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, microglia, mast cells, blood platelets, sweat and salivary glands, dentition, vascular smooth muscle, endothelial cells, skeletal muscle, microglia, astrocytes, and neurons. 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 at molecular, subcellular, and cellular levels: 1) how Orai1 is activated by STIM1 at the single-channel level; 2) how Orai1 signals locally in puncta to generate localized Ca2+ signals; 3) how local Ca2+ signals modulate T cell motility, turning behavior and stopping during immune surveillance. To accomplish these Aims, we have developed and continue to develop new tools for monitoring local Ca2+ signals that will be broadly applicable. Our studies will include electrophysiological analysis, optical imaging of Ca2+ flux through Orai1 channels, and two-photon imaging of in situ cellular motility. Overall, our project will provide fundamental insights into the Orai1 proteins that are currently being targeted for treatment of autoimmune disorders, chronic inflammatory conditions, and neurodegerative diseases such as Alzheimer?s.

Public Health Relevance

Calcium ions (Ca2+) 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 or neurodegerative diseases such as Alzheimer?s. Our goal is to investigate how recently discovered Ca2+ channels found in lymphocytes and many other cell types open and close in response to inter- and intra-cellular signals. A better understanding of molecular and cellular mechanisms involving calcium transport will aid in the development of drugs that can target autoimmune and neurodegerative diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS014609-40
Application #
9936449
Study Section
Neurotransporters, Receptors, and Calcium Signaling Study Section (NTRC)
Program Officer
Leenders, Miriam
Project Start
1978-09-15
Project End
2023-05-31
Budget Start
2020-06-01
Budget End
2021-05-31
Support Year
40
Fiscal Year
2020
Total Cost
Indirect Cost
Name
University of California Irvine
Department
Physiology
Type
Schools of Medicine
DUNS #
046705849
City
Irvine
State
CA
Country
United States
Zip Code
92617
Dong, Tobias X; Othy, Shivashankar; Jairaman, Amit et al. (2017) T-cell calcium dynamics visualized in a ratiometric tdTomato-GCaMP6f transgenic reporter mouse. Elife 6:
Dong, Tobias X; Othy, Shivashankar; Greenberg, Milton L et al. (2017) Intermittent Ca2+ signals mediated by Orai1 regulate basal T cell motility. Elife 6:
Dynes, Joseph L; Amcheslavsky, Anna; Cahalan, Michael D (2016) Genetically targeted single-channel optical recording reveals multiple Orai1 gating states and oscillations in calcium influx. Proc Natl Acad Sci U S A 113:440-5
Amcheslavsky, Anna; Wood, Mona L; Yeromin, Andriy V et al. (2015) Molecular biophysics of Orai store-operated Ca2+ channels. Biophys J 108:237-46
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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
Khadra, Nadine; Bresson-Bepoldin, Laurence; Penna, Aubin et al. (2011) CD95 triggers Orai1-mediated localized Ca2+ entry, regulates recruitment of protein kinase C (PKC) ?2, and prevents death-inducing signaling complex formation. Proc Natl Acad Sci U S A 108:19072-7

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