Transmembrane helix connectivity in Orai1 controls two gates for calcium-dependent transcription

Abstract

The channel Orai1 requires Ca store depletion in the endoplasmic reticulum and an interaction with the Ca sensor STIM1 to mediate Ca signaling. Alterations in Orai1-mediated Ca influx have been linked to several pathological conditions including immunodeficiency, tubular myopathy, and cancer. We screened large-scale cancer genomics data sets for dysfunctional Orai1 mutants. Five of the identified Orai1 mutations resulted in constitutively active gating and transcriptional activation. Our analysis showed that certain Orai1 mutations were clustered in the transmembrane 2 helix surrounding the pore, which is a trigger site for Orai1 channel gating. Analysis of the constitutively open Orai1 mutant channels revealed two fundamental gates that enabled Ca influx: Arginine side chains were displaced so they no longer blocked the pore, and a chain of water molecules formed in the hydrophobic pore region. Together, these results enabled us to identify a cluster of Orai1 mutations that trigger Ca permeation associated with gene transcription and provide a gating mechanism for Orai1.

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

“…Somewhat surprisingly, Frischauf et al (2017) also did not observe the spontaneous thermal pore helix rotations/twisting motions at F99 in their MD simulations, in contrast to our studies (Yamashita et al, 2017;Yeung et al, 2018). These differences may result from divergent computational modeling methodologies, including the choice of force field, homology modeling of the human Orai1 channel, presence of counterions and excess salt (not reported in Frischauf et al, 2017), introduction of bound cholesterol molecules in Frischauf et al (2017), and extent of sampling. However, the lack of noticeable K163-S162 interactions in our simulations is consistent with experimental findings indicating that mutations of R91 or S90 that would be expected to disrupt a putative S90-R91 interaction do not impede STIM1mediated channel gating (Fig.…”

“…1 A) facing the ion permeation pathway, and R91 in particular, control Orai1 gating by repelling Ca 2+ ions or binding Cl − ions to create an anion plug that blocks conduction in closed channels (Zhang et al, 2011;Hou et al, 2012;Frischauf et al, 2017). Channel activation was suggested to occur due to displacement of R91 away from the pore axis, which presumably would lower an electrostatic energy barrier presented by this residue, or via dilation of the inner pore, leading to release of the anion plug (Zhang et al, 2011;Hou et al, 2012;Frischauf et al, 2017). These gating models make specific predictions regarding the consequences of charge neutralization mutations on closed channel stability and channel opening, which we tested as described below.…”

“…Disrupting a putative interaction between R91 and S90 does not interfere with Orai1 gating From the analysis of side-chain fluctuations using MD simulations, a recent study concluded that in the constitutively active Orai1 mutant H134A, R91 side-chains interact with nearby S90 residues from the neighboring subunits (Frischauf et al, 2017). This interaction was postulated to displace R91 away from the pore and drive pore opening (Frischauf et al, 2017). A simple prediction from this model is that mutations at S90 that interrupt this interaction should inhibit STIM1-mediated channel gating.…”

“…Yet, the story is not so simple. In addition to the outer hydrophobic gate described above, several reports have suggested that the CRAC channel pore also harbors a putative second gate, located at the cytoplasmic end of the pore (Zhang et al, 2011;Hou et al, 2012;Frischauf et al, 2017). This idea has its roots in a peculiar feature of the CRAC channel, three rings of positively charged residues formed by R91, K87, and R83 located in the TM1 extension helix, which collectively present a cluster of 18 positive charges in the inner pore.…”

ORAI1 channel gating and selectivity is differentially altered by natural mutations in the first or third transmembrane domain

“…To assess the structural effects of the V107M and T184M mutations on the residues facing the channel pore, we performed MDSs based on the closed conformation of our hORAI1 model, using two ORAI1 mutants previously shown to be constitutively open, H134C and H134S (Frischauf et al . ; Yeung et al . ), as positive controls.…”