Ca2+ mobilization from intracellular stores represents an important cell signalling process1 that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP3), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cyclic ADP ribose cause the release of Ca2+ from sarcoplasmic/endoplasmic reticulum stores by the activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). In contrast, the nature of the intracellular stores targeted by NAADP and the molecular identity of the NAADP receptors remain controversial1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with human TPC1 (also known as TPCN1) and chicken TPC3 (TPCN3) being expressed on endosomal membranes, and human TPC2 (TPCN2) on lysosomal membranes when expressed in HEK293 cells. Membranes enriched with TPC2 show high affinity NAADP binding, and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release by InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but were only attenuated by depleting endoplasmic reticulum Ca2+ stores or by blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger further Ca2+ signals via sarcoplasmic/endoplasmic reticulum. TPCs therefore provide new insights into the regulation and organization of Ca2+ signals in animal cells, and will advance our understanding of the physiological role of NAADP.
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This work was supported by grants from the UK Wellcome Trust and the British Heart Foundation to A.G., J.P. and A.M.E., the US National Institutes of Health to M.X.Z. and J.M., and the American Heart Association to M.X.Z. A.G. was a Wellcome Senior Research Fellow in basic Biomedical Science. The work of A.M.E. was funded by a Wellcome Trust Project Grant (CNW, reference number 070772) and a British Heart Foundation Studentship (PJC, reference number FS/05/050). Part of the work of M.X.Z. was made possible by US National Institutes of Health grant P30-NS045758. We thank T. Kong and W. Li for the HepG2 cell line, O. Ogunbayo for technical assistance, and F. Platt, A. Morgan and M. Viapiano for help with the manuscript.
Author Contributions J.T., R.X. and M.X.Z. cloned TPC1, TPC2 and TPC3. J.T., Y.L., C.W., X.H. and M.X.Z. produced stable cell lines and performed immunostaining and confocal microscopy. X.C., M.R., J.P. and A.G. performed radioligand binding. A.A. and A.G. performed flash photolysis experiments. P.J.C., C.N.W. and A.M.E. performed NAADP dialysis, Ca2+ imaging, shRNA knockdown, western blotting and associated immunocytochemistry and deconvolution microscopy. M.R., K.R., J.P. and A.G. produced and characterized the TPC2 antibody and performed immunostaining and confocal microscopy. K.R. and Y.Z. performed qRT–PCR, Z.P., P.L. and J.M. produced shRNA constructs and studied the effect of NAADP on HepG2 cells. M.R., A.A., L.T., K.-T.C., J.P. and A.G. produced and characterized the Tpc2 knockout mice and carried out β-cell studies. M.X.Z, J.M., A.M.E., J.P. and A.G. wrote the manuscript. All authors discussed the results and commented on the manuscript.
This movie shows a 3D reconstruction of confocal images of HEK293 cells stably expressing HA-tagged humanTPC2 (see file s1 for full Legend).
This movie shows localized Ca2+ wave elicited by NAADP in TPC1-overexpressing cells (see file s1 for full Legend).
This movie shows active movement of TPC1- or TPC2-containing intracellular vesicles (see file s1 for full Legend).
About this article
Phosphatidylinositol-3,5-bisphosphate lipid-binding-induced activation of the human two-pore channel 2
Cellular and Molecular Life Sciences (2018)