Ca2+ influx though CRAC (Ca2+ release activated Ca2+) channels is critical for immune cell functions. Stimulation through receptors depletes intracellular Ca2+ stores and subsequently leads to opening of CRAC channels on the plasma membrane. Recently, Orai1 (CRACM1) and STIM1 were identified as a pore component of the CRAC channel and a mediator between Ca2+ store depletion and CRAC channel opening, respectively. However, much remains to be understood about the molecular composition and the mechanism of CRAC channel regulation. We have used large scale affinity protein purification under store depleted conditions to identify regulators of the CRAC channel. We have identified CRACR2A (Ca2+ release activated Ca2+ channel Regulator 2A) as a novel regulator of CRAC channels using this approach.
The Specific Aims are: (1) To test the hypothesis that Orai1-CRACR2A interaction is critical for the CRAC channel function. The activities of Orai1 mutants incapable of binding to CRACR2A will be analyzed in Orai1-null primary cells. (2) To test the hypothesis that CRACR2A stabilizes the Orai1-STIM1 complex. Our preliminary data suggest that CRACR2A can directly interact with both Orai1 and STIM1. The functions of Orai1 and STIM1 will be examined using CRACR2A knock-down cells. The functional importance of reciprocal interaction of Orai1, STIM1, and CRACR2A will be determined. (3) To test the hypothesis that CRACR2A is critical for the T cell function. Knock-down of CRACR2A in Jurkat T cells decreased Ca2+ entry. We will conditionally target the CRACR2A gene to determine the role of CRACR2A in store-operated Ca2+ entry in peripheral T cells. The experiments on the novel regulator of the CRAC channel are timely because of the very recent discovery of Orai1 and STIM1. In the short term, the proposed experiments should provide new molecular probes for investigating the regulatory mechanism of store-operated Ca2+ entry in mammalian cells, particularly in immune cells. In the long term, we will test the possibility of development of drugs to modulate the function of the CRAC channel;thereby immune activation. Public Health Relevance: We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.

Public Health Relevance

Statement We propose to understand the mechanism of a critical step of immune cell activation, calcium entry via plasma membrane calcium channels through proteomic analysis, further functional analysis, and gene manipulation. We successfully indentified a novel molecule, CRACR2A using these approaches and we will focus to elucidate the novel function of this molecule in the immune system. Our study can benefit development of drugs that can activate or repress immune functions as therapy for immune system related problems such as auto-immune diseases or graft rejection.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI083432-04
Application #
8282753
Study Section
Cellular and Molecular Immunology - A Study Section (CMIA)
Program Officer
Mallia, Conrad M
Project Start
2009-07-01
Project End
2014-06-30
Budget Start
2012-07-01
Budget End
2013-06-30
Support Year
4
Fiscal Year
2012
Total Cost
$377,339
Indirect Cost
$132,314
Name
University of California Los Angeles
Department
Physiology
Type
Schools of Medicine
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Bertin, Samuel; Aoki-Nonaka, Yukari; de Jong, Petrus Rudolf et al. (2014) The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4? T cells. Nat Immunol 15:1055-63
Kim, Kyun-Do; Srikanth, Sonal; Tan, Yossan-Var et al. (2014) Calcium signaling via Orai1 is essential for induction of the nuclear orphan receptor pathway to drive Th17 differentiation. J Immunol 192:110-22
Groenendyk, Jody; Peng, Zhenling; Dudek, Elzbieta et al. (2014) Interplay between the oxidoreductase PDIA6 and microRNA-322 controls the response to disrupted endoplasmic reticulum calcium homeostasis. Sci Signal 7:ra54
Srikanth, Sonal; Kim, Kyun-Do; Gwack, Yousang (2014) Methods to measure cytoplasmic and mitochondrial Ca(2+) concentration using Ca(2+)-sensitive dyes. Methods Enzymol 543:1-20
Srikanth, Sonal; Gwack, Yousang (2013) Molecular regulation of the pore component of CRAC channels, Orai1. Curr Top Membr 71:181-207
Srikanth, Sonal; Gwack, Yousang (2013) Orai1-NFAT signalling pathway triggered by T cell receptor stimulation. Mol Cells 35:182-94
Srikanth, Sonal; Gwack, Yousang (2013) Measurement of intracellular Ca2+ concentration in single cells using ratiometric calcium dyes. Methods Mol Biol 963:3-14
Srikanth, Sonal; Ribalet, Bernard; Gwack, Yousang (2013) Regulation of CRAC channels by protein interactions and post-translational modification. Channels (Austin) 7:
Kim, Kyun-Do; Srikanth, Sonal; Yee, Ma-Khin Win et al. (2011) ORAI1 deficiency impairs activated T cell death and enhances T cell survival. J Immunol 187:3620-30
Srikanth, Sonal; Yee, Ma-Khin Win; Gwack, Yousang et al. (2011) The third transmembrane segment of orai1 protein modulates Ca2+ release-activated Ca2+ (CRAC) channel gating and permeation properties. J Biol Chem 286:35318-28

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