Structures reveal opening of the store-operated calcium channel Orai

Hou, Xiaowei; Burstein, Shana R.; Long, Stephen B.

doi:10.7554/elife.36758

Cited by 82 publications

(191 citation statements)

References 70 publications

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“…Removal of the positive charges individually, pairwise, or as a group (R83S/K87S/R91S [3S] or R83A/K87A/R91A [3A]) did not produce any level of constitutive Orai1 current in the absence of STIM1 (Fig. 2 A; I = −0.16 ± 0.05 pA/pF for the 3S mutant; I = −0.21 ± 0.07 pA/pF for the 3A mutant, where I is current amplitude), consistent with recent observations (Hou et al, 2018) indicating that the removal of some or even all of the basic charges does not destabilize the closed channel state. More surprisingly, neutralization of the basic residues resulted in loss of Orai1 channel activation when STIM1 was coexpressed, with the removal of all inner pore basic charges (3S or 3A) completely abrogating STIM1-mediated Orai1 currents (Fig.…”

Section: Resultssupporting

confidence: 90%

“…Both models predict that structural rearrangement of the inner pore gate (or loss of the anion plug) should destabilize the closed channel state to promote channel opening. However, as first reported by Hou et al (2018), we find that mutations at R91 and the other basic residues do not elicit spontaneous pore opening (Fig. 1 B).…”

Section: Discussionsupporting

confidence: 88%

“…2 C; and Fig. S2 A; see also Hou et al, 2018), indicating that the mutations destabilize the open channel state. The loss of current was not due to impaired protein expression as assessed by Orai1-YFP plasma membrane fluorescence (Fig.…”

Section: Resultsmentioning

confidence: 88%

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The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate

Yamashita

Ing

Yeung

et al. 2019

Journal of General Physiology

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Store-operated Orai1 channels regulate a wide range of cellular functions from gene expression to cell proliferation. Previous studies have shown that gating of Orai1 channels is regulated by the outer pore residues V102 and F99, which together function as a hydrophobic gate to block ion conduction in resting channels. Opening of this gate occurs through a conformational change that moves F99 away from the permeation pathway, leading to pore hydration and ion conduction. In addition to this outer hydrophobic gate, several studies have postulated the presence of an inner gate formed by the basic residues R91, K87, and R83 in the inner pore. These positively charged residues were suggested to block ion conduction in closed channels via mechanisms involving either electrostatic repulsion or steric occlusion by a bound anion plug. However, in contrast to this model, here we find that neutralization of the basic residues dose-dependently abolishes both STIM1-mediated and STIM1-independent activation of Orai1 channels. Molecular dynamics simulations show that loss of the basic residues dehydrates the pore around the hydrophobic gate and stabilizes the pore in a closed configuration. Likewise, the severe combined immunodeficiency mutation, Orai1 R91W, closes the channel by dewetting the hydrophobic stretch of the pore and stabilizing F99 in a pore-facing configuration. Loss of STIM1-gating in R91W and in the other basic residue mutants is rescued by a V102A mutation, which restores pore hydration at the hydrophobic gate to repermit ion conduction. These results indicate that the inner pore basic residues facilitate opening of the principal outer hydrophobic gate through a long-range effect involving hydration of the outer pore.

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Section: Resultssupporting

confidence: 90%

Section: Discussionsupporting

confidence: 88%

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The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate

Yamashita

Ing

Yeung

et al. 2019

Journal of General Physiology

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“…The three currently published crystal and one cryo-EM structure(s) of drosophila melanogaster Orai (dOrai) determine that Orai ion channels form hexameric assemblies [37][38][39] . The Ca 2+ pore is formed by six TM1 domains centered in the middle of the channel complex.…”

Section: Introductionmentioning

confidence: 99%

“…Among the four dOrai structures, one is representative of the closed state 37 . The other three published structures are assumed to constitute open channel variants, as they contain one of the two well-known constitutively activating point mutations H206A (in human Orai1 H134A) 39 and P288L (in human Orai1 P245L) 38 , respectively. Moreover, these structures unveil unique features of the cytosolic strands.…”

Section: Introductionmentioning

confidence: 99%

A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening: Clearance and synergy of Orai1 gating checkpoints controls pore opening

Tiffner

Schober

Hoeglinger

et al. 2020

Preprint

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The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca2+) ion channels represents the store-dependent coupling to the Ca2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of LoF mutations act dominant over most GoF mutations within the same as well as of an adjacent Orai subunit. We further established inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints is required to allow STIM1 coupling and Orai1 pore opening.Graphical Abstract

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Ion Channels as Therapeutic Drug Targets

García¹,

Kaczorowski²

2021

Burger's Medicinal Chemistry and Drug Discovery

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Ion channels comprise a large and diverse family of proteins that control many physiological functions. For this reason, a number of inherited diseases result from mutations in specific genes that alter the functions of given ion channels. Drugs used to treat a variety of pathophysiological conditions derive their benefits from interacting with specific ion channel(s), and efforts to develop novel ion channel drugs that would address unmet clinical needs are ongoing in pharmaceutical, biotech, and academic institutions. During the past period of time, major developments in functional screening technologies have afforded the study of these proteins in high‐density formats. In addition, a large body of information concerning the structure of different ion channels has become available. In some cases, intimate details of the interactions between drugs and ion channels have been obtained, which may help with designing more potent and selective ion channel modulators. Although a number of ion channel modulators have entered clinical development, lack of therapeutic efficacy or toxicity issues have caused termination of the development of many of these agents. Nonetheless, with all accumulated experience and new technological advancements, expectations are high for the successful development of novel ion channel modulators in the future.

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Structures reveal opening of the store-operated calcium channel Orai

Cited by 82 publications

References 70 publications

The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate

The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate

A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening: Clearance and synergy of Orai1 gating checkpoints controls pore opening

Ion Channels as Therapeutic Drug Targets

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