Abstract
Store-operated Ca2+ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca2+-sensing STIM proteins in the endoplasmic reticulum (ER). The nature of this elaborate Orai-gating mechanism has remained enigmatic. Based on the Drosophila 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.
Highlights
Store-operated Ca2؉ entry signals are mediated by plasma membrane Orai channels activated through intermembrane coupling with Ca2؉-sensing STIM proteins in the endoplasmic reticulum (ER)
We observed that concatemers containing 2– 6 Orai1 subunits all give rise to authentic CRAC currents, similar to the expressed Orai1 monomer
The Orai1 dimers reliably assembled into hexameric Orai1 channels
Summary
We compared the function of concatenated Orai channels of varying length [25]. The Orai1-E106D mutation has a shortened side chain that results in altered pore geometry and decreased channel selectivity (28 –30) As expected, this mutation is still able to mediate Ca2ϩ entry (Fig. 2A), and co-expressed with Orai1-WT, the mutant has little effect (Fig. 2B). Removing the charge on the Glu-106 by replacing with Gln (but retaining side-chain length) gives a nonconducting pore (Fig. 2C) In this case, co-expression of Orai1-WT with Orai1-E106Q clearly causes inhibition of Ca2ϩ entry (Fig. 2D). We examined the function of a concatenated hexameric Orai construct containing three pairs of OQ or QO dimers (Fig. 4, E–G) The function of both of these two hexamers was very comparable with that of their respective contributing heterodimers. The results we show here for the two heterohexamers would be consistent with either scheme
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