Store-operated Ca2+ channels (SOCs) generate Ca2+ signals that are critical for many physiological processes ranging from immune cell activation and differentiation to muscle activity, secretion, and motility, and loss of SOC functio in humans leads to a devastating severe combined immunodeficiency with additional myopathy and ectodermal dysplasia. Remarkable progress has been made recently in delineating a diffusion-trap mechanism to explain how these channels are activated. Depletion of Ca2+ from the endoplasmic reticulum (ER) causes STIM1, an ER Ca2+ sensor, to oligomerize, leading to its accumulation at ER-plasma membrane (PM) junctions. At these sites STIM1 binds to Orai1, the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, to trap it and activate local Ca2+ entry across the PM. However, comparatively little is known about the several processes that control the strength of signaling through the CRAC channel after the STIM-Orai complex has formed;these include feedback inhibition via Ca2+-dependent inactivation (CDI), limits on channel open probability imposed by the stoichiometry of STIM-Orai binding, and the dynamics of STIM1 and Orai1 retention at ER-PM junctions. This proposal applies electrophysiology, mutagenesis of STIM1 and tetrameric Orai1 concatemer channels, and superresolution single-particle tracking techniques to understand how these three processes regulate Ca2+ entry through CRAC channels. Recent findings have revealed required roles for STIM1, calmodulin (CaM) and the intracellular II-III loop of Orai1 in the CDI mechanism.
In Aim 1, we will construct concatemeric Orai1 channels with reduced numbers of CaM and STIM1 binding sites to explore how CaM binding, STIM1 binding, and interactions with the II-III loop impact CDI. Several lines of evidence suggest that only a small fraction of CRAC channels at ER-PM junctions are active, even when ER Ca2+ stores are fully depleted, and this large reservoir of dormant channels can be mobilized by the drug 2- aminoethyldiphenyl borate (2-APB).
In Aim 2 we will use 2-APB and CRAC channels with variable numbers of STIM1 binding sites to determine how channels become dormant and how 2-APB recruits them to the active state. Finally, photoactivation studies show that the residence time of STIM1 and Orai1 at the ER-PM junction is rather short, placing limits on the amounts of STIM1 and Orai1 that can accumulate to form active CRAC channel complexes.
In Aim 3 we will apply single-molecule tracking techniques to characterize the diffusion and confinement of STIM1 and Orai1 under various conditions with nanometer precision, and identify the critical protein-protein and protein-lipid interactions that control the mobility and retention of STIM1 and Orai1 at ER-PM junctions. Overall, the results of these studies will increase our understanding of how the strength of store-operated signals is controlled under physiological conditions, and suggest new strategies for up- or down-regulating these signals to provide new treatments for autoimmune and immunodeficiency syndromes.

Public Health Relevance

This proposal seeks to understand the mechanisms that control calcium entry through store-operated calcium channels (SOCs). Mutations that inhibit SOCs lead to a lethal, severe combined immunodeficiency in human patients, showing that store-operated calcium entry is absolutely required for proper immune system function and survival. Thus, a better understanding of the mechanisms that control the rate of calcium entry through SOCs is important for identifying new targets for drug development, with the goal of enhancing function in individuals with compromised immunity, or inhibiting the immune response to treat autoimmune disease (e.g., diabetes, lupus, arthritis) or prevent the rejection of organ transplants.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Special Emphasis Panel (ZRG1-MDCN-B (92))
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Nie, Zhongzhen
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Stanford University
Schools of Medicine
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Yen, Michelle; Lokteva, Ludmila A; Lewis, Richard S (2016) Functional Analysis of Orai1 Concatemers Supports a Hexameric Stoichiometry for the CRAC Channel. Biophys J 111:1897-1907
Mullins, Franklin M; Lewis, Richard S (2016) The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation. J Gen Physiol 147:153-64
Mullins, Franklin M; Yen, Michelle; Lewis, Richard S (2016) Orai1 pore residues control CRAC channel inactivation independently of calmodulin. J Gen Physiol 147:137-52
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Prakriya, Murali; Lewis, Richard S (2015) Store-Operated Calcium Channels. Physiol Rev 95:1383-436
Wu, Minnie M; Covington, Elizabeth D; Lewis, Richard S (2014) Single-molecule analysis of diffusion and trapping of STIM1 and Orai1 at endoplasmic reticulum-plasma membrane junctions. Mol Biol Cell 25:3672-85
Hoover, Paul J; Lewis, Richard S (2011) Stoichiometric requirements for trapping and gating of Ca2+ release-activated Ca2+ (CRAC) channels by stromal interaction molecule 1 (STIM1). Proc Natl Acad Sci U S A 108:13299-304
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