Ca2+ mobilization from intracellular stores represents an important cell signaling process 1 which is regulated, in mammalian cells, by inositol 1,4,5-trisphosphate (InsP3), cyclic ADP ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP). InsP3 and cADPR release Ca2+ from sarco / endoplasmic reticulum (S/ER) stores through activation of InsP3 and ryanodine receptors (InsP3Rs and RyRs). By contrast, the nature of the intracellular stores targeted by NAADP and molecular identity of the NAADP receptors remain controversial 1,2, although evidence indicates that NAADP mobilizes Ca2+ from lysosome-related acidic compartments 3,4. Here we show that two-pore channels (TPCs) comprise a family of NAADP receptors, with TPC1 and TPC3 being expressed on endosomal and TPC2 on lysosomal membranes. Membranes enriched with TPC2 exhibit high affinity NAADP binding and TPC2 underpins NAADP-induced Ca2+ release from lysosome-related stores that is subsequently amplified by Ca2+-induced Ca2+ release via InsP3Rs. Responses to NAADP were abolished by disrupting the lysosomal proton gradient and by ablating TPC2 expression, but only attenuated by depleting ER Ca2+ stores or blocking InsP3Rs. Thus, TPCs form NAADP receptors that release Ca2+ from acidic organelles, which can trigger additional Ca2+ signals via S/ER. 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.

Specialized O 2 -sensing cells exhibit a particularly low threshold to regulation by O 2 supply and function to maintain arterial pO 2 within physiological limits. For example, hypoxic pulmonary vasoconstriction optimizes ventilation-perfusion matching in the lung, whereas carotid body excitation elicits corrective cardio-respiratory reflexes. It is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation in O 2 -sensing cells, thereby mediating, in part, cell activation. However, the mechanism by which this process couples to Ca 2؉ signaling mechanisms remains elusive, and investigation of previous hypotheses has generated contrary data and failed to unite the field. We propose that a rise in the cellular AMP/ATP ratio activates AMP-activated protein kinase and thereby evokes Ca 2؉ signals in O 2 -sensing cells. Co-immunoprecipitation identified three possible AMP-activated protein kinase subunit isoform combinations in pulmonary arterial myocytes, with ␣1␤2␥1 predominant. Furthermore, their tissue-specific distribution suggested that the AMP-activated protein kinase-␣1 catalytic isoform may contribute, via amplification of the metabolic signal, to the pulmonary selectivity required for hypoxic pulmonary vasoconstriction. Immunocytochemistry showed AMPactivated protein kinase-␣1 to be located throughout the cytoplasm of pulmonary arterial myocytes. In contrast, it was targeted to the plasma membrane in carotid body glomus cells. Consistent with these observations and the effects of hypoxia, stimulation of AMPactivated protein kinase by phenformin or 5-aminoimidazole-4-carboxamide-riboside elicited discrete Ca 2؉ signaling mechanisms in each cell type, namely cyclic ADP-ribose-dependent Ca 2؉ mobilization from the sarcoplasmic reticulum via ryanodine receptors in pulmonary arterial myocytes and transmembrane Ca 2؉ influx into carotid body glomus cells. Thus, metabolic sensing by AMP-activated protein kinase may mediate chemotransduction by hypoxia.Specialized O 2 -sensing cells within the body have evolved as vital homeostatic mechanisms that monitor O 2 supply and alter respiratory and circulatory function, as well as the capacity of the blood to transport O 2 . By these means, arterial pO 2 is maintained within physiological limits. Two key systems involved are the pulmonary arteries and the carotid body. Constriction of pulmonary arteries by hypoxia optimizes ventilation-perfusion matching in the lung (1), whereas carotid body excitation by hypoxia initiates corrective changes in breathing patterns via increased sensory afferent discharge to the brain stem (2). Although O 2 -sensitive mechanisms independent of mitochondria may also play a role (3-5), it is generally accepted that relatively mild hypoxia inhibits mitochondrial oxidative phosphorylation and that this underpins, at least in part, cell activation (2, 6 -10). Despite this consensus, the mechanism by which inhibition of mitochondrial oxidative phosphorylation couples to discrete cell-specific Ca 2ϩ signaling ...

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SummaryIn arterial myocytes the Ca 2+ mobilizing messenger NAADP evokes spatially restricted Ca 2+ bursts from a lysosome-related store that are subsequently amplified into global Ca 2+ waves by Ca 2+ -induced Ca 2+ -release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) αlgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5 μm of the nucleus) was ~4-fold greater than in the sub-plasmalemmal (within 1.5 μm of the plasma membrane) and ~2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was ~4-and ~14-fold greater than that in the extraperinuclear and sub-plasmalemmal regions, and ~2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with αlgp120 labelling within the perinuclear region was ~2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between αlgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30μM) abolished global Ca 2+ waves but not Ca 2+ bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca 2+ signalling by NAADP.