Two-pore channels (TPCs) are endolysosomal ion channels implicated in Ca2+ signalling from acidic organelles. The relevance of these ubiquitous proteins for human disease, however, is unclear. Here, we report that lysosomes are enlarged and aggregated in fibroblasts from Parkinson disease patients with the common G2019S mutation in LRRK2. Defects were corrected by molecular silencing of TPC2, pharmacological inhibition of TPC regulators [Rab7, NAADP and PtdIns(3,5)P2] and buffering local Ca2+ increases. NAADP-evoked Ca2+ signals were exaggerated in diseased cells. TPC2 is thus a potential drug target within a pathogenic LRRK2 cascade that disrupts Ca2+-dependent trafficking in Parkinson disease.

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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Background: Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates calcium release from internal acidic stores via two-pore channels (TPCs).Results: A novel photosensitive probe (5-azido-NAADP) identified high affinity NAADP binding sites that interact with, but are distinct from, TPCs.Conclusion: High affinity NAADP-binding proteins complex with TPCs.Significance: This work provides new mechanistic insights into how NAADP regulates calcium release via TPCs.

Background: Calcium homeostasis endoplasmic reticulum protein (CHERP) was originally identified as an integral endoplasmic reticulum membrane protein that regulates intracellular Ca 2ϩ channels. Results: In contrast, we show CHERP binds SR140 in nuclear subdomains. Conclusion: CHERP regulates Ca 2ϩ homeostasis indirectly, not as a binding partner of ryanodine receptors or IP 3 receptors. Significance: These data support an entirely new model for the cellular role of CHERP.