State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

Amcheslavsky, Anna; Safrina, Olga; Cahalan, Michael D.

doi:10.1085/jgp.201411171

Cited by 22 publications

(23 citation statements)

References 33 publications

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“…Other cysteine mutants of TM1 residues (E81, G73, and R66) homologous to Orai1 pore-lining residues made Orai3 channels insensitive to 2-APB-activation. Orai3 TM3 residue E165 was found to only assist in pore formation of the 2-APB-activated, but not store-operated, Orai3 channel (59). These data suggest that the store-operated (i.e., STIM-activated) Orai channel conduction pathway differs from that of the 2-APB-activated Orai3 channel pore.…”

Section: Calcium-dependent Inactivationmentioning

confidence: 80%

Molecular Biophysics of Orai Store-Operated Ca2+ Channels

Amcheslavsky

Wood

Yeromin

et al. 2015

Biophysical Journal

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Upon endoplasmic reticulum Ca 2þ store depletion, Orai channels in the plasma membrane are activated directly by endoplasmic reticulum-resident STIM proteins to generate the Ca 2þ -selective, Ca 2þ release-activated Ca 2þ (CRAC) current. After the molecular identification of Orai, a plethora of functional and biochemical studies sought to compare Orai homologs, determine their stoichiometry, identify structural domains responsible for the biophysical fingerprint of the CRAC current, identify the physiological functions, and investigate Orai homologs as potential therapeutic targets. Subsequently, the solved crystal structure of Drosophila Orai (dOrai) substantiated many findings from structure-function studies, but also revealed an unexpected hexameric structure. In this review, we explore Orai channels as elucidated by functional and biochemical studies, analyze the dOrai crystal structure and its implications for Orai channel function, and present newly available information from molecular dynamics simulations that shed light on Orai channel gating and permeation.

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Section: Calcium-dependent Inactivationmentioning

confidence: 80%

Molecular Biophysics of Orai Store-Operated Ca2+ Channels

Amcheslavsky

Wood

Yeromin

et al. 2015

Biophysical Journal

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“…Although 2-APB does indeed potently block Ca 2+ influx via the Orai1 and Orai2 subtypes of CRAC channels, it has the opposite effect on the Orai3 family member. Several groups have described the ability of 2-APB to allosterically stabilize the open-gated conformation of Orai3 channels and change their permeability properties into nonselective cation channels that facilitate fluxes of Ca 2+ and monovalent cations 54–59 . The ability of 2-APB to stimulate increased cytosolic [Ca 2+ ] in our LPS-primed BMDC model suggests that these cells express significant levels of Orai3 channels and this is supported by our preliminary western blot analyses of BMDC and BMDM.…”

Section: Discussionmentioning

confidence: 99%

K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling

Katsnelson

Rucker

Russo

et al. 2015

The Journal of Immunology

237

182

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Perturbation of intracellular ion homeostasis is a major cellular stress signal for activation of NLRP3 inflammasome signaling that results in caspase-1 mediated production of IL-1β and pyroptosis. However, the relative contributions of decreased cytosolic [K+] versus increased cytosolic [Ca2+] remain disputed and incompletely defined. We investigated roles for elevated cytosolic [Ca2+] in NLRP3 activation and downstream inflammasome signaling responses in primary murine dendritic cells and macrophages in response to two canonical NLRP3 agonists (ATP and nigericin) that facilitate primary K+ efflux by mechanistically distinct pathways or the lysosome-destabilizing agonist Leu-Leu-O-methyl ester (LLME). The study provides three major findings relevant to this unresolved area of NLRP3 regulation. First, increased cytosolic [Ca2+] was neither a necessary nor sufficient signal for the NLRP3 inflammasome cascade during activation by endogenous ATP-gated P2X7 receptor channels, the exogenous bacterial ionophore nigericin, or the lysosomotropic agent LLME. Second, agonists for three Ca2+-mobilizing G protein-coupled receptors (formyl peptide receptor/FPR; P2Y2 purinergic receptor/P2Y2R; calcium-sensing receptor/CaSR) expressed in murine dendritic cells were ineffective as activators of rapidly induced NLRP3 signaling when directly compared to the K+ efflux agonists. Third, the intracellular Ca2+ buffer, BAPTA, and the channel blocker, 2-aminoethoxydiphenyl borate (2-APB), widely used reagents for disruption of Ca2+-dependent signaling pathways, strongly suppressed nigericin-induced NLRP3 inflammasome signaling via mechanisms dissociated from their canonical or expected effects on Ca2+ homeostasis. The results indicate that the ability of K+ efflux agonists to activate NLRP3 inflammasome signaling can be dissociated from changes in cytosolic [Ca2+] as a necessary or sufficient signal.

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“…And its actions on CRAC activity, that are mediated by different isoforms of STIM and Orai, has been intensively examined (15–23, 32, 51). However, current understandings of the molecular basis of its actions are mainly constrained on its activations (22, 23, 52–57), while the possible mechanisms of its inhibitory effects were poorly understood (15, 16, 21). …”

Section: Discussionmentioning

confidence: 99%

Molecular mechanisms underlying inhibition of STIM1-Orai1-mediated Ca2+ entry induced by 2-aminoethoxydiphenyl borate

Wei

Zhou

Sun

et al. 2016

Pflugers Arch - Eur J Physiol

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Store operated Ca2+ entry (SOCE) mediated by STIM1 and Orai1 is crucial for Ca2+ signaling and homeostasis in most cell types. 2-aminoethoxydiphenyl borate (2-APB) is a well described SOCE inhibitor but its action remains largely elusive. Here we show that 2-APB does not affect the dimeric state of STIM1, but enhances the intramolecular coupling between the coiled-coil 1 (CC1) and STIM-Orai Activating Region (SOAR) of STIM1, with subsequent reduction in the formation of STIM1 puncta in the absence of Orai1overexpression. 2-APB also inhibits Orai1 channels, directly inhibiting Ca2+ entry through the constitutively active, STIM1-independent Orai1 mutants, Orai1-P245T and Orai1-V102A. When unbound with STIM1, the constitutively active Orai1-V102C mutant is not inhibited by 2-APB. Thus we used Orai1-V012C as a tool to examine whether 2-APB can also inhibit the coupling between STIM1 and Orai1. We find that the functional coupling between STIM1 and Orai1-V102C is inhibited by 2-APB. This inhibition on coupling is indirect, arising from 2-APB’s action on STIM1, and it is most likely mediated by gating residues in Orai1 N terminus. Overall, our finding of this two-direct-site inhibition may help better understanding of Orai1-activation by STIM1 and future drug design.

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State-dependent block of Orai3 TM1 and TM3 cysteine mutants: Insights into 2-APB activation

Abstract: Residue E165, in transmembrane helix 3, participates in formation of the dilated pore of the 2-APB–activated Orai3 channel but not that of the more selective store-operated Orai3 pore.

Cited by 22 publications

References 33 publications

Molecular Biophysics of Orai Store-Operated Ca2+ Channels

Molecular Biophysics of Orai Store-Operated Ca2+ Channels

K+ Efflux Agonists Induce NLRP3 Inflammasome Activation Independently of Ca2+ Signaling

Molecular mechanisms underlying inhibition of STIM1-Orai1-mediated Ca2+ entry induced by 2-aminoethoxydiphenyl borate

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