TY - JOUR
T1 - The basic residues in the Orai1 channel inner pore promote opening of the outer hydrophobic gate
AU - Yamashita, Megumi
AU - Ing, Christopher E.
AU - Yeung, Priscilla See Wai
AU - Maneshi, Mohammad M.
AU - Pomès, Régis
AU - Prakriya, Murali
N1 - Funding Information:
Molecular simulations conducted in this work were enabled by supercomputing resources and support provided by SciNet and Compute Canada (www.computecanada.ca). This work was supported by National Institutes of Health grants NS057499 and GM114210 (to M. Prakriya), Canadian Institutes of Health Research grant MOP130461 (to R. Pomès), and National Institutes of Health Predoctoral Fellowship F31NS101830 (to P.S.-W.
Funding Information:
Molecular simulations conducted in this work were enabled by supercomputing resources and support provided by SciNet and Compute Canada (www.computecanada.ca). This work was supported by National Institutes of Health grants NS057499 and GM114210 (to M. Prakriya), Canadian Institutes of Health Research grant MOP130461 (to R. Pom?s), and National Institutes of Health Predoctoral Fellowship F31NS101830 (to P.S.-W. Yeung). Northwestern University?s Center for Advanced Microscopy is supported by National Institutes of Health grant NCRR 1S10 RR031680.
Funding Information:
Yeung). Northwestern University’s Center for Advanced Microscopy is supported by National Institutes of Health grant NCRR 1S10 RR031680. The authors declare no competing financial interests.
Publisher Copyright:
© 2019 Yamashita et al.
PY - 2020/1/6
Y1 - 2020/1/6
N2 - 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.
AB - 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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U2 - 10.1085/JGP.201912397
DO - 10.1085/JGP.201912397
M3 - Article
C2 - 31816637
AN - SCOPUS:85076289239
SN - 0022-1295
VL - 152
SP - 1
EP - 14
JO - Journal of General Physiology
JF - Journal of General Physiology
IS - 1
ER -