TPC2 is a Novel NAADP-Sensitive Intracellular Ca2+-Release Channel with Unique Gating Characteristics

We have recently demonstrated that human TPC2 is an ion-channel equipped with the specialised gating and conduction properties required to fulfil the role of the lysosomal NAADP-sensitive Ca2+-release channel (Pitt et al., 2010 J.Biol.Chem. In press). We now examine the mechanisms that underlie NAADP activation of TPC2 channels after reconstitution of purified, recombinant TPC2 into artificial bilayers. We find that TPC2 channels gate in three basic modes: 1. The constitutively-active state, 2. The NAADP-activated state and 3. The NAADP-inactivated state. In the constitutively-active state, TPC2 can open, albeit with very low Po, in the apparent absence of activating ligands. Reducing cytosolic or luminal [Ca2+] from 10µM to <1nM does not abolish channel openings. The NAADP-activated state is observed at low [NAADP] (<1µM) and in this state, the affinity and efficacy of NAADP are crucially dependent on luminal pH and [Ca2+] whereas cytosolic Ca2+ (≤ 10 µM) does not increase Po. A unique characteristic of the NAADP-activated state is episodic gating where multiple channels open and close simultaneously in a synchronized manner. The NAADP-inactivated state is observed at high [NAADP], typically ≥1mM. In this state, constitutive channel openings are abolished and Po becomes zero indicating the presence of high affinity activation and lower affinity inactivation sites. An important finding of this study is that, unlike other major classes of Ca2+-release channel (RyR and IP3R), activation of TPC2 is not heavily dependent on a rise in cytosolic [Ca2+]. We predict that the unique, episodic coupled gating behaviour of TPC2 plays an important role in enabling TPC2 to provide a flexible Ca2+-release system capable of wide variations in the magnitude and time-dependence of Ca2+ fluxes from lysosomal stores.Supported by the BHF and Wellcome Trust.