Inhibition of a receptor-operated calcium channel in pig aortic microsomes by cyclic GMP-dependent protein kinase.
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ABSTRACT:
SUBMITTER: Blayney LM
PROVIDER: S-EPMC1149835 | biostudies-other | 1991 Feb
REPOSITORIES: biostudies-other
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Project description:Receptor-operated Ca2+ channels were characterized by their ability to decrease steady-state ATP-dependent Ca2+ accumulation into pig aortic microsomes. The vasoconstrictor agents noradrenaline, angiotensin II and adenosine 5'-[alpha beta-methylene]triphosphate (pp[CH2]pA) all decreased Ca2+ accumulation only when sonicated into vesicles (to allow access to receptor sites) and in the presence of guanosine 5'-[beta gamma-imido]triphosphate to activate transducing G-proteins. The effect of noradrenaline was inhibited by the alpha 2 antagonist yohimbine, but not by the alpha 1 antagonist prazosin. The effect of none of the agonists was reversed by diltiazem. SK&F 96365 (an inhibitor of receptor-mediated Ca2+ influx into intact cells) reversed the effect of noradrenaline, but not that of pp[CH2]pA, which suggests that at least two receptor-operated channels may be present in this preparation.
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Project description:ORAI1 is the pore-forming subunit of the calcium release-activated calcium (CRAC) channel, a store-operated channel that is central to Ca(2+) signaling in mammalian cells. Electrophysiological data have shown that the acidic residues E106 in transmembrane helix 1 (TM1) and E190 in TM3 contribute to the high selectivity of ORAI1 channels for Ca(2+). We have examined the pore architecture of the ORAI1 channel using ORAI1 proteins engineered to contain either one or two cysteine residues. Disulfide cross-linking shows that ORAI1 assembles as a tetramer or a higher oligomer with TM1 centrally located. Cysteine side chains projecting from TM1 at position 88, 95, 102, or 106 cross-link efficiently to the corresponding side chain in a second ORAI1 monomer. Cysteine residues at position 190 or at surrounding positions in TM3 do not cross-link. We conclude that E106 residues in wild-type ORAI1 are positioned to form a Ca(2+) binding site in the channel pore and that E190 interacts less directly with ions traversing the pore. The cross-linking data further identify a relatively rigid segment of TM1 adjacent to E106 that is likely to contribute to the selectivity filter.
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Project description:The current optogenetic toolkit lacks a robust single-component Ca2+-selective ion channel tailored for remote control of Ca2+ signaling in mammals. Existing tools are either derived from engineered channelrhodopsin variants without strict Ca2+ selectivity or based on the stromal interaction molecule 1 (STIM1) that might crosstalk with other targets. Here, we describe the design of a light-operated Ca2+ channel (designated LOCa) by inserting a plant-derived photosensory module into the intracellular loop of an engineered ORAI1 channel. LOCa displays biophysical features reminiscent of the ORAI1 channel, which enables precise optical control over Ca2+ signals and hallmark Ca2+-dependent physiological responses. Furthermore, we demonstrate the use of LOCa to modulate aberrant hematopoietic stem cell self-renewal, transcriptional programming, cell suicide, as well as neurodegeneration in a Drosophila model of amyloidosis.
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Project description:Store-operated Ca(2+) entry through the plasma membrane Ca(2+) release-activated Ca(2+) (CRAC) channel in mammalian T cells and mast cells depends on the sensor protein stromal interaction molecule 1 (STIM1) and the channel subunit ORAI1. To study STIM1-ORAI1 signaling in vitro, we have expressed human ORAI1 in a sec6-4 strain of the yeast Saccharomyces cerevisiae and isolated sealed membrane vesicles carrying ORAI1 from the Golgi compartment to the plasma membrane. We show by in vitro Ca(2+) flux assays that bacterially expressed recombinant STIM1 opens wild-type ORAI1 channels but not channels assembled from the ORAI1 pore mutant E106Q or the ORAI1 severe combined immunodeficiency (SCID) mutant R91W. These experiments show that the STIM1-ORAI1 interaction is sufficient to gate recombinant human ORAI1 channels in the absence of other proteins of the human ORAI1 channel complex, and they set the stage for further biochemical and biophysical dissection of ORAI1 channel gating.
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Project description:Orai channels mediate store-operated Ca2+ signals crucial in regulating transcription in many cell types, and implicated in numerous immunological and inflammatory disorders. Despite their central importance, controversy surrounds the basic subunit structure of Orai channels, with several biochemical and biophysical studies suggesting a tetrameric structure yet crystallographic evidence indicating a hexamer. We systematically investigated the subunit configuration of the functional Orai1 channel, generating a series of tdTomato-tagged concatenated Orai1 channel constructs (dimers to hexamers) expressed in CRISPR-derived ORAI1 knock-out HEK cells, stably expressing STIM1-YFP. Surface biotinylation demonstrated that the full-length concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca2+ entry. Moreover, each Orai1 concatemer mediated Ca2+ currents with inward rectification and reversal potentials almost identical to those observed with expressed Orai1 monomer. In Orai1 tetramers, subunit-specific replacement with Orai1 E106A "pore-inactive" subunits revealed that functional channels utilize only the N-terminal dimer from the tetramer. In contrast, Orai1 E106A replacement in Orai1 hexamers established that all the subunits can contribute to channel formation, indicating a hexameric channel configuration. The critical Ca2+ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca2+ ion within the pore. Thus, multiple E106A substitutions in the Orai1 hexamer may promote an alternative "trimer-of-dimers" channel configuration in which the C-terminal E106A subunits are excluded from the hexameric core. Our results argue strongly against a tetrameric configuration for Orai1 channels and indicate that the Orai1 channel functions as a hexamer.
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Project description:The store-operated calcium (Ca2+) channel Orai governs Ca2+ influx through the plasma membrane of many non-excitable cells in metazoans. The channel opens in response to the depletion of Ca2+ stored in the endoplasmic reticulum (ER). Loss- and gain-of-function mutants of Orai cause disease. Our previous work revealed the structure of Orai with a closed pore. Here, using a gain-of-function mutation that constitutively activates the channel, we present an X-ray structure of Drosophila melanogaster Orai in an open conformation. Well-defined electron density maps reveal that the pore is dramatically dilated on its cytosolic side in comparison to the slender closed pore. Cations and anions bind in different regions of the open pore, informing mechanisms for ion permeation and Ca2+ selectivity. Opening of the pore requires the release of cytosolic latches. Together with additional X-ray structures of an unlatched-but-closed conformation, we propose a sequence for store-operated activation.
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Project description:Alcohol during brain development leads to the widespread neuronal death observed in fetal alcohol spectrum disorders (FASD). In comparison, the mature brain is less vulnerable to alcohol. Studies into maturation-acquired alcohol resistance uncovered a protective mechanism that reduces alcohol-induced neuronal death through nitric oxide-cGMP-cyclic GMP-dependent protein kinase (NO-cGMP-cGK) signaling. However, the downstream processes underlying this neuroprotection remain unclear. Alcohol can disrupt levels of intracellular calcium ([Ca2+]i) in vulnerable neuronal populations to trigger cell death in both in vivo and in vitro models of FASD. Since cGK has been demonstrated to regulate and inhibit intracellular Ca2+ release, we examined the hypothesis that cGK confers alcohol resistance by preventing [Ca2+]i disruptions. Alcohol resistance, determined by neuronal survival after 24?h of alcohol exposure, was examined in primary cerebellar granule neuron (CGN) cultures derived from 5 to 7?day-old neonatal mice with an activator, 8-Br-cGMP, and/or an inhibitor, Rp-8-pCPT-cGMPS, of cGK signaling. Intracellular Ca2+ responses to alcohol were measured by ratiometric Ca2+ imaging in Fura-2-loaded CGN cultures after 8-Br-cGMP treatment. Our results indicate that activating cGK with 8-Br-cGMP before alcohol administration provided neuroprotection, which the cGK inhibitor, Rp-8-pCPT-cGMPS, blocked. Alcohol exposure elevated [Ca2+]i, whereas 8-Br-cGMP pretreatment reduced both the level of the alcohol-induced rise in [Ca2+]i as well as the number of cells that responded to alcohol by increasing [Ca2+]i. These findings associate alcohol resistance, mediated by cGK signaling, to reduction of the persistent and toxic increase in [Ca2+]i from alcohol exposure.
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Project description:ObjectiveThe physiologic significance of the nitric oxide (NO)/cGMP signaling pathway in islets is unclear. We hypothesized that cGMP-dependent protein kinase type I (cGKI) is directly involved in the secretion of islet hormones and glucose homeostasis.Research design and methodsGene-targeted mice that lack cGKI in islets (conventional cGKI mutants and cGKI? and I? rescue mice [?/?RM] that express cGKI only in smooth muscle) were studied in comparison to control (CTR) mice. cGKI expression was mapped in the endocrine pancreas by Western blot, immuno-histochemistry, and islet-specific recombination analysis. Insulin, glucagon secretion, and cytosolic Ca²(+) ([Ca²(+)](i)) were assayed by radioimmunoassay and FURA-2 measurements, respectively. Serum levels of islet hormones were analyzed at fasting and upon glucose challenge (2 g/kg) in vivo.ResultsImmunohistochemistry showed that cGKI is present in ?- but not in ?-cells in islets of Langerhans. Mice that lack ?-cell cGKI had significantly elevated fasting glucose and glucagon levels, whereas serum insulin levels were unchanged. High glucose concentrations strongly suppressed the glucagon release in CTR mice, but had only a moderate effect on islets that lacked cGKI. 8-Br-cGMP reduced stimulated [Ca²(+)](i) levels and glucagon release rates of CTR islets at 0.5 mmol/l glucose, but was without effect on [Ca²(+)](i) or hormone release in cGKI-deficient islets.ConclusionsWe propose that cGKI modulates glucagon release by suppression of [Ca²(+)](i) in ?-cells.
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