Molecular Mechanisms of STIM1-Mediated Orai-1 Channel Activation

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