A family of membrane embedded ATP-powered ion pumps, known as Secretory Pathway Ca2+, Mn2+-ATPases (SPCA), is conserved from yeast to human. SPCA pumps supply the Golgi lumen with ions essential for protein processing, sorting and glycosylation reactions. There is emerging evidence that SPCA pumps are also critically important for cytoplasmic Ca2+ signaling events and Mn2+ homeostasis. In this proposal, we will investigate three unique, physiologically distinct and clinically relevant functions of SPCA pumps.
In Aim 1, we will test the hypothesis that SPCA1 contributes to manganese clearance through bile. We will use liver-specific, shRNA mediated knockdown of SPCA1 in a murine model to evaluate a role in manganese detoxification. Using a polarized, hepatocyte derived cell line, we will investigate the role of a novel Golgi Mn2+ sensor in SPCA1 trafficking, and identify molecular determinants for endosomal localization of SPCA1.
In Aim 2, we will determine the mechanism of an unconventional interaction between SPCA2 and the Orai1 ion channel using fluorescence and electrophysiological approaches, as well as an innovative yeast expression strategy. N- and C-terminal SPCA2 domains with dominant negative or constitutively active properties will be evaluated for functional interactions with ion channels.
In Aim 3, we seek to understand the physiological role of pump-channel interactions in eliciting robust calcium influx at the plasma membrane. We will follow up on preliminary studies showing that a calcium handling module of pumps, channels, buffers and sensors, including SPCA2, is coordinately induced upon lactation. A unique, 3-dimensional model of lactating mammary epithelial cells will be used to determine if SPCA2 interacts with and activates Orai channels for effective calcium secretion into milk. We will assess the function of a novel, truncated C-terminal SPCA2 transcript, specifically regulated by MIST1, a bHLH transcription factor that shows overlapping expression with SPCA2. Finally, we will examine how dysregulation of this calcium module in tumor cells contributes to distinct modes of cell proliferation and migration underlying cancer progression.

Public Health Relevance

Calcium and manganese ions are essential for many aspects of biology, and must be regulated tightly to avoid toxicity. Excess manganese must be cleared by the liver to avoid neurotoxicity resembling Parkinson's disease. Unregulated calcium levels result in excessive proliferation associated with tumor growth and metastasis. This study investigates molecular pathways by which these critical ions are transported.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry and Biophysics of Membranes Study Section (BBM)
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Chin, Jean
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Johns Hopkins University
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Dang, Donna; Prasad, Hari; Rao, Rajini (2017) Secretory pathway Ca2+ -ATPases promote in vitro microcalcifications in breast cancer cells. Mol Carcinog 56:2474-2485
Dang, Donna; Rao, Rajini (2016) Calcium-ATPases: Gene disorders and dysregulation in cancer. Biochim Biophys Acta 1863:1344-50
Kondapalli, Kalyan C; Llongueras, Jose P; Capilla-González, Vivian et al. (2015) A leak pathway for luminal protons in endosomes drives oncogenic signalling in glioblastoma. Nat Commun 6:6289
Prasad, Hari; Rao, Rajini (2015) Applying knowledge of autism to brain cancer management: what do we know? Future Oncol 11:1847-50
Prasad, Hari; Rao, Rajini (2015) The Na+/H+ exchanger NHE6 modulates endosomal pH to control processing of amyloid precursor protein in a cell culture model of Alzheimer disease. J Biol Chem 290:5311-27
Cross, Brandie M; Breitwieser, Gerda E; Reinhardt, Timothy A et al. (2014) Cellular calcium dynamics in lactation and breast cancer: from physiology to pathology. Am J Physiol Cell Physiol 306:C515-26
Feng, Ming-Ye; Rao, Rajini (2013) New insights into store-independent Ca(2+) entry: secretory pathway calcium ATPase 2 in normal physiology and cancer. Int J Oral Sci 5:71-4
Cross, Brandie M; Hack, Anniesha; Reinhardt, Timothy A et al. (2013) SPCA2 regulates Orai1 trafficking and store independent Ca2+ entry in a model of lactation. PLoS One 8:e67348
Patenaude, Cassandra; Zhang, Yongqiang; Cormack, Brendan et al. (2013) Essential role for vacuolar acidification in Candida albicans virulence. J Biol Chem 288:26256-64
Leitch, Sharon; Feng, Mingye; Muend, Sabina et al. (2011) Vesicular distribution of Secretory Pathway Ca²+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells. Biometals 24:159-70

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