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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM062142-10
Application #
8402802
Study Section
Biochemistry and Biophysics of Membranes Study Section (BBM)
Program Officer
Chin, Jean
Project Start
2001-06-01
Project End
2015-11-30
Budget Start
2012-12-01
Budget End
2013-11-30
Support Year
10
Fiscal Year
2013
Total Cost
$304,844
Indirect Cost
$116,669
Name
Johns Hopkins University
Department
Physiology
Type
Schools of Medicine
DUNS #
001910777
City
Baltimore
State
MD
Country
United States
Zip Code
21218
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Prasad, Hari; Rao, Rajini (2015) Applying knowledge of autism to brain cancer management: what do we know? Future Oncol 11:1847-50
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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
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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
Feng, Mingye; Grice, Desma M; Faddy, Helen M et al. (2010) Store-independent activation of Orai1 by SPCA2 in mammary tumors. Cell 143:84-98

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