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Mapping the Functional Anatomy of Orai1 Transmembrane Domains for CRAC Channel Gating

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Mapping the Functional Anatomy of Orai1 Transmembrane Domains for CRAC Channel Gating Mapping the functional anatomy of Orai1 transmembrane domains for CRAC channel gating Priscilla S.-W. Yeunga, Megumi Yamashitaa, Christopher E. Ingb,c, Régis Pomèsb,c, Douglas M. Freymannd, and Murali Prakriyaa,1 aDepartment of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL 60611; bMolecular Medicine, The Hospital for Sick Children Research Institute, Toronto, ON, Canada M5G 0A4; cDepartment of Biochemistry, University of Toronto, Toronto, ON, Canada M5S 1A8; and dDepartment of Biochemistry and Molecular Genetics, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 Edited by Michael D. Cahalan, University of California, Irvine, CA, and approved April 17, 2018 (received for review October 23, 2017) Store-operated Orai1 channels are activated through a unique contains four transmembrane helices (TMs) arranged in concen- inside-out mechanism involving binding of the endoplasmic re- tric layers, with the centrally located TM1 helices lining the pore, + ticulum Ca2 sensor STIM1 to cytoplasmic sites on Orai1. Although TM2 and TM3 in the next layer, and TM4 forming the most ex- atomic-level details of Orai structure, including the pore and puta- ternal, lipid-exposed segment (7). Recent studies indicated that tive ligand binding domains, are resolved, how the gating signal is the channel gate is formed, at least in part, by the combination of communicated to the pore and opens the gate is unknown. To ad- two hydrophobic pore residues, F99 and V102, which regulate the dress this issue, we used scanning mutagenesis to identify 15 resi- closed–open transition (8–11). STIM1 binding is proposed to open – dues in transmembrane domains (TMs) 1 4 whose perturbation this gate by inducing a modest rotation of the pore helix to move activates Orai1 channels independently of STIM1. Cysteine accessi- the bulky F99 side chains away from the pore axis to lower the bility analysis and molecular-dynamics simulations indicated that energy barrier for ion conduction (11). constitutive activation of the most robust variant, H134S, arises A major unresolved question in this process is the molecular from a pore conformational change that opens a hydrophobic gate mechanism by which the STIM1 gating signal is communicated to to augment pore hydration, similar to gating evoked by STIM1. the Orai1 pore. Although not considered in most early studies, Mutational analysis of this locus suggests that H134 acts as steric there is increasing evidence from genetic disease association studies PHYSIOLOGY brake to stabilize the closed state of the channel. In addition, atomic in human patients that the transmembrane domains of Orai1 may packing analysis revealed distinct functional contacts between the TM1 pore helix and the surrounding TM2/3 helices, including one set be critically involved in this process. In particular, several reports mediated by a cluster of interdigitating hydrophobic residues and have described patients with tubular aggregate myopathy, throm- another by alternative ridges of polar and hydrophobic residues. bocytopenia, and congenital miosis caused by gain-of-function – Perturbing these contacts via mutagenesis destabilizes STIM1- (GOF) Orai1 mutations located within the TM domains (12 15). – mediated Orai1 channel gating, indicating that these bridges be- In addition, recent structure function analysis of several GOF tween TM1 and the surrounding TM2/3 ring are critical for convey- mutations including that of a putative “hinge” at the base of TM4 ing the gating signal to the pore. These findings help develop a (16), a Pro residue in TM4 (17), and several residues cataloged in a framework for understanding the global conformational changes cancer genomics database (18), provide more direct support for and allosteric interactions between topologically distinct domains involvement of the non–pore-lining TMs in Orai1 gating. These that are essential for activation of Orai1 channels. findings suggest that, as seen in many ligand-gated channels (19), CRAC channels | Orai1 | STIM1 | calcium | store-operated calcium entry Significance + + a2 release-activated Ca2 (CRAC) channels mediate store- Store-operated Orai1 channels mediate transcriptional, pro- + + Coperated Ca2 entry, an essential mechanism for Ca2 influx liferative, and effector-cell programs in many cells. Mutations in in many cells that is triggered in response to depletion of en- Orai1 that block channel activation or evoke constitutive channel + doplasmic reticulum (ER) Ca2 stores (1). Opening of CRAC activity are known to cause debilitating diseases in humans such + channels evokes local and global increases in intracellular Ca2 , as immunodeficiency, autoimmunity, myopathy, and thrombo- + which not only refills ER Ca2 stores but also drives numerous cytopenia. However, our understanding of the underlying mo- effector functions such as gene expression, cell proliferation, lecular mechanisms of these diseases is limited by fundamental secretion of inflammatory mediators, and cell migration (1, 2). gaps in how Orai1 channels are gated. Here, we map key func- The importance of CRAC channels for human health is high- tional interactions between the transmembrane domains of lighted by mutations in CRAC channel proteins that give rise to Orai1 and identify several contacts that are critical for conveying severe immunodeficiency, autoimmunity, muscle weakness, skin the STIM1 gating signal to the pore. Our findings illuminate and tooth defects, and thrombocytopenia (3, 4). important allosteric interactions between topologically distinct Activation of CRAC channels by store depletion occurs through domains of Orai1 and help elucidate the molecular underpin- a unique inside-out mechanism whose broad contours are now nings of disease-causing mutations. well established. In the first step of the process, depletion of ER 2+ 2+ Author contributions: P.S.-W.Y., M.Y., C.E.I., R.P., D.M.F., and M.P. designed research; Ca stores mobilizes the ER Ca sensor STIM1 into its active P.S.-W.Y., M.Y., C.E.I., R.P., D.M.F., and M.P. performed research; D.M.F. contributed state, which exposes a catalytic domain in its cytoplasmic region, new reagents/analytic tools; P.S.-W.Y., M.Y., C.E.I., R.P., D.M.F., and M.P. analyzed data; known as the CRAC activation domain (CAD) (5) or STIM1- and P.S.-W.Y., M.Y., C.E.I., R.P., D.M.F., and M.P. wrote the paper. Orai1 activating region (SOAR) (6). STIM1 then oligomerizes The authors declare no conflict of interest. and migrates from the bulk ER to the ER–plasma membrane This article is a PNAS Direct Submission. junctions (1). In the next step, the CAD/SOAR domain of Published under the PNAS license. STIM1 directly binds to the CRAC channel pore subunit Orai1, 1To whom correspondence should be addressed. Email: [email protected]. setting into motion conformational changes in the Orai1 protein This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. that culminate in the opening of the channel gate. At the struc- 1073/pnas.1718373115/-/DCSupplemental. tural level, each Orai1 subunit of the hexameric CRAC channel www.pnas.org/cgi/doi/10.1073/pnas.1718373115 PNAS Latest Articles | 1of10 Downloaded by guest on September 29, 2021 local conformational changes at the agonist binding site (16, 20, 21) bular aggregate myopathy with additional symptoms including are allosterically relayed to the distally located channel gate in the thrombocytopenia and miosis (12–15). The identification of pore (8, 11) through an unknown pathway. GOF mutations in the non–pore-lining segments suggests that The discovery of pathological human mutations, along with these domains of Orai1 may play a greater role in channel ac- the increasing evidence from the structure–function studies de- tivation than previously appreciated. To begin addressing this scribed above, prompted us to examine the structural basis of question, we started our study with an unbiased mutagenesis how the non–pore-lining TMs regulate channel activation. By screen to identify residues in the TMs that could regulate using scanning mutagenesis, state-dependent accessibility analy- Orai1 channel activation. There are 130 transmembrane residues sis, molecular-dynamics (MD) simulations, and atomic packing in the human Orai1 sequence based on comparison with the analysis, we generated a functional map of key interactions be- crystal structure of highly homologous Drosophila melanogaster tween the transmembrane domains of Orai1 that are critical for Orai (dOrai) protein [Protein Data Bank (PDB) ID code conveying the gating signal to the CRAC channel pore. 4HKR)] (7), with three endogenous Cys residues (C126, C145, C196) in TM2 and TM3. We sequentially mutated each of the Results non-Cys Orai1 transmembrane residues to generate 127 DNA Scanning Mutagenesis of Orai1 TMs Reveals Numerous Constitutively constructs, each containing a single Cys mutation. These mutants Active CRAC Channels. Several recent studies have described GOF were transfected into HEK293 cells in the absence of STIM1 and missense Orai1 mutations in TMs 2–4, with some linked to tu- tested by whole-cell mode patch clamping. Cys substitutions were A TM1 (Pore Helix) TM2 TM3 TM4 F199C Q108C L119C A235C K198C L120C A236C V197C V107C I121C W196C I237C Extracellular E106C A122C C195 A238C F123C L194C S239C V105C L193C S124C T240C V192C A125C M104C V191C T241C C126 A103C E190C I242C A189C T127C M243C V102C T128C L188C F187C V244C V129C M101C L186C P245C L130C L185C F246C A100C V131C T184C G247C G183C F99C A132C I182C L248C V133C V181C G98C I249C H134C T180C F250C S97C L135C S179C I251C F136C F178C L96C A177C V252C A137C W176C F253C L138C L95C A175C Intracellular A254C M139C L174C A94C I140C E173C V255C I172C S93C S141C H256C H171C T142C F257C T92C R170C C143 H169C Y258C R91C S144C M168C R259C R167C L145C S260C S90C E166C P146C L261C C C H165C C C S89C N N147C N P164C N V262C N 0 -10 -20 -30 -40 -50 0 -10 -20 -30 0 -5 -10 -15 0 -5 -10 -15 I (pA/pF) I (pA/pF) I (pA/pF) I (pA/pF) B Top View Top View Side Views Pore Pore Pore 90° 180° TM1 Helix TM2 Helix TM3 Helix TM4 Helix Cytosolic Extensions Fig.
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