Pi 3 5 p2

Human TPC2 WT and polymorphic TPC2 variants (C-terminally fused to YFP) were transiently transfected into HEK293 cells using TurboFect Transfection Reagent (Thermo Fisher, Waltham, MA, USA). For whole-endolysosomal patch-clamp recordings, isolated intact endolysosomes from HEK293 cells were manually isolated after vacuolin treatment for at least 2 h. Currents were recorded using an EPC-10 patch-clamp amplifier and PatchMaster acquisition software (HEKA, www.heka.com, accessed on 13 January 2024). Data were digitized at 40 kHz and filtered at 2.8 kHz. Cytoplasmic solution contained 140 mM potassium methanesulfonate (KMSA), 5 mM KOH, 4 mM NaCl, 0.39 mM CaCl₂, 1 mM EGTA, and 20 mM HEPES (pH adjusted with KOH to 7.2). Luminal solution was 140 mM NaMSA, 5 mM KMSA, 2 mM CaMSA, 1 mM CaCl₂, 10 mM HEPES, and 10 mM MES (pH adjusted with MSA to 4.6). For measurements with PI(3,5)P2 (water-soluble diC8 form, from Echelon Biosciences, Salt Lake City, UT, USA), PI(3,5)P2 was added to the cytoplasmic solution to give a final concentration of 1µM. 500 ms voltage ramps from −100 to +100 mV were applied every 5 s from a holding potential of 0 mV. Current amplitudes at −100 mV were determined from individual ramp current recordings, and current density was calculated by dividing by cell capacitance. All recordings were performed at 23–25 °C.