NAADP mobilizes calcium from acidic organelles through two-pore channels

Abstract

Ca(2+) mobilization from intracellular stores represents an important cell signalling process that is regulated, in mammalian cells, by inositol-1,4,5-trisphosphate (InsP(3)), cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP(3) and cyclic ADP ribose cause the release of Ca(2+) from sarcoplasmic/endoplasmic reticulum stores by the activation of InsP(3) and ryanodine receptors (InsP(3)Rs and RyRs). In contrast, the nature of the intracellular stores targeted by NAADP and the molecular identity of the NAADP receptors remain controversial, although evidence indicates that NAADP mobilizes Ca(2+) from lysosome-related acidic compartments. 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 Ca(2+) release from lysosome-related stores that is subsequently amplified by Ca(2+)-induced Ca(2+) release by InsP(3)Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but were only attenuated by depleting endoplasmic reticulum Ca(2+) stores or by blocking InsP(3)Rs. Thus, TPCs form NAADP receptors that release Ca(2+) from acidic organelles, which can trigger further Ca(2+) signals via sarcoplasmic/endoplasmic reticulum. TPCs therefore provide new insights into the regulation and organization of Ca(2+) signals in animal cells, and will advance our understanding of the physiological role of NAADP.

Figure 1. Tissue and subcellular expression of mammalian TPC2

a, Hydropathy plot of human TPC2. Window size = 9 a.a. b, Evolutionary relationships of TPCs with single-pore and four-pore domain channels. h, human; d, dog; r, rat; at, Arabidopsis thaliana. c, Northern blot analysis of TPC2 expression in human tissues. d, Colocalization of endogenous TPC2 with LAMP-2 in untransfected HEK293 cells. e and f, Colocalization of HA-hTPC2 with LAMP-2 (e) and LysoTracker (f) in hTPC2 stable cell line. Right panel in f shows stacked image of overall LysoTracker accumulation relative to expressed HA-hTPC2 (3D projections, supplementary Movie 1). Scale bars = 10 μm.

Figure 2. [³²P]NAADP binding to TPC2

a, Specific binding for membranes from wild type HEK293 and hTPC2 cells (n = 3). b, Depletion of hTPC2 by immunoprecipitation abolished [³²P]NAADP binding. The supernatant was depleted of hTPC2 (i) and [³²P]NAADP binding (ii) by anti-HA but not by rat IgG (control) antibody; hTPC2 (i) and [³²P]NAADP binding (iii) appeared in the pellet with anti-HA only. c and d, Representative ligand competition assay for membranes prepared from hTPC2 cells (c) and mouse liver (d). The maximal specific binding for hTPC2 membranes ranged from 167.6 to 300 dpm and for mouse liver from 1,000 to 1,600 dpm.

Figure 3. TPC2 expression and NAADP-evoked Ca²⁺ signaling

a-c, Effect of photoreleased NAADP on Fluo3 fluorescence in hTPC2 (a) and wild type HEK293 cells (b). c, Means ± SEM of peak response. H: heparin, R: ryanodine. * p < 0.05 different from hTPC2 only. d-f, Effect of intracellular dialysis of NAADP on Fura2 ratio in hTPC2 (d) and wild type (e, the cell within dashed lines) cells: upper panels pseudocolour images, lower panels ratio against time. Time scale bars = 20 s. f, Means ± SEM (except n = 2) of peak response. * p < 0.05 different from hTPC2, 10 nM NAADP. Baf: bafilomycin, scrbld: scrambled. Number of cells in parentheses.

Figure 4. Pancreatic β cells from TPC2 knockout mice are NAADP insensitive

a, Approximate position of the gene trap vector in Tpcn2 gene. b, PCR analysis of genomic DNA. +/+, wild type; +/-, heterozygote; -/-, homozygote. c, RT-PCR products from wild type and mutant Tpcn2 mRNAs with approximate positions of amplicons indicated by red bars; numbers indicate exons (see supplementary information for details). d and e, Cation currents at -70 mV evoked by intracellular dialysis of 100 nM NAADP in pancreatic β-cells isolated from wild type (d) and TPC2 knockout (e) mice. Left, representative traces from single cells; right, overlaid traces for 5 cells.