TPC2 is a novel NAADP-sensitive Ca2+ release channel, operating as a dual sensor of luminal pH and Ca2+.

School of Physiology and Pharmacology, Medical Sciences Building, and Center for Nanoscience and Quantum Information, University of Bristol, Bristol BS8 1TD, United Kingdom.
Journal of Biological Chemistry (Impact Factor: 4.6). 11/2010; 285(45):35039-46. DOI: 10.1074/jbc.M110.156927
Source: PubMed

ABSTRACT Nicotinic acid adenine dinucleotide phosphate (NAADP) is a molecule capable of initiating the release of intracellular Ca(2+) required for many essential cellular processes. Recent evidence links two-pore channels (TPCs) with NAADP-induced release of Ca(2+) from lysosome-like acidic organelles; however, there has been no direct demonstration that TPCs can act as NAADP-sensitive Ca(2+) 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 Ca(2+) that will enable it to act as a Ca(2+) 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 [Ca(2+)] 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 Ca(2+) release is obtained from the use of Ned-19, the selective blocker of cellular NAADP-induced Ca(2+) 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.

  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Organelle ion homeostasis within the endo-lysosomal system is critical for physiological functions. TPCs (Two-Pore Channels) are cation channels that reside in endo-lysosomal organelles and over-expression results in endo-lysosomal trafficking defects. However, the impact of lack of TPC expression on endo-lysosomal trafficking is unknown. Here, we characterize Tpcn1 expression in two transgenic mouse lines (Tpcn1(XG716) and Tpcn1(T159)) and show expression of a novel evolutionarily conserved Tpcn1B transcript from an alternative promoter, raising important questions regarding the status of Tpcn1 expression in mice recently described as Tpcn1 knockouts. We show that the transgenic Tpcn1(T159) line lacks expression of both Tpcn1 isoforms in all tissues analysed. Using mouse embryonic fibroblasts (MEFs) from Tpcn1(-/-) and Tpcn2(-/-) animals we show that lack of Tpcn1 or Tpcn2 expression has no significant impact on resting endo-lysosomal pH or morphology. However, differential effects were observed in endo-lysosomal function upon loss of Tpcn1 or Tpcn2 expression; thus, while Tpcn1(-/-) MEFs have impaired trafficking of cholera toxin from the plasma membrane to the Golgi, Tpcn2(-/-) MEFs show slower kinetics of ligand-induced PDGFRβ degradation, which is dependent on trafficking to lysosomes. Our findings indicate that TPC1 and TPC2 have important, but distinct roles in the endo-lysosomal pathway.
    Molecular and Cellular Biology 08/2014; 34(21). DOI:10.1128/MCB.00113-14 · 5.04 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Egg activation at fertilization is an excellent process for studying calcium regulation. Nicotinic acid adenine dinucleotide-phosphate (NAADP), a potent calcium messenger, is able to trigger calcium release, likely through two-pore channels (TPCs). Concomitantly, a family of ectocellular enzymes, the ADP-ribosyl cyclases (ARCs), has emerged as being able to change their enzymatic mode from one of nucleotide cyclization in formation of cADPR to a base-exchange reaction in the generation of NAADP. Using sea star oocytes we gain insights into the functions of endogenously expressed TPCs and ARCs in the context of the global calcium signals at fertilization. Three TPCs and one ARC were found in the sea star (Patiria miniata) that were localized in the cortex of the oocytes and eggs. PmTPCs were localized in specialized secretory organelles called cortical granules, and PmARCs accumulated in a different, unknown, set of vesicles, closely apposed to the cortical granules in the egg cortex. Using morpholino knockdown of PmTPCs and PmARC in the oocytes, we found that both calcium regulators are essential for early embryo development, and that knockdown of PmTPCs leads to aberrant construction of the fertilization envelope at fertilization and changes in cortical granule pH. The calcium signals at fertilization are not significantly altered when individual PmTPCs are silenced, but the timing and shape of the cortical flash and calcium wave are slightly changed when the expression of all three PmTPCs is perturbed concomitantly, suggesting a cooperative activity among TPC isoforms in eliciting calcium signals that may influence localized physiological activities.
    Development 11/2014; DOI:10.1242/dev.113563 · 6.27 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The organellar targeting of Two Pore Channels (TPCs) and their capacity to associate as homo and hetero dimers may be critical to endolysosome signalling. A more detailed understanding of the functional association of vertebrate TPC1, TPC2 and TPC3 is therefore necessary. We report here that, when stably expressed in HEK293 cells, human (h)TPC1 and chicken (c)TPC3 are specifically targeted to different subpopulations of endosomes, human (h)TPC2 to lysosomes, and rabbit (r)TPC3 to both endosomes and lysosomes. Intracellular dialysis of NAADP evoked a Ca2+ transient in HEK293 cells that stably over-expressed hTPC1, hTPC2 and rTPC3, but not in cells that stably expressed cTPC3. The Ca2+ transients induced in cells that over-expressed endosome targeted hTPC1 were abolished upon depletion of acidic Ca2+ stores by bafilomycin A1, but remained unaffected following depletion of endoplasmic reticulum (ER) stores by thapsigargin. By contrast, Ca2+ transients induced via the lysosome targeted hTPC2 and endolysosome targeted rTPC3 were abolished by bafilomycin A1 and markedly attenuated by thapsigargin. NAADP induced marked Ca2+ transients in HEK293 cells that stably co-expressed hTPC2 with hTPC1 or cTPC3, but failed to evoke any such response in cells that co-expressed interacting hTPC2 and rTPC3 subunits. We conclude therefore that: (1) All three TPC subtypes may support Ca2+ signalling from their designate acidic stores; (2) lysosome but not endosome targeted TPCs support coupling to the ER.
    Journal of Biological Chemistry 12/2014; 290(2). DOI:10.1074/jbc.M114.610493 · 4.60 Impact Factor


Available from
May 23, 2014