The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1

Derler, Isabella; Plenk, Peter; Fahrner, Marc; Muik, Martin; Jardín, Isaac; Schindl, Rainer; Gruber, Hermann J.; Gröschner, Klaus; Romanin, Christoph

doi:10.1074/jbc.m113.501510

Cited by 103 publications

(231 citation statements)

References 38 publications

(79 reference statements)

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“…We analyzed the ion selectivity of Orai1 and SS-Orai1 V102A mutants by quantifying the reversal potentials of mutant channel currents in a calcium-containing solution. In agreement with earlier studies [28,31], we found that V102A Orai1 mediates a non-selective constant cation current when expressed alone (V rev = 19.6 ± 5.2 mV), while co-expression of the V102A mutant channel with STIM1 produced a rightward shift in current reversal potential (V rev = 45.5 ± 5.6 mV), indicating that the interaction with STIM1 affects its ion selectivity ( Figure 4E and 4G). A similar rightward shift in current reversal potential was recorded for cells expressing SS-Orai1 V102A (V rev = 43.9 ± 6.5, Figure 4F and 4G).…”

Section: Mutations In the Orai1 Tm4-cbd Linker Abolish Activation Butsupporting

confidence: 79%

“…The loss of activity did not result from incorrect protein folding or loss of PM expression, as the constitutively activating V102A mutation restored conductance to both SS-Orai1 ∆C 273-301 and ∆Ν 1-77 Orai1-SS ( Figure 2G). In view of the earlier demonstration that interaction of soluble S1C with the Orai1 NBD is essential for gating and that a similar NBD truncation significantly reduces this interaction in vitro [28], the lack of activation of ∆Ν Orai1 by either co-expressed or C-terminally tethered S1C likely arises from a disruption of S1C binding to the truncated NBD. Similarly, loss of activation of ∆C Orai1 by N-terminally tethered S1C likely arises from a disruption of S1C binding to the truncated CBD.…”

Section: Direct Role In Crac Channel Activation For Interaction Of S1mentioning

confidence: 99%

“…A recent study found that N-terminal truncations of Orai1 to residue S75 retained STIM1-dependent channel activity, but that further deletion past this point abolished activity [28]. In light of the differences in efficacy in Orai1 activation between the full-length STIM1 and CAD, we first confirmed this key observation and found that the truncation mutant (Orai1 ∆N1-76) displays normal PM expression but could not be activated by S1C ( Figures 2E and Supplementary information, Figure S3).…”

Section: Direct Role In Crac Channel Activation For Interaction Of S1mentioning

confidence: 99%

“…Dimerization of STIM1 ER luminal domains triggers extensive conformational changes in the cytoplasmic domains that lead to the exposure of CAD [20,[22][23][24][25][26]. CAD binds with low affinity to a site in the Orai1 N terminal (NBD, residues 73-87; Figure 1A) and with higher affinity to a site in the Orai1 C terminal (CBD, residues 267-292; Figure 1A) [14,15,28,29]. The interaction of activated STIM1 with Orai1 results in clustering of STIM1-Orai1 at sites of ER- PM interface and in pore opening of CRAC channels that mediates localized and selective calcium influx into cells [3,9,[30][31][32][33][34][35].…”

Section: Introductionmentioning

confidence: 99%

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Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

2015

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Calcium flux through store-operated calcium entry is a major regulator of intracellular calcium homeostasis and various calcium signaling pathways. Two key components of the store-operated calcium release-activated calcium channel are the Ca 2+-sensing protein stromal interaction molecule 1 (STIM1) and the channel pore-forming protein Orai1. Following calcium depletion from the endoplasmic reticulum, STIM1 undergoes conformational changes that unmask an Orai1-activating domain called CAD. CAD binds to two sites in Orai1, one in the N terminal and one in the C terminal. Most previous studies suggested that gating is initiated by STIM1 binding at the Orai1 N-terminal site, just proximal to the TM1 pore-lining segment, and that binding at the C terminal simply anchors STIM1 within reach of the N terminal. However, a recent study had challenged this view and suggested that the Orai1 C-terminal region is more than a simple STIM1-anchoring site. In this study, we establish that the Orai1 C-terminal domain plays a direct role in gating. We identify a linker region between TM4 and the C-terminal STIM1-binding segment of Orai1 as a key determinant that couples STIM1 binding to gating. We further find that Proline 245 in TM4 of Orai1 is essential for stabilizing the closed state of the channel. Taken together with previous studies, our results suggest a dual-trigger mechanism of Orai1 activation in which binding of STIM1 at the N-and C-terminal domains of Orai1 induces rearrangements in proximal membrane segments to open the channel.

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Section: Mutations In the Orai1 Tm4-cbd Linker Abolish Activation Butsupporting

confidence: 79%

Section: Direct Role In Crac Channel Activation For Interaction Of S1mentioning

confidence: 99%

Section: Direct Role In Crac Channel Activation For Interaction Of S1mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

2015

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“…Also, in the previous studies and our own studies, the linkers are placed immediately upstream of the Orai1 N terminus of each conjoined Orai1 subunit. This N-terminal segment has ϳ70 amino acids that are redundant in the function of Orai1 channels (35,36), and secondary structure analysis reveals little predicted secondary structure. These flexible 70 amino acids may therefore assist in linking the Orai1 units, making it still less clear why the 6-versus the 36-amino acid linker would selectively affect the function of Orai1 concatemers.…”

Section: Design and Expression Of Orai1 Concatemers In A Nullmentioning

confidence: 99%

The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Cai

Zhou

Nwokonko

et al. 2016

Journal of Biological Chemistry

102

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Edited by Roger ColbranOrai channels mediate store-operated Ca 2؉ 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 fulllength concatemers were surface membrane-expressed. Unexpectedly, Orai1 dimers, trimers, tetramers, pentamers, and hexamers all mediated similar and substantial store-operated Ca 2؉ entry. Moreover, each Orai1 concatemer mediated Ca 2؉ 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 Ca 2؉ selectivity filter-forming Glu-106 residue may mediate Orai1 channel assembly around a central Ca 2؉ 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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Gating and regulation of the calcium release‐activated calcium channel: Recent progress from experiments and molecular modeling

Huo

Dong

2020

Biopolymers

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Calcium release-activated calcium (CRAC) channels are highly calcium ion (Ca 2+)-selective channels in the plasma membrane. The transient drop of endoplasmic reticulum Ca 2+ level activates its calcium sensor stromal interaction molecule (STIM) and then triggers the gating of the CRAC channel pore unit Orai. This process involves a variety of activities of the immune system. Therefore, understanding how the activation and regulation of the CRAC channel can be accomplished is essential. Here we briefly summarize the recent progress on Orai gating and its regulation by 2-aminoethoxydiphenylborate (2-APB) obtained from structural biology studies, biochemical and electrophysiological measurements, as well as molecular modeling. Indeed, integration between experiments and computations has further deepened our understanding of the channel gating and regulation.

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The Extended Transmembrane Orai1 N-terminal (ETON) Region Combines Binding Interface and Gate for Orai1 Activation by STIM1

Cited by 103 publications

References 38 publications

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

Critical role for Orai1 C-terminal domain and TM4 in CRAC channel gating

The Orai1 Store-operated Calcium Channel Functions as a Hexamer

Gating and regulation of the calcium release‐activated calcium channel: Recent progress from experiments and molecular modeling

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