The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores - PubMed (original) (raw)

The two-pore channel TPCN2 mediates NAADP-dependent Ca(2+)-release from lysosomal stores

Xiangang Zong et al. Pflugers Arch. 2009 Sep.

Abstract

Second messenger-induced Ca(2+)-release from intracellular stores plays a key role in a multitude of physiological processes. In addition to 1,4,5-inositol trisphosphate (IP(3)), Ca(2+), and cyclic ADP ribose (cADPR) that trigger Ca(2+)-release from the endoplasmatic reticulum (ER), nicotinic acid adenine dinucleotide phosphate (NAADP) has been identified as a cellular metabolite that mediates Ca(2+)-release from lysosomal stores. While NAADP-induced Ca(2+)-release has been found in many tissues and cell types, the molecular identity of the channel(s) conferring this release remained elusive so far. Here, we show that TPCN2, a novel member of the two-pore cation channel family, displays the basic properties of native NAADP-dependent Ca(2+)-release channels. TPCN2 transcripts are widely expressed in the body and encode a lysosomal protein forming homomers. TPCN2 mediates intracellular Ca(2+)-release after activation with low-nanomolar concentrations of NAADP while it is desensitized by micromolar concentrations of this second messenger and is insensitive to the NAADP analog nicotinamide adenine dinucleotide phosphate (NADP). Furthermore, TPCN2-mediated Ca(2+)-release is almost completely abolished when the capacity of lysosomes for storing Ca(2+) is pharmacologically blocked. By contrast, TPCN2-specific Ca(2+)-release is unaffected by emptying ER-based Ca(2+) stores. In conclusion, these findings indicate that TPCN2 is a major component of the long-sought lysosomal NAADP-dependent Ca(2+)-release channel.

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Figures

Fig. 1

Fig. 1

Properties of two-pore channels. aUpper panel Transmembrane topology of TPCN1 and TPCN2. The predicted N-glycosylation sites are marked by red forks. Lower panel Schematic representation of the primary sequence of TPCN1 and TPCN2. The degree of sequence identity within the N- and C-termini, the two transmembrane building blocks and the interdomain linker is indicated. b Mouse multiple tissue northern blots of TPCN1 (left panel) and TPCN2 (right panel) demonstrate expression in all tissues investigated. c EGFP-TPCN1 (upper panel) and EGFP-TPCN2 (lower panel) channels expressed in HEK293 cells are localized intracellularly (green TPCN channels; red membrane marker, blue nuclei; scale bar 5 µm). d Western blots with lysates from HEK293 cells containing myc-tagged TPCN1 (left panel), wild-type TPCN2 (middle) panel, and a glycosylation-deficient TPCN2 double-mutant (TPCN-2Q; right panel). Ten micrograms of protein were applied per lane. e Lysates of HEK293 cells cotransfected with myc-tagged TPCN2 and EGFP-tagged TPCN2 (lanes 1–3) or myc-tagged TPCN1 and EGFP-tagged TPCN2 (lanes 6–8) were immunoprecipitated with anti-myc antibody (lanes 2, 5, 7) or anti-GST (control, lanes 3 and 8), blotted and probed with anti-GFP. Lanes 1, 6 input, lane 4 negative control

Fig. 2

Fig. 2

TPCN2 is localized in the lysosomes. Immunocytochemistry of TPCN2 in COS-7 cells. a Strong TPCN2 staining (green) is observed in intracellular compartments. By contrast, TPCN2 is absent from the plasma membrane (visualized in red by a specific maker). b After permeabilization, HA-tagged TPCN2 is detected intracellularily. c HA-tagged TPCN2 is not detected in the membrane of non permeabilized cells. d–f Colocalization of TPCN2 (green) and the ER marker protein calnexin (red). Most TPCN2-positive structures (d) costaine for endoplasmic reticulum (e), yielding yellow in the overlay (f). g–i Colocalization of TPCN2 (green) and lamp1 (red). Most TPCN2-positive structures (g) costained for lysosomes (h), yielding yellow in the overlay (i). (bars 10 µm)

Fig. 3

Fig. 3

NAADP triggers Ca2+-release in HEK293 cells transfected with TPCN2. Application of 30 nM NAADP via the patch pipette induces Ca2+-release from internal stores in cells transfected with TPCN2-EGFP (a) but not in cells transfected with EGFP alone (b, upper panel) or cells transfected with TPCN1-EGFP (b, lower panel). In cells transfected with TPCN2-EGFP, NADP (30 nM; negative control) did not induce Ca2+-release (b, middle panel). The arrows in a, b indicate the start of cell perfusion. c Population data for experiments performed in (a, b). d Dose–response relationship of NAADP. All values are given as mean ± SEM. Number of cells measured is indicated in brackets in (c) and (d)

Fig. 4

Fig. 4

TPCN2 mediates NAADP-dependent Ca2+-release from lysosomes. a In HEK293 cells transfected with TPCN2-EGFP, preincubation (45 min) with 100 nM bafilomycin almost completely abolished NAADP (30 nM)-induced Ca2+-release (black line). Red line control without pretreatment. b In HEK293 cells transfected with TPCN2-EGFP, preincubation (15 min) with 1 µM thapsigargin did not reduce NAADP-sensitive Ca2+-release (black line). Red line same control as in (a). c Effect of thapsigargin (1 µM) on the IP3 (3 µM)-induced Ca2+-release. Experiments were performed either with (black line) or without (red line) thapsigargin preincubation (1 µM; 15 min). Arrows in (a–c) indicate the start of cell perfusion. d Population data for experiments shown in (a–c). Number of cells measured is indicated in brackets. Tg thapsigargin; Baf bafilomycin; IP3 Inositol-1,4,5-trisphosphate. Fluorescent ratio were normalized for better comparison

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References

    1. {'text': '', 'ref_index': 1, 'ids': [{'type': 'DOI', 'value': '10.1096/fj.05-5538fje', 'is_inner': False, 'url': 'https://doi.org/10.1096/fj.05-5538fje'}, {'type': 'PubMed', 'value': '16585058', 'is_inner': True, 'url': 'https://pubmed.ncbi.nlm.nih.gov/16585058/'}\]}
    2. Beck A, Kolisek M, Bagley LA, Fleig A, Penner R (2006) Nicotinic acid adenine dinucleotide phosphate and cyclic ADP-ribose regulate TRPM2 channels in T lymphocytes. Faseb J 20:962–964 - PubMed
    1. {'text': '', 'ref_index': 1, 'ids': [{'type': 'DOI', 'value': '10.1074/jbc.M203224200', 'is_inner': False, 'url': 'https://doi.org/10.1074/jbc.m203224200'}, {'type': 'PubMed', 'value': '12223470', 'is_inner': True, 'url': 'https://pubmed.ncbi.nlm.nih.gov/12223470/'}\]}
    2. Berridge G, Dickinson G, Parrington J, Galione A, Patel S (2002) Solubilization of receptors for the novel Ca2+-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate. J Biol Chem 277:43717–43723 - PubMed
    1. {'text': '', 'ref_index': 1, 'ids': [{'type': 'DOI', 'value': '10.1038/35036035', 'is_inner': False, 'url': 'https://doi.org/10.1038/35036035'}, {'type': 'PubMed', 'value': '11413485', 'is_inner': True, 'url': 'https://pubmed.ncbi.nlm.nih.gov/11413485/'}\]}
    2. Berridge MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11–21 - PubMed
    1. {'text': '', 'ref_index': 1, 'ids': [{'type': 'DOI', 'value': '10.1161/01.RES.0000047507.22487.85', 'is_inner': False, 'url': 'https://doi.org/10.1161/01.res.0000047507.22487.85'}, {'type': 'PubMed', 'value': '12480818', 'is_inner': True, 'url': 'https://pubmed.ncbi.nlm.nih.gov/12480818/'}\]}
    2. Boittin FX, Galione A, Evans AM (2002) Nicotinic acid adenine dinucleotide phosphate mediates Ca2+ signals and contraction in arterial smooth muscle via a two-pool mechanism. Circ Res 91:1168–1175 - PubMed
    1. {'text': '', 'ref_index': 1, 'ids': [{'type': 'DOI', 'value': '10.1073/pnas.85.21.7972', 'is_inner': False, 'url': 'https://doi.org/10.1073/pnas.85.21.7972'}, {'type': 'PMC', 'value': 'PMC282335', 'is_inner': False, 'url': 'https://pmc.ncbi.nlm.nih.gov/articles/PMC282335/'}, {'type': 'PubMed', 'value': '2973058', 'is_inner': True, 'url': 'https://pubmed.ncbi.nlm.nih.gov/2973058/'}\]}
    2. Bowman EJ, Siebers A, Altendorf K (1988) Bafilomycins: a class of inhibitors of membrane ATPases from microorganisms, animal cells, and plant cells. Proc Natl Acad Sci U S A 85:7972–7976 - PMC - PubMed

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