Store-operated Ca2+ entry (SOCE) underlies numerous cellular processes throughout the body and initiates signaling cascades in T lymphocytes that cause changes in motility, secretion of cytolytic granules, cytokine release, and cell proliferation. The channels that underlie SOCE have been identified recently through RNA interference (RNAi) screening as a conserved family of four transmembrane-spanning proteins named Orai that are activated by STIM proteins in the ER membrane. Isoforms of these proteins are expressed throughout the body in a tissue-specific manner. Important cellular functions of Orai1 have been identified in lymphocytes, mast cells, blood platelets, sweat and salivary glands, dentition, vascular smooth muscle, endothelial cells, and skeletal muscle. In the immune system, STIM1 and Orai1 mediate antigen-induced Ca2+ signaling, motility inhibition at the site of antigen presentation, secretion of cytolytic granules by CD8+ T cells and NK cells, and gene expression responses that lead to cytokine release and cell proliferation. STIM and Orai proteins are being developed as targets for treatment of autoimmune diseases and prevention of transplant rejection. Our overall goal is to understand how Orai channels function at the molecular and cellular level. Orai channels in the plasma membrane are unrelated to other known ion channels and have unusual characteristics that distinguish them, including a very high degree of selectivity for Ca2+, low single-channel conductance, and activation by binding of a small cytosolic domain of the STIM protein. Moreover, the human Orai1 and Orai3 proteins differ in their activation requirements and tissue distribution. In this project, we have three goals. We seek to understand: 1) how Orai1 and Orai3 are activated by diverse stimuli to form ion channels with variable subunit stoichiometry, ion permeation, and gating characteristics;2) how STIM1 molecules in the ER activate Orai1 subunits in the PM;and 3) how Orai1 channels regulate motility and formation of the immunological synapse in T lymphocytes. To accomplish these Aims, we will develop new tools for monitoring local Ca2+ signals that will be broadly applicable. Our studies will include electrophysiological analysis of gating and ion permeation, optical imaging of Ca2+ flux through Orai1 channels, and cellular assays of motility and immunological synapse formation between T cells and antigen-presenting cells. Overall, our project will provide fundamental insights into the Orai1 proteins that are currently being targeted for treatment of autoimmune disorders and chronic inflammatory conditions.

Public Health Relevance

Calcium ions play key roles during the immune response and in many other physiological processes in the body;Ca2+ dysregulation can cause autoimmune disorders such as multiple sclerosis and type 1 diabetes, or severe combined immunodeficiency that renders patients susceptible to lethal infections. Our goal is to investigate how recently discovered Ca2+ channels found in lymphocytes and many other cell types open and close in response to intracellular signals. A better understanding of molecular and cellular mechanisms involving calcium transport will aid in the development of drugs that can target autoimmune diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
2R01NS014609-34
Application #
8506041
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Stewart, Randall R
Project Start
1978-09-15
Project End
2018-03-31
Budget Start
2013-04-01
Budget End
2014-03-31
Support Year
34
Fiscal Year
2013
Total Cost
$550,953
Indirect Cost
$171,241
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92697
Amcheslavsky, Anna; Safrina, Olga; Cahalan, Michael D (2014) State-dependent block of Orai3 TM1 and TM3 cysteine mutants: insights into 2-APB activation. J Gen Physiol 143:621-31
Amcheslavsky, Anna; Safrina, Olga; Cahalan, Michael D (2013) Orai3 TM3 point mutation G158C alters kinetics of 2-APB-induced gating by disulfide bridge formation with TM2 C101. J Gen Physiol 142:405-12
Greenberg, Milton L; Yu, Ying; Leverrier, Sabrina et al. (2013) Orai1 function is essential for T cell homing to lymph nodes. J Immunol 190:3197-206
Newton, Ryan H; Leverrier, Sabrina; Srikanth, Sonal et al. (2011) Protein kinase D orchestrates the activation of DRAK2 in response to TCR-induced Ca2+ influx and mitochondrial reactive oxygen generation. J Immunol 186:940-50
Demuro, Angelo; Penna, Aubin; Safrina, Olga et al. (2011) Subunit stoichiometry of human Orai1 and Orai3 channels in closed and open states. Proc Natl Acad Sci U S A 108:17832-7
Zhang, Shenyuan L; Yeromin, Andriy V; Hu, Junjie et al. (2011) Mutations in Orai1 transmembrane segment 1 cause STIM1-independent activation of Orai1 channels at glycine 98 and channel closure at arginine 91. Proc Natl Acad Sci U S A 108:17838-43
Krummel, Matthew F; Cahalan, Michael D (2010) The immunological synapse: a dynamic platform for local signaling. J Clin Immunol 30:364-72
Cahalan, Michael D; Chandy, K George (2009) The functional network of ion channels in T lymphocytes. Immunol Rev 231:59-87
Lioudyno, Maria I; Kozak, J Ashot; Penna, Aubin et al. (2008) Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation. Proc Natl Acad Sci U S A 105:2011-6
Zhang, Shenyuan L; Yeromin, Andriy V; Zhang, Xiang H-F et al. (2006) Genome-wide RNAi screen of Ca(2+) influx identifies genes that regulate Ca(2+) release-activated Ca(2+) channel activity. Proc Natl Acad Sci U S A 103:9357-62

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