TPC2-A1-N is a novel lipophilic, membrane-permeable subtype-selective TPC2 small molecule agonist that activates endogenous TPC2 to moderately increase lysosomal pH in untransfected cells in a time-dependent manner, and can be used to assay the differential function of TPC2 channels in intact cells.

Ca2+:10.5 μM (EC50), Ca2+ current response from TPC2:10.5 μM (EC50)

Two-pore channels (TPC1-3) are ancient members of the voltage-gated ion channel superfamily, expressed throughout the endo-lysosomal system, and regulate the trafficking of various cargoes. TPC2 mediates different physiological and potentially pathophysiological effects depending on its activation, with its ion selectivity being agonist-dependent. TPC2 uniquely conducts different ions in response to different activating ligands. TPC2-A1-N (10 μM) reproducibly evokes Ca2+ signals, achieving its plateau faster than TPC2-A1-P, with an EC50 of 7.8 μM in cells stably expressing TPC2L11A/L12A. TPC2-A1-N (10 μM) induces Ca2+ influx through TPC2 in TPC2L11A/L12A-expressing cells but not in TPC2L11A/L12A/L265P-expressing cells. TPC2-A1-N responses can be selectively blocked by Tetrandrine, Raloxifene, and Fluphenazine by removing extracellular Ca2+[1]. In endo-lysosomal patch-clamp experiments, TPC2-A1-N (30 μM) elicits Na+ currents in vacuolin-enlarged endo-lysosomes isolated from HEK293 cells transiently expressing human TPC2 (hTPC2) but not in cells expressing TPC1[1]. Furthermore, TPC2-A1-N (30 μM) induces larger currents in endo-lysosomes from cells expressing the gain-of-function TPC2M484L variant compared to the wild-type isoform, with an EC50 of 0.6 μM for TPC2-A1-N[1].