Orai1 Concatemers Reveal a Hexameric Orai1 Channel Assembly

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Monday, February 29, 2016

We investigated the effects of simulated acute ischemia on L-type Ca2þ current, ICa-L (pO2 < 5 mmHg, pH 7.4 to 6.8) in short-term cultures (4-7 days) of rat neonatal cardiomyocytes (rN-CM), voltage-clamped in the perforated patch (amphotericin B) configuration. Two types ICa-L were found in these cultures: cells with rapidly inactivating (tau ~ 10 ms) and cells with slowly inactivating ICa (tau > 40ms). The hypoxic suppression of ICa-L showed variable kinetics: It stabilized at ~20% within ~10s in cells with rapid inactivation of ICa-L while in cells with slower inactivation suppression of up to 40% developed over tens of seconds and appeared to involve SR or mitochondria. In both cell types the suppressive effect of acidosis (pH 7.4 to 6.7) on the quickly inactivating ICa-L was larger (~ 40%) and faster than the 25% observed for the slowly inactivating ICaL. These suppressive effects were blunted by phosphorylation (100 nM isoproterenol). The effects of hypoxia and acidosis were additive in cells with quickly inactivating ICa-L, but not in cells where ICa inactivated slowly. Acidosis always slowed the activation of the ICa-L by ~ 25%. Genetically engineered Ca2þ probes, that were targeted to RyR and mitochondria showed that Ca2þ m- domains of RyR were more strongly affected than global cytosolic [Ca2þ] by both hypoxia and low pH, while mitochondrial [Ca2þ] signals were unchanged by acidosis, but were strongly inhibited by hypoxia. Our results suggest that different mechanisms with distinct kinetics mediate the oxygen- and pHsensing of the L-type Ca2þ channel, and consequently the influx of Ca2þ, handling of cytosolic Ca2þ by SR and mitochondria and the conditioning of the cells to acute hypoxic and acidosis stresses. Supported by NIH Ro-1 HL 107600. 1308-Pos Board B285 STIM-Orai Interaction in Schistosoma Mansoni Indicates the Existence of Functional Store-Operated Calcium Entry in the Parasite Ana Eliza Zeraik, Marina Gabriel Fontes, Jose Luiz Souza Lopes, Ana Paula, Ulian Araujo, Ricardo DeMarco. Physics Institute of Sao Carlos (IFSC), Sao Paulo University (USP), Sa˜o Carlos, Brazil. Store-operated calcium entry (SOCE) is a conserved calcium pathway in eukaryotes, although not yet studied in Schistosoma mansoni, the main causative agent of schistosomiasis in America. The proteins STIM1 (calcium sensor) and Orai1 (calcium channel) are the central components of SOCE and the recently published genome of this parasite encodes orthologs of these two proteins, indicating that this Ca2þ pathway is present in S. mansoni. STIM comprises several domains, including three cytosolic coiled coil domains (CC). The first CC (STIM_1CC) is predicted to interact with the other two CC domains in the resting state, and upon activation by calcium depletion, the channel activating domain (CAD), comprised by the second and third CC, is exposed to interact with the C-terminus of Orai, opening the channel. The aim of the present work is to characterize the SOCE pathway in S. mansoni, through the expression of the recombinant proteins from this pathway and interaction assays among STIM and Orai domains, in order to get insights into the mechanism of action of this calcium channel in platyhelminths. S. mansoni STIM_1CC (SmSTIM_1CC), SmSTIM_CAD and SmOrai-C were obtained by recombinant expression in E. coli. Pull down and Isotermic Titration calorimetry experiments showed that SmOrai-C interacts with SmSTIM_CAD in vitro, indicating that the SOCE pathway might be functional in Schistosomes. Additionally, pull down and surface plasmon resonance experiments indicated that SmSTIM_1CC and SmSTIM_CAD also interact with each other, which may consist in a mechanism of autoinhibition of the channel in the resting state, as reported for other organisms. Therefore, the mechanisms by which SmStim activates SmOrai appear highly conserved and the characterization of a new calcium channel for this important human parasite sheds light into possible new targets against schistosomiasis. 1309-Pos Board B286 Inhibitor of Store-Operated Calcium Entry YM58483 Shows Similar Pharmacological Profile to Non-Steroidal Anti-Inflammatory Drug Celecoxib Alexandra S. Gherghina, Aurelien Boillat, Shihab Shah, Nikita Gamper. School of Biomedical Sciences, University of Leeds, Leeds, United Kingdom. Here we show that trifluoromethyl pyrazole derivatives YM58483 (inhibitor of store-operated Ca2þ entry, SOCE) and a non-steroidal anti-inflammatory drug and COX2 inhibitor celecoxib demonstrate similar pharmacological profiles that might explain reported analgesic efficacy of both compounds,

particularly in the inflammatory pain conditions. Thus Fura2 Ca2þ imaging demonstrated that both compounds inhibited SOCE in both HEK293 cells and cultured dorsal root ganglia (DRG) neurons. Thus, in Ca2þ add-back experiments after the store depletion with 1 mM thapsigargin in HEK293 cells both YM58483 and celecoxib inhibited SOCE with IC50 values of 0.41 mM and 1.02 mM, respectively. In similar experiments in cultured DRG neurons both drugs significantly inhibited SOCE, but celecoxib was less efficacious as compared to YM58483. Using total internal reflection fluorescence (TIRF) microscopy we show that thapsigargin induced movement of GFPlabelled endoplasmic reticulum towards the plasma membrane in HEK293 cells, an effect that was prevented by both YM58483 and celecoxib. Since many inflammatory mediators excite nociceptive sensory neurons via G protein coupled receptor cascades involving Ca2þ release from the intracellular stores, SOCE inhibitory activity of both drugs may contribute to their analgesic efficacy in inflammatory conditions. Interestingly, celecoxib was recently shown to augment the activity of inhibitory Kv7 (KCNQ) channels, an effect that also contributes to the analgesic properties of celecoxib. Remarkably, YM58483 also augmented recombinant Kv7.4 and Kv7.5 but was ineffective to augment Kv7.2/Kv7.3 currents. Taken together, our data suggest that both YM58483 and celecoxib inhibit SOCE and augment M currents with both effects potentially contributing to the analgesic properties of these drugs. 1310-Pos Board B287 Zinc Inhibits Orai1-Mediated Calcium Signals in Esophageal Cancer Cells Sangyong Choi1, Chaochu Cui2, Yanhong Luo3, Sun Hee Kim1, Jae-Kyun Ko4, Jianjie Ma5, Irina Korichneva6, Zui Pan7. 1 Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA, 2Sun Yat-Sen University Cancer Institute, Guangzhou, China, 3Department of Endocrinology, The Children’s Hospital of Chongqing Medical University, Chongqing, China, 4Department of Physiology and Biophysics, Rutgers University-Robert Wood Johnson Medical School, Piscataway, NJ, USA, 5Department of Surgery, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA, 6University of Picardie Jules Verne, Amiens, France, 7Department of Internal Medicine, Davis Heart and Lung Research Institute, The Ohio State University Wexner Medical Center, Columbus, OH, USA. Intracellular Ca2þ signals, such as oscillations, regulate proliferation, migration and other cellular events in cancer cells. Zinc (Zn), a major dietary micronutrient, plays important roles in chemoprevention. However, the molecular mechanisms underlying Zn-preventive function as well as the crosstalk with Ca2þ signaling are still unclear. We previously demonstrated that Orai1, a store-operated Ca2þ entry (SOCE) channel, is highly expressed in esophageal squamous cell carcinoma (ESCC) compared to adjacent normal tissues from patients, and the elevated expression of Orai1 is strongly associated with poor prognosis. The present study shows that Zn can greatly inhibit cell proliferation and Orai1-mediated SOCE in ESCC cells. We also found that the inhibitory function of Zn on SOCE is redox sensitive; reducing agent DTT could recover the full activity of Orai1-mediated SOCE. Based on the topology information of Orai1, we hypothesized that a histidine residue in the linker region between two transmembranes of Orai1 (H113) as well as three cysteine residues may play critical roles in the Zn-inhibitory effects. The data showed that, in the cells containing H113A-mutated Orai1, the Zn-inhibitory effect is vanished whereas Zn treatment reduces SOCE in the cell expressing wildtype Orai1. Similarly, the ESCC cells expressing any of three mutations in cysteine residues (C126A, C143A and C195A) of Orai1 displayed partial loss of the Zn-inhibitory function. These results suggest that the H113 and three cysteine residues likely bind Zn, which modulate Zn-inhibitory functions on the Orai1-mediated SOCE in ESCC cells. This study reveals how dietary Zn inhibits SOCE and thus Zn and Ca2þ signals may cross-talk, which in turn regulates cancer cell proliferation and migration in esophageal epithelia. Further investigations are required to search for novel and effective prevention strategies as well as therapeutic options targeting on Zn and Ca2þ signaling in ESCC. 1311-Pos Board B288 Orai1 Concatemers Reveal a Hexameric Orai1 Channel Assembly Xiangyu Cai, Yandong Zhou, Xianming Wang, Natalia Loktionova, Robert Nwokonko, Mohamed Trebak, Donald Gill. Penn State University, Hershey, PA, USA. The multimeric assembly of the CRAC channel has remained a contentious issue despite strong crystallographic evidence of a hexameric structure for

Monday, February 29, 2016 both Drosophila and human Orai1. Recent studies suggested concatemeric tetramers of Orai1 mediate authentic CRAC current whereas equivalent hexameric concatemers form only non-selective cation channels. We expressed concatenated human Orai1 constructs with intervening 36-aa linkers. We observed that expression of dimers, trimers, tetramers, pentamers and hexamers of human Orai1 in HEK cells stably expressing STIM1 (HEK-STIM1 cells), all gave rise to similar high levels of Ca2þ entry. All constructs were C-terminally tagged with tdTomato and all were observed to be exclusively PM-expressed. Each construct also gave similar Ca2þ entry when expressed in MEFS derived from Orai1 knockout mice, the MEFS stably expressing STIM1. Expressed in HEK-STIM1 cells, the Orai1 dimers, trimers, tetramers and hexamers all gave rise to authentic CRAC channel activity with similar inward rectification and reversal potential. Substituting the pore-inactive E106A mutant in tetramers, we observed that the initial two N-terminal residues of the tetramer are crucial for channel activity. The remaining two C-terminal residues in tetramers are inconsequential for function. In hexamers, the position of inactive mutants in the concatemer are not specific. Substitution with a single E106A mutant monomer in the hexamer has the same effect in reducing channel function at each position. Substitution with two E106A residues results in little remaining channel function. We interpret these results to reveal that hexamers are likely the true functional Orai1 channel unit. Tetramers likely feed dimers into a hexameric structure, with the two C-terminal residues outside the hexameric ring, explaining why these last two residues in the tetramer can be E106A without loss of activity. Certainly, the hexameric concatemer gives a fully functional CRAC channel. 1312-Pos Board B289 STIM1-Induced Clustering of Orai1 Channels Robert Nwokonko, Yandong Zhou, Xiangyu Cai, Natalia Loktionova, Xianming Wang, Donald Gill. Penn State University, Hershey, PA, USA. Store operated Ca2þ entry is an evolutionarily conserved mechanism in all eukaryotic cells. Decreased ER Ca2þ promotes a transmembrane conformational shift and unfolding of the cytoplasmic STIM1 domain to expose the STIM-Orai Activating Region (SOAR) of STIM1. SOAR1 couples to and activates PM Orai1 channels, mediating Ca2þ entry signals. The F394H mutation in SOAR prevents it coupling with and activating Orai1. SOAR concatemer-dimers containing a single F394H mutated SOAR unit, fully couple to and activate Orai1, suggesting only one functional monomer of the STIM1 dimer is required for coupling to and activating Orai1 channels, consistent with a stoichiometry of one STIM1 dimer binding to each Orai1 monomer in the hexameric Orai1 channel. Using high-resolution fluorescence imaging approaches, we reveal the existence of dense clusters of YFP-tagged concatenated SOAR-dimers dependent on Orai1-His stably expressed in HEK cells. These clusters are absent in cells expressing heterodimers of concatenated-SOAR dimers containing one single F394H-SOAR residue. Concatenated-SOAR dimers with both monomers mutated with F394H, give no visible clustering. Using HEK cells stably expressing Orai1 C-terminally labeled with CFP, wildtype SOAR dimers did not undergo clustering. Thus, the CFP-label appears to sterically block cluster formation mediated by SOAR dimers. 2-APB is able overcome the inhibitory effect of the F394H mutation in SOAR. Interestingly, 2-APB induced recovery of Orai1-dependent clustering of SOAR concatemer dimers heteromeric for the F394H mutation. The results suggest that the second Orai1 binding site within a SOAR dimer can interact with adjacent Orai1 channels to form clusters of Orai1 channels in the PM. This theory is corroborated by recent electron microscopy data showing Orai1 channels spaced approximately the distance of a SOAR1 dimer. Physiologically, Orai1 cluster formation in ER-PM junctions may be important enhancing activation and deactivation kinetics of Orai1 channels and/or amplifying local Ca2þ signals. 1313-Pos Board B290 Activation Mechanism of the Calcium Release-Activated Calcium Channel Revealed by the Gating Competence of Constitutively Open Orai Mutants Hao Dong. Kuang Yaming Honors School, Nanjing University, Nanjing, China. Calcium release-activated calcium (CRAC) channels in the plasma membrane are integral membrane proteins that is critical in cellular signaling by generating the sustained influx of calcium. The crystal structure of Orai [1], the pore unit of a CRAC channel, provides the first insight into the architecture of this transmembrane protein at atomic level. However, the gating mechanism of CRAC channel remains elusive. Previously, we used the crystal struc-

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ture as the starting point to compare the wild type protein and its V174A mutant in fully hydrated lipid bilayers. We identified that both pore-waters and counterions are actively involved in regulating the channel conductance [2-3]. Nevertheless, the intrinsic flexibility of the channel, especially the pore-forming helices, is more relevant to its gating triggered by the endoplasmic reticulum calcium sensor, the stromal interaction molecule (STIM). In this work, we probe the channel’s conduction properties by exploring the wild type Orai with a more hydrated pore, as well as the STIM1independent constitutively open Orai mutants, G170P and P288A, with combined molecular dynamics and free-energy calculations. The insertion of water molecules is to study the inherent rigidity of the pore, and mutations target at conserved residues on and off the pore-forming helices; all of the three systems were found to have significant pore expansion. In accordance with the motions of the pore-lining residues, ion permeation through the pore of the mutant has a much lower free energy barrier than that in the wild type protein. Therefore the open state structures of Orai obtained from computer simulations provide models at atomic level to study the STIMactivated CRAC gating. [1] Science, 2012, 338, 1308-1313. [2] PNAS, 2013, 110, 1733217337. [3] J. Phys. Chem. B, 2014, 118, 96689676. 1314-Pos Board B291 Molecular Mechanisms of STIM1-Mediated Orai-1 Channel Activation Zainab Haydari, Hengameh Shams, Mohammad R.K. Mofrad. University of California Berkeley, Berkeley, CA, USA. Immune response mechanisms at a cellular level are mostly triggered by a decrease in the concentration of Ca2þ within the endoplasmic reticulum (ER), followed by the opening of the calcium release-activated calcium (CRAC) channels that leads to increased intracellular Ca2þ concentrations. Recently, the stromal interaction molecule (STIM) was determined to be the ER Ca2þ sensor that activates the channel in response to the depletion of intracellular calcium content. However, the molecular details of STIM interaction with Orai that causes the channel opening are not yet known. Understanding these molecular processes would shed light on this signal transduction pathway and opens up new doors for tapping into such a mechanism with profound biological and clinical implications. CRAC channels in the plasma membrane are integral membrane proteins that play a central role in cellular signaling by generating the calcium influx. Orai protein is a pore subunit of the channel, which has 3 homologs (Orai-1, Orai2, and Orai-3). We develop all-atomic molecular dynamics models of the STIM1/Orai-1 complex with the aim to demonstrate how the binding of STIM1 to C-terminus of Orai-1 will result in a conformational change in Orai-1 protein complex leading to the activation of CRAC channels. Different mutations of Orai-1 C-terminus are considered and the influence of factors such as ER tension on the enhancement of this conformational change is explored in accordance with experimental evidences. 1315-Pos Board B292 Impact of STIM1 R304W Mutant on Intra- and Intermolecular Cytosolic Coiled-Coil Interactions Marc Fahrner, Michael Stadlbauer, Martin Muik, Christoph Romanin. University of Linz, Institute for Biophysics, Linz, Austria. STIM1 and Orai1 are key components of the Ca2þ-release activated Ca2þ (CRAC) current which plays an important role in a broad range of cellular/physiological processes including T cell activation as well as cell proliferation, growth and apoptosis. Activation of Orai1 - the CRAC channel forming subunit - occurs via a physical interaction with the ER resident Ca2þ sensor protein STIM1. Upon ER Ca2þ store depletion, STIM1 undergoes extensive structural rearrangements resulting in an activated extended conformation. Interhelical rearrangements between the three cytosolic STIM1 coiled-coil (CC) domains drive this process therefore exposing SOAR/CAD allowing puncta formation and coupling to Orai1 in the cell periphery. Two years ago, the genetically inherited Stormorken Syndrome disease has been linked to the constitutively active human STIM1 R304W mutant (first published by Nesin et al.). In the present study, we focused on intra- and inter-molecular interactions specifically between the three cytosolic STIM1 CC domains comparing the STIM1 R304W mutant to the wildtype system as well as the constitutively active STIM1 L251S mutant employing electrophysiology, the YFP-OASF-CFP-FRET sensor and the recently described FRET based FIRE system. Our results revealed new insights into the mechanism how the disease related mutant R304W yields constitutive activity pointing