Expression of ORAI1, a Plasma Membrane Resident Subunit of the CRAC Channel, in Rodent and Non-rodent Species

Guzman, Roberto E.; Valente, Eliane G.; Pretorius, Jim; Pacheco, Efrain; Qi, Meiying; Bennett, Brian D.; Fong, David; Lin, Fen‐Fen; Bi, Vivian; McBride, Helen J.

doi:10.1369/0022155414554926

Cited by 21 publications

(28 citation statements)

References 38 publications

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“…These results provide evidence that the fractions designated "heavy microsomes" and "light microsomes" are enriched in microsomes derived from the ER, and that the fraction designated "PM" is enriched in microsomes derived from the PM. to the glycosylated form of Orai1 [16,41] and is similar to the size of endogenous Orai1 observed previously in hepatocytes and liver cells [18] and in other mammalian cell types [42,43]. Relative to whole liver, STIM1 was enriched in the heavy and light microsomal fractions, with no detectable enrichment in the PM fraction or in the low speed pellet (Fig.…”

Section: And Insupporting

confidence: 84%

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

et al. 2018

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Ca entry through store-operated Ca channels (SOCs) in the plasma membrane (PM) of hepatocytes plays a central role in the hormonal regulation of liver metabolism. SOCs are composed of Orai1, the channel pore protein, and STIM1, the activator protein, and are regulated by hormones and reactive oxygen species (ROS). In addition to Orai1, STIM1 also interacts with several other intracellular proteins. Most previous studies of the cellular functions of Orai1 and STIM1 have employed immortalised cells in culture expressing ectopic proteins tagged with a fluorescent polypeptide such as GFP. Little is known about the intracellular distributions of endogenous Orai1 and STIM1. The aims are to determine the intracellular distribution of endogenous Orai1 and STIM1 in hepatocytes and to identify novel STIM1 binding proteins. Subcellular fractions of rat liver were prepared by homogenisation and differential centrifugation. Orai1 and STIM1 were identified and quantified by western blot. Orai1 was found in the PM (0.03%), heavy (44%) and light (27%) microsomal fractions, and STIM1 in the PM (0.09%), and heavy (85%) and light (13%) microsomal fractions. Immunoprecipitation of STIM1 followed by LC/MS or western blot identified peroxiredoxin-4 (Prx-4) as a potential STIM1 binding protein. Prx-4 was found principally in the heavy microsomal fraction. Knockdown of Prx-4 using siRNA, or inhibition of Prx-4 using conoidin A, did not affect Ca entry through SOCs but rendered SOCs susceptible to inhibition by HO. It is concluded that, in hepatocytes, a considerable proportion of endogenous Orai1 and STIM1 is located in the rough ER. In the rough ER, STIM1 interacts with Prx-4, and this interaction may contribute to the regulation by ROS of STIM1 and SOCs.

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Section: And Insupporting

confidence: 84%

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

et al. 2018

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“…Isolated protein samples were heated to 65°C in Laemmli sample buffer (Bio-Rad) containing 0.1% mercaptoethanol and run on 4–20% SDS-polyacrylamide gradient gels (Bio-Rad). Protein was transferred onto nitrocellulose membrane and Orai1 was detected using 1:7500 purified monoclonal primary antibodies provided by Amgen (m266.1) [ 37 ] and 1:10000 peroxidase-labeled secondary antibody (GE Healthcare Life Sciences). GAPDH was blotted as a loading control.…”

Section: Methodsmentioning

confidence: 99%

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding

2015

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Store-operated CRAC channels regulate a wide range of cellular functions including gene expression, chemotaxis, and proliferation. CRAC channels consist of two components: the Orai proteins (Orai1-3), which form the ion-selective pore, and STIM proteins (STIM1-2), which form the endoplasmic reticulum (ER) Ca2+ sensors. Activation of CRAC channels is initiated by the migration of STIM1 to the ER-plasma membrane (PM) junctions, where it directly interacts with Orai1 to open the Ca2+-selective pores of the CRAC channels. The recent elucidation of the Drosophila Orai structure revealed a hexameric channel wherein the C-terminal helices of adjacent Orai subunits associate in an anti-parallel orientation. This association is maintained by hydrophobic interactions between the Drosophila equivalents of human Orai1 residues L273 and L276. Here, we used mutagenesis and chemical cross-linking to assess the nature and extent of conformational changes in the self-associated Orai1 C-termini during STIM1 binding. We find that linking the anti-parallel coiled-coils of the adjacent Orai1 C-termini through disulfide cross-links diminishes STIM1-Orai1 interaction, as assessed by FRET. Conversely, prior binding of STIM1 to the Orai1 C-terminus impairs cross-linking of the Orai1 C-termini. Mutational analysis indicated that a bend of the Orai1 helix located upstream of the self-associated coils (formed by the amino acid sequence SHK) establishes an appropriate orientation of the Orai1 C-termini that is required for STIM1 binding. Together, our results support a model wherein the self-associated Orai1 C-termini rearrange modestly to accommodate STIM1 binding.

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“… 109 – 111 Of note, high Orai1 expression in the brain implicates its role in the nervous system. 112 Various mechanisms, such as glycosylation and pH sensing, and proteins, including golli proteins and septins, can further regulate SOCE. 103 Through the formation of ER:plasma membrane microdomains, the ER—an intracellular Ca 2+ store—becomes functionally linked to the extracellular supply of Ca 2+ .…”

Section: Er Ca 2+ Regulation and The Impact On Paimentioning

confidence: 99%

Endoplasmic reticulum–mitochondria interplay in chronic pain: The calcium connection

et al. 2020

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Chronic pain is a debilitating condition that affects roughly a third to a half of the world’s population. Despite its substantial effect on society, treatment for chronic pain is modest, at best, notwithstanding its side effects. Hence, novel therapeutics are direly needed. Emerging evidence suggests that calcium plays an integral role in mediating neuronal plasticity that underlies sensitization observed in chronic pain states. The endoplasmic reticulum and the mitochondria are the largest calcium repositories in a cell. Here, we review how stressors, like accumulation of misfolded proteins and oxidative stress, influence endoplasmic reticulum and mitochondria function and contribute to chronic pain. We further examine the shuttling of calcium across the mitochondrial-associated membrane as a mechanism of cross-talk between the endoplasmic reticulum and the mitochondria. In addition, we discuss how endoplasmic reticulum stress, mitochondrial impairment, and calcium dyshomeostasis are implicated in various models of neuropathic pain. We propose a novel framework of endoplasmic reticulum–mitochondria signaling in mediating pain hypersensitivity. These observations require further investigation in order to develop novel therapies for chronic pain.

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Expression of ORAI1, a Plasma Membrane Resident Subunit of the CRAC Channel, in Rodent and Non-rodent Species

Cited by 21 publications

References 38 publications

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

Evidence for the interaction of peroxiredoxin-4 with the store-operated calcium channel activator STIM1 in liver cells

Conformational Changes in the Orai1 C-Terminus Evoked by STIM1 Binding

Endoplasmic reticulum–mitochondria interplay in chronic pain: The calcium connection

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