Luminal Ca2+ is a Major Sensitiser of Two-Pore Channels to NAADP

Abstract

It has been suggested that two-pore domain channels (TPCs) are the NAADP receptors responsible for NAADP-mediated Ca2+-release from lysosome-related stores yet there is evidence that NAADP could also regulate RyR channels. We have therefore compared the effects of NAADP on native RyR1, RyR2 and purified human TPC2, reconstituted into artificial membranes under identical experimental conditions. Similar to RyR channels, we find that TPC2 behaves as an ion-channel permeable to both monovalent (300±14pS; symmetrical 210 mM K+; SD; n=3) and divalent cations (15±2pS; 10μM cis/50 mM trans Ca2+;SD; n=5) with no evidence for anion permeability (in a 210mM trans: 510mM cis KCl gradient, the reversal potential coincides with the calculated value for a channel ideally selective for cations (Erev=-23mV)). Addition of trans NAADP had no effect on TPC2, but cis application induced marked channel activation. In symmetrical 210mM K+ and 10μM Ca2+, NAADP dose-dependently activated TPC2 channels with an EC50 of 500nM. Addition of 200μM trans Ca2+ significantly increased the sensitivity of TPC2, shifting the EC50 to 5nM. We have previously demonstrated that ligand-activation of RyR channels is also highly sensitive to luminal Ca2+ and therefore we have investigated how NAADP affects RyR1 and RyR2 in the presence of sensitising levels of luminal Ca2+. Addition of NAADP (≤1μM) did not affect RyR2 Po but slightly activated RyR1 (1μM NAADP increased Po from 0.022±0.035 to 0.106±0.147; SD, n=5). In contrast, larger increases in TPC2 Po (0.001±0.002 to 0.4±0.2; SD, n=3, P<0.05) could be elicited with much lower NAADP concentrations (10nM). Our study is the first to show that animal TPCs form functional, Ca2+-permeable ion-channels. We also provide further evidence that TPC2 is capable of mediating NAADP-sensitive Ca2+-release from acidic organelles but do not rule out a role for RyR1.BHF supported