Figure 1: Direct and specific activation of lysosomal TRPML1 channels by ROS.
From: MCOLN1 is a ROS sensor in lysosomes that regulates autophagy
(a) Representative time course of whole-endolysosome TRPML1-mediated currents (ITRPML1, open circles, measured at −140 mV) activated by bath application of ChT (75 μM), followed by a 4-min washout and then ML-SI3 (50 μM) application. ITRPML1 was recorded from an enlarged vacuole isolated from COS-1 cells overexpressing EGFP–TRPML1. The currents were elicited by repeated voltage ramps (−140 to +140 mV; 200 ms) with a 4-s inter-step interval. (b) Representative traces of basal (blue), ChT-activated (magenta) and ML-SI3-inhibited (black) ITRPML1 at the three time points indicated in a. Only a portion of the voltage protocol is shown; holding potential was 0 mV. (c) Dose-dependence of ChT activation (n=4–8 patches for each data point). (d) Oxidant-specific activation of ITRPML1. Active oxidants included ChT (150 μM), NaOCl (3 mM), N-chlorosuccinimide (NCS, 500 μM), thimerosal (TMS, 50 μM), H2O2 (10 mM) and t-butyl hydroperoxide (TBHP, 1 mM). Representative traces are in Supplementary Fig. 2. Other tested oxidants included cysteine-specific oxidants (DTNB and DTNP, both at 100 μM), an NO-donor (SNAP, 100 μM) and a reactive lipid (4-HNE, 300 μM). The effects of oxidants were normalized to that of ML-SA1 (20 μM). Numbers of patches tested for each oxidant are shown in brackets. (e) Representative traces of DTNP-insensitive (red), ChT-activated (magenta) ITRPML1. (f) ChT induced single-channel openings in an inside-out patch isolated from TRPML1-4A-expressing cells. (g) The single-channel conductance of ChT- and ML-SA1-activated ITRPML1. (h) ChT activated mTRPML1 specifically, but not mTRPML2, mTRPML3, zTRPML1.1 or mTPC2. Numbers of patches tested for each constructs are shown in brackets. (i) Insensitivity of zTRPML1.1 to ChT. (j) Insensitivity of whole-endolysosome TRPML1Va currents to ChT. (k,l) ChT activated endogenous whole-endolysosome ITRPML1 in WT but not TRPML1 KO mouse macrophages. Data are presented as mean±s.e.m. in c,d and h.
