The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation |
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Authors: | Yaping Lin-Moshier Michael V Keebler Robert Hooper Michael J Boulware Xiaolong Liu Dev Churamani Mary E Abood Timothy F Walseth Eugen Brailoiu Sandip Patel Jonathan S Marchant |
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Institution: | aDepartment of Pharmacology, University of Minnesota, Minneapolis, MN, 55455;;bDepartment of Cell and Developmental Biology, University College London, London WC1E 6BT, United Kingdom; and;cDepartments of Anatomy, Cell Biology, and Pharmacology and the Center for Substance Abuse, Temple University School of Medicine, Philadelphia, PA, 19140 |
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Abstract: | 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 Ca2+ homeostasis, trafficking, and membrane organization. Among these interactors, TPCs were resolved to scaffold Rab GTPases and regulate endomembrane dynamics in an isoform-specific 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 Ca2+ release was also impaired using either a Rab binding-defective 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.Two-pore channels (TPCs) are an ancient family of intracellular ion channels and a likely ancestral stepping stone in the evolution of voltage-gated Ca2+ and Na+ channels (1). Architecturally, TPCs resemble a halved voltage-gated Ca2+/Na+ channel with cytosolic NH2 and COOH termini, comprising two repeats of six transmembrane spanning helices with a putative pore-forming domain between the fifth and sixth membrane-spanning 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 Ca2+ 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 Ca2+ 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)P2. 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 interactome establishes a useful community resource as a “rosetta stone” for interrogating the cell biology of TPCs and their regulation. The dataset reveals a predomination of links between TPCs and effectors controlling membrane organization and trafficking, relevant for disease states involving lysosomal proliferation where TPC functionality may be altered (14). |
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Keywords: | Ca2+ signaling lysosome Xenopus |
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