Increases in Ca2+ resulting from activation of intracellular Ca2+ channels regulate many physiological events. Dysfunction of these Ca2+ channels is involved in neurodegenerative and neurological disease, as well as exocrine, cardiac abnormalities and cancer. To appreciate how cell functions are controlled by Ca2+ signals, and how pathological cues subvert their function, we must understand how both the distribution and the properties (the 'functional architecture') of intracellular Ca2+ channels controls the patterning of Ca2+ signals. Here, we investigate the structural and functional coupling between two intracellular Ca2+-permeable channels that are activated by distinct second messengers: (i) nicotinic acid adenine dinucleotide phosphate (NAADP) which activates the recently discovered two-pore channels (TPCs) within endolysosomes, and (ii) inositol trisphosphate (IP3), which activates IP3 receptors (IP3Rs) in the endoplasmic reticulum. Despite localization in separate organelles, the activity of these Ca2+ channels is intimately related: a functional coupling garnering increasing attention in neurodegenerative disorders involving lysosomal proliferation. Here, by defining the mammalian TPC interactome we provide insight into two key unknowns: (i) how the functional architecture between TPCs and IP3Rs is established and (ii) the molecular identity of the NAADP receptor (NAADP-R, part of the TPC complex). Both are key pieces of knowledge for designing new drugs to modify this coupling. Our six person team, combining chemical, proteomic, molecular and live cell imaging expertise, will resolve: (1) Whether TPCs are Rab effectors? TPCs associate with a clade of Rab GTPases. We will define how TPC channels act as a node for coupling Ca2+ signaling to endolysosomal trafficking and fusion events. (2) How TPC/IP3R activity is coordinated between discrete organelles. We will use novel molecular insight from the TPC interactome to interrogate the functional architecture of TPCs/IP3Rs at membrane contact sites, and uncover how dysregulation triggers lysosomal proliferation. (3) Identify the NAADP-R. We have designed and optimized a novel bifunctional photoprobe to unmask the NAADP-R within the TPC interactome. This is a key roadblock, hampering knowledge of TPC activation. The broad significance of this work is in understanding principles controlling ion channel dynamics, and thereby the kinetics of Ca2+ signals that control compartmentalized cellular and system-levels outcomes. Such data will aid our understanding of the role of ubiquitous Ca2+ signaling pathways in health and disease.

Public Health Relevance

All cell types use changes in calcium concentration to regulate their behavior. It is very important that these changes in cellular calcium are well controlled: excessive rises in calcium contribute to neuronal cell death following a stroke, and even subtle changes contribute to chronic conditions such as neurodegeneration. Here, we will define the interrelationship between two families of calcium channels that regulate intracellular calcium with the long term goal of exploiting these channels, and their interactions, as therapeutic targets.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
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Nie, Zhongzhen
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University of Minnesota Twin Cities
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Lin-Moshier, Yaping; Keebler, Michael V; Hooper, Robert et al. (2014) The Two-pore channel (TPC) interactome unmasks isoform-specific roles for TPCs in endolysosomal morphology and cell pigmentation. Proc Natl Acad Sci U S A 111:13087-92
Lin-Moshier, Yaping; Marchant, Jonathan S (2013) The Xenopus oocyte: a single-cell model for studying Ca2+ signaling. Cold Spring Harb Protoc 2013:
Lin-Moshier, Yaping; Marchant, Jonathan S (2013) Nuclear microinjection to assess how heterologously expressed proteins impact Ca2+ signals in Xenopus oocytes. Cold Spring Harb Protoc 2013:
Subramanian, Veedamali S; Subramanya, Sandeep B; Ghosal, Abhisek et al. (2013) Modulation of function of sodium-dependent vitamin C transporter 1 (SVCT1) by Rab8a in intestinal epithelial cells: studies utilizing Caco-2 cells and Rab8a knockout mice. Dig Dis Sci 58:641-9
Lin-Moshier, Yaping; Marchant, Jonathan S (2013) A rapid Western blotting protocol for the Xenopus oocyte. Cold Spring Harb Protoc 2013:
Subramanian, Veedamali S; Nabokina, Svetlana M; Patton, Joseph R et al. (2013) Glyoxalate reductase/hydroxypyruvate reductase interacts with the sodium-dependent vitamin C transporter-1 to regulate cellular vitamin C homeostasis. Am J Physiol Gastrointest Liver Physiol 304:G1079-86
Lin-Moshier, Yaping; Sebastian, Peter J; Higgins, Leeann et al. (2013) Re-evaluation of the role of calcium homeostasis endoplasmic reticulum protein (CHERP) in cellular calcium signaling. J Biol Chem 288:355-67
Zhang, Dan; Liu, Xiaolong; Chan, John D et al. (2013) Characterization of a flatworm inositol (1,4,5) trisphosphate receptor (IP?R) reveals a role in reproductive physiology. Cell Calcium 53:307-14
Walseth, Timothy F; Lin-Moshier, Yaping; Jain, Pooja et al. (2012) Photoaffinity labeling of high affinity nicotinic acid adenine dinucleotide phosphate (NAADP)-binding proteins in sea urchin egg. J Biol Chem 287:2308-15
Lin-Moshier, Yaping; Walseth, Timothy F; Churamani, Dev et al. (2012) Photoaffinity labeling of nicotinic acid adenine dinucleotide phosphate (NAADP) targets in mammalian cells. J Biol Chem 287:2296-307

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