Pore properties of Orai1 calcium channel dimers and their activation by the STIM1 ER calcium sensor

Abstract

Store-operated Ca entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Orai structure, mammalian Orai1 channels are hexamers comprising three dimeric subunit pairs. We utilized concatenated Orai1 dimers to probe the function of key domains within the channel pore and gating regions. The Orai1-E106Q selectivity-filter mutant, widely considered a dominant pore blocker, was surprisingly nondominant within concatenated heterodimers with Orai1-WT. The Orai1-E106Q/WT heterodimer formed STIM1-activated nonselective cation channels with significantly enlarged apparent pore diameter. Other Glu-106 substitutions entirely blocked the function of heterodimers with Orai1-WT. The hydrophobic pore-lining mutation V102C, which constitutively opens channels, was suppressed by Orai1-WT in the heterodimer. In contrast, the naturally occurring R91W pore-lining mutation associated with human immunodeficiency was completely dominant-negative over Orai-WT in heterodimers. Heterodimers containing the inhibitory K85E mutation extending outward from the pore helix gave an interesting partial effect on both channel activation and STIM1 binding, indicating an important allosteric link between the cytosolic Orai1 domains. The Orai1 C-terminal STIM1-binding domain mutation L273D powerfully blocked STIM1-induced channel activation. The Orai1-L273D/WT heterodimer had drastically impaired STIM1-induced channel gating but, unexpectedly, retained full STIM1 binding. This reveals the critical role of Leu-273 in transducing the STIM1-binding signal into the allosteric conformational change that initiates channel gating. Overall, our results provide important new insights into the role of key functional domains that mediate STIM1-induced gating of the Orai1 channel.

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

“…In line with preserved STIM1 mediated activation of Orai1 H134W-Orai1, also Orai1 H134W-Orai1 S239C exhibited store-dependent activation upon co-expression with STIM1 to a similar extent as for Orai1-Orai1 H134A (Fig 6 f, h). Interestingly, also Orai1 K85E-Orai1 H134A displayed store-operated currents (Fig 6 g), in accordance with the activity of Orai1 K85E-Orai1 dimer published in Cai et al 63 . In contrast, Orai1 E149K -Orai1 H134A and Orai1 L174D -Orai1 H134A remained inactive in the presence of STIM1 (Fig 6 g, h).…”

“…Initially, we generated dimers with one wild-type subunit and one subunit, containing the constitutive MTR-GoF-H134A or -P245L mutation. In contrast to the strong constitutive activity of these point mutations in the homomer, in the heteromer, they allow only store-operated activation in the presence of STIM1 ( Fig 6 h; Supp Fig 10) in line with the store-dependent activation of an Orai1 dimer containing one wild-type and one Orai1 V102A subunit 63 . This indicates that at least more than three GoF mutations within a hexamer are required to induce constitutive activity.…”

“…(62). Both wild type and Orai mutants were embedded in a pure POPC membrane composed by 388 lipids using the CHARMM-GUI web interface (63). The TIP3p (64) model is used to represent water molecules.…”

The native ORAI channel trio underlies the diversity of Ca2+ signaling events

“…7). Recent studies showed that co-expression of STIM1 with a concatenated heterodimer consisting of WT ORAI1 and ORAI1-E106A in ORAI-SKO cells generated SOCE and membrane currents that were substantially reduced compared with a concatenated heterodimer composed of WT ORAI1 and ORAI1-E106Q 30 . These results suggest that ORAI isoforms heteromerize under native conditions and that Ca 2+ oscillations can be sustained by a minuscule amount of Ca 2+ entry that is below the detectable limit of Fura2.…”

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

“…[38] However, it is still very challenging to establish the structural basis for the formation of the Orai-STIM complex at the activated state, as the fragmented information available is not enough to facilitate spelling out the complete picture of this complicated molecular machinery. [39][40][41][42][43][44][45][46] The pore protein Orai usually assembles as homomultimers, though heterooligomers were also observed. When overexpressed, the hetero-pentameric assembly of subunits could be formed that three Orai1 subunits and two Orai3 subunits integrate into an arachidonic acid-activated channel.…”