PMC

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)

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)

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

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