Peter J. Calcraft1, Abdelilah Arredouani2, Zui Pan3, Xiaotong Cheng2, Jisen Tang4, Margarida Ruas2, Katja Rietdorf2, Peihui Lin3, John Parrington2, Jianjie Ma3, A. Mark Evans1, Antony Galione2, Michael X. Zhu4. 1University of Edinburgh, Edinburgh, United Kingdom, 2University of Oxford, Oxford, United Kingdom, 3UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ, USA, 4The Ohio State University, Columbus, OH, USA. Two-pore channels (TPCs) are novel members of the superfamily of voltagegated ion channels. Their predicted structures indicate 2-fold symmetry with a total of 12 putative transmembrane (TM) a-helices. Sequence homology and membrane topology analyses suggest that TPCs may represent evolutionary intermediates from the single domain 6-TM architecture Kþ and non-selective cation channels to the four-repeat 24-TM structure of voltage-gated Ca2þ and Naþ channels. Three genes (TPCN1-3) exist in most vertebrates but their functions remain elusive. We now show that TPC1 and TPC3 are expressed on the membrane of different endosome populations while TPC2 is expressed on the membrane of lysosomes. We provide functional data showing that TPC2 is a target of nicotinic acid adenine dinucleotide phosphate (NAADP), a potent Ca2þ mobilizing messenger that evokes Ca2þ release from acidic organelles rather than from the sarco/endoplasmic reticulum. Thus, microsomal membranes enriched with TPC2 exhibit similar high affinity binding with NAADP as native NAADP receptors. In response to NAADP, cells overexpressing TPC2 exhibit enhanced intracellular Ca2þ release and more efficient coupling to IP3 receptors to evoke global Ca2þ transients. These effects were blocked by disrupting lysosomal Hþ gradient or RNAi-mediated silencing of TPC2 expression. Our findings provide for the first time a molecular basis for further detailed characterization of the regulatory mechanisms and physiological functions of NAADP-mediated signaling and, in addition, suggest a general role for TPCs in Ca2þ mobilization, Ca2þ homeostasis, and Ca2þ signaling from endosomal/lysosomal compartments of vertebrate cells, which are known to be important for diverse functions in many physiological systems.