Figure 1: PI(3,5)P2 reversibly activated hTPC1 channel expressed in vacuoles isolated from Arabidopsis mesophyll cells.
From: The human two-pore channel 1 is modulated by cytosolic and luminal calcium
(a) Confocal image of a representative EGFP-positive vacuole showing expression of hTPC1 proteins on its membrane. Scale bar 10 μm. (b,c) Time course of current amplitude (b) recorded in response to bath application of 90 nM PI(3,5)P2; each point represents the steady-state current at +40 mV. The time course of current activation reflects the speed of solution exchange. Time points indicated by numbers correspond to the current traces, shown in C lower panel, in control (1), in the presence (2) and after wash out (3) of PI(3,5)P2. Currents were elicited by the voltage profile shown in c upper panel. (d) PI(3,5)P2 but not NAADP activates hTPC1 channels on Arabidopsis vacuoles. Summary plot of currents densities elicited at +40 mV by PI(3,5)P2 (90 nM, n = 57) or NAADP (100 nM, n = 6) on EGFP-tagged vacuoles (hTPC1). PI(3,5)P2 was ineffective when applied to untrasformed vacuoles (control, n = 53). (e) Dose-dependence activation of PI(3,5)P2-mediated current. For each vacuole, responses were normalized to the value obtained in the presence of 90 nM PI(3,5)P2 (n ranging from 3 to 6). Data were fitted with the Michaelis-Menten function Imax/([PI(3,5)P2] +Km), with half-maximal activation (Km) of 15 ± 3 vM at +40 mV and 18 ± 3 vM at −40 mV. Imax was 1.16 ± 0.04 and 1.13 ± 0.03 respectively at + and −40 mV.
