (∆V) of 20 mV for 0.5 s were used to elicit ITPC2 in A-D. HP = 0 mV. Unless otherwise indicated, symmetric (bath/cytosol vs pipette/lumen) Na+ (150 mM) solutions were used for all whole-endolysosome recordings, and PI(3,5)P2 (0.3 µM), LyNa-VA1.2 (100 µM), and LyNA1 (300 µM) were bath- applied to induce ITPC2. (B–D) Representative ITPC2 step currents activated by PI(3,5)P2 (B), LyNa-VA1.2 (C), and LyNA1 (D). (E) Representative normalized I-V plots based on the instantaneous currents activated by various agonists. (F) Rectification index, calculated as the ratio of the current amplitudes between +80 and −80 mV, of PI(3,5)P2-, LyNa-VA1.2-, and LyNA1- activated ITPC2. (G) The inactivation of ITPC2 at −120 mV was quantified as the ratio of current amplitudes at 10 vs. 500 ms, based on step currents in B, (C) and D. (H) Voltage steps from −120 to 100 mV (∆V = 20 mV) for 1 s were used to elicit tail currents at −120 mV shown in (I) and (J). (I) The tail currents of PI(3,5)P2- evoked whole-endolysosome ITPC2. (J) The tail currents of LyNa-VA1.2- activated whole-endolysosome ITPC2. Arrows in (I) and (J) indicate where the currents were measured to calculate the channel conductance (G = I/V). (K) Normalized G-V curves of PI(3,5)P2- and LyNa-VA1.2- activated ITPC2. LyNa-VA1.2 activated ITPC2 in a voltage dependent manner with a V1/2 = −20.3 ± 3.5 mV (n = 5 patches). For panels F and G, individual data and Mean ± S.E.M. are presented. ***, p<0.001. Individual data for (F) and (G) are presented in Figure 2—source data 1.