Store-operated calcium entry through calcium release–activated calcium (CRAC) channels replenishes intracellular calcium stores and plays a critical role in cellular calcium signaling. CRAC channels are activated by tightly regulated interaction between the endoplasmic reticulum (ER) calcium sensor STIM proteins and plasma membrane (PM) Orai channels. Our current understanding of the role of STIM–Orai-dependent calcium signals under physiologically relevant conditions remains limited in part due to a lack of spatiotemporally precise methods for direct manipulation of endogenous CRAC channels. Here, we report the synthesis and characterization of azoboronate light-operated CRAC channel inhibitors (LOCIs) that allow for a dynamic and fully reversible remote modulation of the function of native CRAC channels using ultraviolet (UV) and visible light. We demonstrate the use of LOCI-1 to modulate gene expression in T lymphocytes, cancer cell seeding at metastatic sites, and pain-related behavior.
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Endoplasmic Reticulum Calcium Mediates Drosophila Wing Development
The temporal dynamics of morphogen presentation impacts transcriptional responses and tissue patterning (1). However, the mechanisms controlling morphogen release are far from clear. We found that inwardly rectifying potassium (Irk) channels regulate endogenous transient increases in intracellular calcium and Bone Morphogenetic Protein (BMP/Dpp) release for Drosophila wing development (2). Inhibition of Irk channels reduces BMP/Dpp signaling, and ultimately disrupts wing morphology (2, 3). Ion channels impact development of several tissues and organisms in which BMP signaling is essential (2-15). In neurons and pancreatic beta cells, Irk channels modulate membrane potential to affect intracellular Ca++ to control secretion of neurotransmitters and insulin (15-21). Based on Irk activity in neurons, we hypothesized that electrical activity controls endoplasmic reticulum Ca++ release into the cytoplasm to regulate the release of BMP. To test this hypothesis, we reduced expression of proteins that control endoplasmic reticulum calcium (Stim, Orai, SERCA, SK, and Best2) and documented wing phenotypes. We found that reduced Stim and SERCA function decreases amplitude and frequency of endogenous calcium transients in the wing disc and reduced Dpp/BMP release in the wing disc. Together, our results suggest control of endoplasmic reticulum is required for Dpp/BMP release.
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- Award ID(s):
- 1945916
- PAR ID:
- 10499556
- Publisher / Repository:
- Mary Ann Liebert
- Date Published:
- Journal Name:
- Bioelectricity
- Volume:
- 5
- Issue:
- 4
- ISSN:
- 2576-3105
- Page Range / eLocation ID:
- 290 to 306
- Subject(s) / Keyword(s):
- calcium, transients, endoplasmic reticulum, SERCA, Stim, Orai, bestrophin, Drosophila wing development, wing venation, BMP, bone morphogenic protein
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Synaptic plasticity relies on rapid, yet spatially precise signaling to alter synaptic strength. Arc is a brain enriched protein that is rapidly expressed during learning-related behaviors and is essential for regulating metabotropic glutamate receptor-mediated long-term depression (mGluR-LTD). We previously showed that disrupting the ubiquitination capacity of Arc enhances mGluR-LTD; however, the consequences of Arc ubiquitination on other mGluR-mediated signaling events is poorly characterized. Here we find that pharmacological activation of Group I mGluRs with S-3,5-dihydroxyphenylglycine (DHPG) increases Ca2+release from the endoplasmic reticulum (ER). Disrupting Arc ubiquitination on key amino acid residues enhances DHPG-induced ER-mediated Ca2+release. These alterations were observed in all neuronal subregions except secondary branchpoints. Deficits in Arc ubiquitination altered Arc self-assembly and enhanced its interaction with calcium/calmodulin-dependent protein kinase IIb (CaMKIIb) and constitutively active forms of CaMKII in HEK293 cells. Colocalization of Arc and CaMKII was altered in cultured hippocampal neurons, with the notable exception of secondary branchpoints. Finally, disruptions in Arc ubiquitination were found to increase Arc interaction with the integral ER protein Calnexin. These results suggest a previously unknown role for Arc ubiquitination in the fine tuning of ER-mediated Ca2+signaling that may support mGluR-LTD, which in turn, may regulate CaMKII and its interactions with Arc.
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The standard model for Ca 2+ oscillations in insulin-secreting pancreatic β cells centers on Ca 2+ entry through voltage-activated Ca 2+ channels. These work in combination with ATP-dependent K + channels, which are the bridge between the metabolic state of the cells and plasma membrane potential. This partnership underlies the ability of the β cells to secrete insulin appropriately on a minute-to-minute time scale to control whole body plasma glucose. Though this model, developed over more than 40 years through many cycles of experimentation and mathematical modeling, has been very successful, it has been challenged by a hypothesis that calcium-induced calcium release from the endoplasmic reticulum through ryanodine or inositol trisphosphate (IP3) receptors is instead the key driver of islet oscillations. We show here that the alternative model is in fact incompatible with a large body of established experimental data and that the new observations offered in support of it can be better explained by the standard model.more » « less
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Abstract Small‐molecule allosteric activators of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA) hold promise as novel experimental tools to manipulate intracellular calcium concentrations and as therapeutic agents to treat medical conditions associated with elevated cytosolic calcium levels. Here, we synthesized and characterized 20 analogs of the known allosteric SERCA activator CDN1163 and tested their ability to stimulate SERCA activity. The structures of the compounds varied in the alkyl group of the parent scaffold's ether moiety as well as in the composition of the nitrogenous aromatic ring system. The most active compounds exhibited potencies in the sub‐micromolar range while increasing enzyme activity by more than 25 %. The observed structure‐activity relationships indicated that bulky alkyl groups in the ether moiety along with a quinoline ring methyl substituent were beneficial for activity. Replacement of the quinoline by a pyrimidine ring system reduced activity. To conceive a potential mechanism of action, we generated a molecular model of the transition state of SERCA when undergoing the rate‐limiting step of its catalytic cycle. Subsequent blind docking with CDN1163 identified a high‐affinity binding site close to the enzyme's ATP binding pocket, suggesting that the activators may accelerate SERCA's catalytic cycle by aiding in ATP binding and positioning.
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null (Ed.)Abstract Neurons in the association cortices are particularly vulnerable in cognitive disorders such as schizophrenia and Alzheimer’s disease, while those in primary visual cortex remain relatively resilient. This review proposes that the special molecular mechanisms needed for higher cognitive operations confer vulnerability to dysfunction, atrophy, and neurodegeneration when regulation is lost due to genetic and/or environmental insults. Accumulating data suggest that higher cortical circuits rely on magnified levels of calcium (from NMDAR, calcium channels, and/or internal release from the smooth endoplasmic reticulum) near the postsynaptic density to promote the persistent firing needed to maintain, manipulate, and store information without “bottom-up” sensory stimulation. For example, dendritic spines in the primate dorsolateral prefrontal cortex (dlPFC) express the molecular machinery for feedforward, cAMP–PKA–calcium signaling. PKA can drive internal calcium release and promote calcium flow through NMDAR and calcium channels, while in turn, calcium activates adenylyl cyclases to produce more cAMP–PKA signaling. Excessive levels of cAMP–calcium signaling can have a number of detrimental effects: for example, opening nearby K + channels to weaken synaptic efficacy and reduce neuronal firing, and over a longer timeframe, driving calcium overload of mitochondria to induce inflammation and dendritic atrophy. Thus, calcium–cAMP signaling must be tightly regulated, e.g., by agents that catabolize cAMP or inhibit its production (PDE4, mGluR3), and by proteins that bind calcium in the cytosol (calbindin). Many genetic or inflammatory insults early in life weaken the regulation of calcium–cAMP signaling and are associated with increased risk of schizophrenia (e.g., GRM3 ). Age-related loss of regulatory proteins which result in elevated calcium–cAMP signaling over a long lifespan can additionally drive tau phosphorylation, amyloid pathology, and neurodegeneration, especially when protective calcium binding proteins are lost from the cytosol. Thus, the “genie” we need for our remarkable cognitive abilities may make us vulnerable to cognitive disorders when we lose essential regulation.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1089/bioe.2022.0036
