Nicotinic acid adenine dinucleotide phosphate (NAADP) is a widespread and potent calcium-mobilizing messenger that is highly unusual in activating calcium channels located on acidic stores. However, the molecular identity of the target protein is unclear. In this study, we show that the previously uncharacterized human two-pore channels (TPC1 and TPC2) are endolysosomal proteins, that NAADP-mediated calcium signals are enhanced by overexpression of TPC1 and attenuated after knockdown of TPC1, and that mutation of a single highly conserved residue within a putative pore region abrogated calcium release by NAADP. Thus, TPC1 is critical for NAADP action and is likely the long sought after target channel for NAADP.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a ubiquitous messenger proposed to stimulate Ca2+ release from acidic organelles via two-pore channels (TPCs). It has been difficult to resolve this trigger event from its amplification via endoplasmic reticulum Ca2+ stores, fuelling speculation that archetypal intracellular Ca2+ channels are the primary targets of NAADP. Here, we redirect TPC2 from lysosomes to the plasma membrane and show that NAADP evokes Ca2+ influx independent of ryanodine receptors and that it activates a Ca2+-permeable channel whose conductance is reduced by mutation of a residue within a putative pore. We therefore uncouple TPC2 from amplification pathways and prove that it is a pore-forming subunit of an NAADP-gated Ca2+ channel.

A multistage therapeutic community treatment system has been instituted in the Delaware correctional system, and its effectiveness has captured the attention of the National Institutes of Health, the Department of Justice, members ofCongress, and the White House. Treatment occurs in a three-stage system, with each phase corresponding to the client's changing correctional status-incarceration, work release, and parole. In this paper, 18 month follow-up data are analyzed for those who receivedtreatment in: (/) a prison-based therapeutic community only, (2) a work release therapeutic communityfollowed by aftercare, and (3) the prison-based therapeutic community followed by the work release therapeutic community and aftercare. These groups are compared with a no-treatment group. Those receiving treatment in the two-stage (work release and aftercare) and three-stage (prison, work release, and aftercare) models had significantly lower rates of drug relapse and criminal recidivism, even when adjustedfor other risk factors. The results support the effectivenessofa multistage therapeutic community model for drug-involved offenders, and the importance of a work release transitional therapeutic community as a component ofthis model. is an AssociateScientist at the Center for Drug and AlcoholStudies and coprincipal investigatoron the "Ongoing Studies" project. He has authored or co-authored more than 40 articleson the epidemiology, etiology,prevention, and treatment ofsubQance abuse. COtTord A. Butzin, Ph.D., is also an Associate Scientist at the Center for Drug and Alcohol Studies. He was previousiyChiefof the ResearchConsulting Divisionat WMordHall MedicalCenter in San Antonio, and before that he was on the faculty at Duke University. Robert M. Hooper, Ph.D., Executive Directorof Substance Abuse Treatment, CorrectionalMedicalSystems,has been involved in justice programming nationallyfor the paQ 2.5 years, Over the laQ 8 )'\larshe has developedintensive abuse tJeatment programs withthe Delaware model receivingnational attention.

The two-pore channels (TPC1 and TPC2) belong to an ancient family of intracellular ion channels expressed in the endolysosomal system. Little is known about how regulatory inputs converge to modulate TPC activity, and proposed activation mechanisms are controversial. Here, we compiled a proteomic characterization of the human TPC interactome, which revealed that TPCs complex with many proteins involved in Ca 2+ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoformspecific manner. TPC2, but not TPC1, caused a proliferation of endolysosomal structures, dysregulating intracellular trafficking, and cellular pigmentation. These outcomes required both TPC2 and Rab activity, as well as their interactivity, because TPC2 mutants that were inactive, or rerouted away from their endogenous expression locale, or deficient in Rab binding, failed to replicate these outcomes. Nicotinic acid adenine dinucleotide phosphate (NAADP)-evoked Ca 2+ release was also impaired using either a Rab bindingdefective TPC2 mutant or a Rab inhibitor. These data suggest a fundamental role for the ancient TPC complex in trafficking that holds relevance for lysosomal proliferative scenarios observed in disease. Ca 2+ signaling | lysosome | Xenopus T wo-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca 2+ and Na + channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca 2+ /Na + channel with cytosolic NH 2 and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membranespanning regions. Since their discovery in vertebrate systems, many studies have investigated the properties of these channels (2-7) that may support such a lengthy evolutionary pedigree. In this context, demonstration that (i) the two human TPC isoforms (TPC1 and TPC2) are uniquely distributed within the endolysosomal system (2, 3) and that (ii) TPC channel activity is activated by the Ca 2+ mobilizing molecule nicotinic acid adenine dinucleotide phosphate (NAADP) (4-6) generated considerable excitement that TPCs function as effectors of this mercurial second messenger long known to trigger Ca 2+ release from "acidic stores." The spectrum of physiological activities that have been linked to NAADP signaling over the last 25 years (8, 9) may therefore be realized through regulation of TPC activity. However, recent studies have questioned the idea that TPCs are NAADP targets (10, 11), demonstrating instead that TPCs act as Na + channels regulated by the endolysosomal phosphoinositide PI(3,5)P 2 . Such controversy (12, 13) underscores how little we know about TPC regulatory inputs and the dynamic composition of TPC complexes within cells.Here, to generate unbiased insight into the cell biology of the TPC complex, we report a proteomic analysis of human TPCs. The TPC interactom...

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Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent and widespread calcium-mobilizing messenger, the properties of which have been most extensively described in sea urchin eggs. The molecular basis for calcium release by NAADP, however, is not clear and subject to controversy. Recent studies have provided evidence that members of the two-pore channel (TPC) family in mammals are the long sought after target channels for NAADP. Here, we show that the TPC3 gene, which has yet to be functionally characterized, is present throughout the deuterostome lineage but is a pseudogene in humans and other primates. We report the molecular cloning of the complete ancestral TPC gene family from the sea urchin and demonstrate that all three isoforms localize to acidic organelles to mediate NAADP-dependent calcium release. Our data highlight the functional divergence of this novel gene family during deuterostome evolution and provide further evidence that NAADP mediates calcium release from acidic stores through activation of TPCs.

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