TPC2 Is a Novel NAADP-sensitive Ca2+ Release Channel, Operating as a Dual Sensor of Luminal pH and Ca2+

Abstract

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca2+ required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca2+ from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca2+ release channels. Controversial evidence also proposes ryanodine receptors as the primary target of NAADP. We show that TPC2, the major lysosomal targeted isoform, is a cation channel with selectivity for Ca2+ that will enable it to act as a Ca2+ release channel in the cellular environment. NAADP opens TPC2 channels in a concentration-dependent manner, binding to high affinity activation and low affinity inhibition sites. At the core of this process is the luminal environment of the channel. The sensitivity of TPC2 to NAADP is steeply dependent on the luminal [Ca2+] allowing extremely low levels of NAADP to open the channel. In parallel, luminal pH controls NAADP affinity for TPC2 by switching from reversible activation of TPC2 at low pH to irreversible activation at neutral pH. Further evidence earmarking TPCs as the likely pathway for NAADP-induced intracellular Ca2+ release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca2+ release. Ned-19 antagonizes NAADP-activation of TPC2 in a non-competitive manner at 1 μm but potentiates NAADP activation at nanomolar concentrations. This single-channel study provides a long awaited molecular basis for the peculiar mechanistic features of NAADP signaling and a framework for understanding how NAADP can mediate key physiological events.

High susceptibility to fatty liver disease in two-pore channel 2-deficient mice

“…NAADP-dependent currents in WT lysosomes displayed a bell-shaped dose-response relationship (Fig. 1d) as reported previously 25,26 .…”

“…Independent of what the nature of the endogenous ligand of these channels might be, there is substantial evidence that on activation TPCs mediate the release of Ca 2 þ from lysosomal stores. Patch-clamp 21,22,24 , lipid bilayer and calcium imaging experiments 18,25,26 indicate that TPCs are Ca 2 þ permeable channels and, hence, may directly confer Ca 2 þ release from endosomes/lysosomes.…”

Organelle-specific Subunit Interactions of the Vertebrate Two-pore Channel Family

“…The fact that NAADP-dependent Ca 2ϩ signaling is enhanced by the expression of hTPC1, hTPC2, and rTPC3, respectively, provides further support for the view that TPCs are of fundamental importance to this process (11), irrespective of the controversy surrounding their ability to conduct Ca 2ϩ (9,10,13). That hTPC1 was entirely restricted to different subpopulations of endosomes, hTPC2 to lysosomes, and rTPC3 to both endosomes and lysosomes provided us with the opportunity to further assess the capacity for organellar coupling to the ER by CICR.…”

Lysosomes Integrate Metabolic-Inflammatory Cross-talk in Primary Macrophage Inflammasome Activation

“…Consistent with this notion, IL-1␤ release was reduced significantly by 2-APB, U18666A, and NED-19, all of which disrupt the release or handling of intracellular calcium stores. U18666A and NED-19 specifically modulate acidic calcium stores (23,24). Similar to the lysosomal acidification inhibitors, these calcium-modulating compounds reduced the level of IL-1␤ mRNA.…”

“…8F). To further investigate the role of lysosomal calcium in this response, we took advantage of the compound NED-19, which selectively inhibits the NAADPactivated calcium channel that resides on the lysosome (24). NED-19-treated pMACs also secreted less IL-1␤ and produced less IL-1␤ mRNA compared with vehicle-treated cells, providing further evidence that lysosomal calcium contributes to lipotoxic inflammasome activation (Fig.…”