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.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a messenger that regulates calcium release from intracellular acidic stores. Although several channels, including two-pore channels (TPC), ryanodine receptor (RYR) and mucolipin (TRP-ML1) have been implicated in NAADP regulation of calcium signaling, the NAADP receptor has not been identified. In this study, the photoaffinity probe, [32P]–5-azido-NAADP ([32P]–5-N3-NAADP), was used to study NAADP binding proteins in extracts from NAADP responsive Jurkat T-lymphocytes. [32P]–5-N3-NAADP photolabeling of Jurkat S100 cytosolic fractions resulted in the labeling of at least ten distinct proteins. Several of these S100 proteins, including a doublet at 22/23 kDa and small protein at 15 kDa displayed selectivity for NAADP as the labeling was protected by inclusion of unlabeled NAADP, whereas the structurally similar NADP required much higher concentrations for protection. Interestingly, the labeling of several S100 proteins (60, 45, 33 and 28 kDa) was stimulated by low concentrations of unlabeled NAADP, but not by NADP. The effect of NAADP on the labeling of the 60 kDa protein was biphasic, peaking at 100 nM with a five-fold increase and displaying no change at 1 µM NAADP. Several proteins were also photolabeled when the P100 membrane fraction from Jurkat cells was examined. Similar to the results with S100, a 22/23 kDa doublet and a 15 kDa protein appeared to be selectively labeled. NAADP did not increase the labeling of any P100 proteins as it did in the S100 fraction. The photolabeled S100 and P100 proteins were successfully resolved by two-dimensional gel electrophoresis. [32P]–5-N3-NAADP photolabeling and two-dimensional electrophoresis should represent a suitable strategy in which to identify and characterize NAADP binding proteins.

NAADP is a potent Ca2+ mobilizing messenger in a variety of cells but its molecular mechanism of action is incompletely understood. Accumulating evidence indicates that the poorly characterized two-pore channels (TPCs) in animals are NAADP sensitive Ca2+-permeable channels. TPCs localize to the endo-lysosomal system but are functionally coupled to the better characterized endoplasmic reticulum Ca2+ channels to generate physiologically relevant complex Ca2+ signals. Whether TPCs directly bind NAADP is not clear. Here we discuss the idea based on recent studies that TPCs are the pore-forming subunits of a protein complex that includes tightly associated, low molecular weight NAADP-binding proteins.

Mammalian cells obtain vitamin B1 (thiamin) from their surrounding environment and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. Most of TPP is then transported into the mitochondria via a carrier-mediated process that involves the mitochondrial thiamin pyrophosphate transporter (MTPPT). Knowledge about the physiological parameters of the MTPP-mediated uptake process, MTPPT targeting and the impact of clinical mutations in MTPPT in patients with Amish lethal microcephaly and neuropathy and bilateral striatal necrosis are not fully elucidated, and thus, were addressed in this study using custom-made 3H-TPP as a substrate and mitochondria isolated from mouse liver and human-derived liver HepG2 cells. Results showed 3H-TPP uptake by mouse liver mitochondria to be pH-independent, saturable (Km = 6.79±0.53 µM), and specific for TPP. MTPPT protein was expressed in mouse liver and HepG2 cells, and confocal images showed a human (h)MTPPT-GFP construct to be targeted to mitochondria of HepG2 cells. A serial truncation analysis revealed that all three modules of hMTPPT protein cooperated (although at different levels of efficiency) in mitochondrial targeting rather than acting autonomously as independent targeting module. Finally, the hMTPPT clinical mutants (G125S and G177A) showed proper mitochondrial targeting but displayed significant inhibition in 3H-TPP uptake and a decrease in level of expression of the MTPPT protein. These findings advance our knowledge of the physiology and cell biology of the mitochondrial TPP uptake process. The results also show that clinical mutations in the hMTPPT system impair its functionality via affecting its level of expression with no effect on its targeting to mitochondria.