Figure 2

EDME is a potent and selective blocker of TRPML1. (a) Representative concentration-effect relationships for Ca²⁺ increases (Fluo-4) in response to different concentrations of EDME on HEK293 cells stably expressing different TRP channels including hTRPML1∆NC, 2, 3, or TPC2, respectively. Corresponding activators for each TRP channel are listed in parentheses. (b–e) Concentration-effect relationships obtained from whole-cell patch-clamp measurements showing effect of EDME (b, d) and the previously described non-selective TRPML blocker ML-SI3 (c, e) on hTRPML1L15/16A, L577/578A (pH 4.6; n = 6, each), hTRPML2 (pH 7.4; n = 4, each), and hTRPML3 (pH 7.4; n = 3, each) in the presence of 30 µM ML-SA1. Current recording was done with WinWCP5.2.7 (University of Strathclyde, UK) software, and analysis was done with the help of a customized Igor pro program (WaveMetrics). (f) Endolysosomal patch-clamp experiment showing effect of EDME on WT hTRPML1 after activation with ML-SA1 (10 µM). (g) Statistical analysis for experiments as shown in (f) (luminal pH 4.6; n = 3, each). P-values were calculated by one-way ANOVA followed by Tukey’s post hoc test. **p-value < 0.01. (h) Endolysosomal patch-clamp experiment showing effect of EDME on WT hTRPML1 after activation with the endogenous ligand PI(3,5)P₂ (1 µM). (i) Endolysosomal patch-clamp experiment showing effect of EDME (10 µM) on TRPML1 endogenously expressed in mouse alveolar macrophages after activation with ML-SA1 (10 µM). All endolysosomal patch-clamp experiments were analyzed using PatchMaster acquisition software (https://www.heka.com/) and OriginPro 6.1 (https://www.originlab.com/). All statistical analysis was done using GraphPadPrism software (https://www.graphpad.com/scientific-software/prism/).