Rainer Schindl

BioTechMed-Graz

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Christoph Romanin

Johannes Kepler University of Linz

Martin Muik

Johannes Kepler University of Linz

Marc Fahrner

Johannes Kepler University of Linz

Isabella Derler

Johannes Kepler University of Linz

Irene Frischauf

Johannes Kepler University of Linz

Klaus Groschner

Medical University of Graz

Tony Schmidt

BioTechMed-Graz

Reinhard Fritsch

Johannes Kepler University of Linz

Linda Waldherr

Medical University of Graz

Judith Bergsmann

Johannes Kepler University of Linz

Cooperative Institutions

Inserm

162

Centre National de la Recherche Scientifique

139

University of Michigan–Ann Arbor

126

Harvard University

120

National Institutes of Health

Chinese Academy of Sciences

Medical University of Graz

Istituti di Ricovero e Cura a Carattere Scientifico

Johannes Kepler University of Linz

Consejo Superior de Investigaciones Científicas

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Mechanisms of STIM1 Activation of Store-Independent Leukotriene C₄-Regulated Ca²⁺ Channels

Molecular and Cellular Biology (2013)

Xuexin Zhang José C. González‐Cobos Rainer Schindl Martin Muik Brian Ruhle

We recently showed, in primary vascular smooth muscle cells (VSMCs), that the platelet-derived growth factor activates canonical store-operated Ca2+ entry and Ca2+ release-activated Ca2+ currents encoded by Orai1 and STIM1 genes. However, thrombin activates store-independent Ca2+ selective channels contributed by both Orai3 and Orai1. These store-independent Orai3/Orai1 channels are gated by cytosolic leukotriene C4 (LTC4) and require STIM1 downstream LTC4 action. However, the source of LTC4 and the signaling mechanisms of STIM1 in the activation of this LTC4-regulated Ca2+ (LRC) channel are unknown. Here, we show that upon thrombin stimulation, LTC4 is produced through the sequential activities of phospholipase C, diacylglycerol lipase, 5-lipo-oxygenease, and leukotriene C4 synthase. We show that the endoplasmic reticulum-resident STIM1 is necessary and sufficient for LRC channel activation by thrombin. STIM1 does not form sustained puncta and does not colocalize with Orai1 either under basal conditions or in response to thrombin. However, STIM1 is precoupled to Orai3 and Orai3/Orai1 channels under basal conditions as shown using Forster resonance energy transfer (FRET) imaging. The second coiled-coil domain of STIM1 is required for coupling to either Orai3 or Orai3/Orai1 channels and for LRC channel activation. We conclude that STIM1 employs distinct mechanisms in the activation of store-dependent and store-independent Ca2+ entry pathways.

Orai1

TRPC3

Leukotriene C4

10.1128/mcb.00554-13

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Citations (55)

Twisting gating residues in the Orai pore

Cell Calcium (2020)

Daniel Bonhenry Romana Schober Rainer Schindl

Funnel

Orai1

10.1016/j.ceca.2020.102323

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Citations (2)

Protein–Protein Interactions in TRPC Channel Complexes

Frontiers in neuroscience (2006)

Petra Eder Klaus Groschner Rainer Schindl Christoph Romanin

Screening for homologues of the Drosophila trp (transient receptor potential) gene product has uncovered a large family of membrane proteins of which the closest relatives to the Drosophila protein have been assigned to the canonical or classical subfamily (TRPC). The prominent physiological function of these proteins, as delineated from heterologous expression and knockdown experiments in native cells, appears to be sensing phospholipase C (PLC)–derived stimuli and conversion of this input into cellular Ca2+ signals. Another common feature of TRPC proteins is the ability to form cation-conducting pore structures. Thus, the role of TRPCs in PLC-dependent Ca2+ signaling has been attributed to the formation of regulated Ca2+ entry channels. Despite the existence of this unifying principle of TRPC signal transduction, an unforeseen complexity of the roles of TRPC proteins within the cellular Ca2+ signaling network emerged. The contribution of TRPC proteins to cellular Ca2+homeostasis may involve formation of a wide variety of different Ca2+ entry pathways that contain distinct TRPC homo-or heteromultimeric pore structures, along with a multitude of regulatory proteins and scaffolds. Here, we summarize current knowledge on protein–protein interactions that are of potential significance for the formation and function of native TRPC channel complexes and highlight recent concepts regarding the role of these interactions for cellular control of plasma membrane cation conductances and cellular Ca2+ signaling.

10.1201/9781420005844.ch24

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Citations (6)

Store-Independent Orai1/3 Channels Activated by Intracrine LeukotrieneC ₄

Circulation Research (2013)

José C. González‐Cobos Xuexin Zhang Wei Zhang Brian Ruhle Rajender K. Motiani

Through largely unknown mechanisms, Ca(2+) signaling plays important roles in vascular smooth muscle cell (VSMC) remodeling. Orai1-encoded store-operated Ca(2+) entry has recently emerged as an important player in VSMC remodeling. However, the role of the exclusively mammalian Orai3 protein in native VSMC Ca(2+) entry pathways, its upregulation during VSMC remodeling, and its contribution to neointima formation remain unknown.The goal of this study was to determine the agonist-evoked Ca(2+) entry pathway contributed by Orai3; Orai3 potential upregulation and role during neointima formation after balloon injury of rat carotid arteries.Ca(2+) imaging and patch-clamp recordings showed that although the platelet-derived growth factor activates the canonical Ca(2+) release-activated Ca(2+) channels via store depletion in VSMC, the pathophysiological agonist thrombin activates a distinct Ca(2+)-selective channel contributed by Orai1, Orai3, and stromal interacting molecule1 in the same cells. Unexpectedly, Ca(2+) store depletion is not required for activation of Orai1/3 channel by thrombin. Rather, the signal for Orai1/3 channel activation is cytosolic leukotrieneC4 produced downstream thrombin receptor stimulation through the catalytic activity of leukotrieneC4 synthase. Importantly, Orai3 is upregulated in an animal model of VSMC neointimal remodeling, and in vivo Orai3 knockdown inhibits neointima formation.These results demonstrate that distinct native Ca(2+)-selective Orai channels are activated by different agonists/pathways and uncover a mechanism whereby leukotrieneC4 acts through hitherto unknown intracrine mode to elicit store-independent Ca(2+) signaling that promotes vascular occlusive disease. Orai3 and Orai3-containing channels provide novel targets for control of VSMC remodeling during vascular injury or disease.

Neointima

Orai1

TRPC3

10.1161/circresaha.111.300220

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Citations (114)

A series of Orai1 gating checkpoints in transmembrane and cytosolic regions requires clearance for CRAC channel opening: Clearance and synergy of Orai1 gating checkpoints controls pore opening

bioRxiv (Cold Spring Harbor Laboratory) (2020)

Adéla Tiffner Romana Schober Carmen Höglinger Daniel Bonhenry Saurabh Pandey

Abstract The initial activation step in gating of ubiquitously expressed Orai1 Calcium (Ca 2+ ) ion channels represents the store-dependent coupling to the Ca 2+ sensor protein STIM1. An array of constitutively active Orai1 mutants gave rise to the hypothesis that STIM1 mediated Orai1 pore opening is accompanied by a global conformational change of all Orai TM helices within the channel complex. Here, we prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that a global, opening-permissive allosteric communication of TM helices is indispensable for pore opening and requires clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in middle and cytosolic extended TM domain regions. Our findings are based on a library of double point mutants that contain each one loss-of-function (LoF) with one gain-of-function (GoF) point mutation in a series of possible combinations. We demonstrated that an array of LoF mutations act dominant over most GoF mutations within the same as well as of an adjacent Orai subunit. We further established inter- and intramolecular salt-bridge interactions of Orai subunits as a core element of an opening-permissive Orai channel architecture. Collectively, clearance and synergistic action of all these gating checkpoints is required to allow STIM1 coupling and Orai1 pore opening. Graphical Abstract

Orai1

10.1101/2020.07.16.207183

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Citations (2)

Blockage of Store-Operated Ca2+ Influx by Synta66 is Mediated by Direct Inhibition of the Ca2+ Selective Orai1 Pore

Cancers (2020)

Linda Waldherr Adéla Tiffner Deepti Mishra Matthias Sallinger Romana Schober

The Ca2+ sensor STIM1 and the Ca2+ channel Orai1 that form the store-operated Ca2+ (SOC) channel complex are key targets for drug development. Selective SOC inhibitors are currently undergoing clinical evaluation for the treatment of auto-immune and inflammatory responses and are also deemed promising anti-neoplastic agents since SOC channels are linked with enhanced cancer cell progression. Here, we describe an investigation of the site of binding of the selective inhibitor Synta66 to the SOC channel Orai1 using docking and molecular dynamics simulations, and live cell recordings. Synta66 binding was localized to the extracellular site close to the transmembrane (TM)1 and TM3 helices and the extracellular loop segments, which, importantly, are adjacent to the Orai1-selectivity filter. Synta66-sensitivity of the Orai1 pore was, in fact, diminished by both Orai1 mutations affecting Ca2+ selectivity and permeation of Na+ in the absence of Ca2+. Synta66 also efficiently blocked SOC in three glioblastoma cell lines but failed to interfere with cell viability, division and migration. These experiments provide new structural and functional insights into selective drug inhibition of the Orai1 Ca2+ channel by a high-affinity pore blocker.

Orai1

Docking (animal)

10.3390/cancers12102876

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Citations (33)

STIM1 couples to ORAI1 via an intramolecular transition into an extended conformation

The EMBO Journal (2011)

Martin Muik Marc Fahrner Rainer Schindl Peter B. Stathopulos Irene Frischauf

Orai1

10.1038/emboj.2011.79

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Citations (219)

CRAC channel opening is determined by a series of Orai1 gating checkpoints in the transmembrane and cytosolic regions

Journal of Biological Chemistry (2020)

Adéla Tiffner Romana Schober Carmen Höglinger Daniel Bonhenry Saurabh Pandey

The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca

Orai1

10.1074/jbc.ra120.015548

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Citations (24)

STIM1/Orai1 coiled-coil interplay in the regulation of store-operated calcium entry

Nature Communications (2013)

Peter B. Stathopulos Rainer Schindl Marc Fahrner Le Zheng Geneviève M. C. Gasmi-Seabrook

Orai1 calcium channels in the plasma membrane are activated by stromal interaction molecule-1 (STIM1), an endoplasmic reticulum calcium sensor, to mediate store-operated calcium entry (SOCE). The cytosolic region of STIM1 contains a long putative coiled-coil (CC)1 segment and shorter CC2 and CC3 domains. Here we present solution nuclear magnetic resonance structures of a trypsin-resistant CC1–CC2 fragment in the apo and Orai1-bound states. Each CC1–CC2 subunit forms a U-shaped structure that homodimerizes through antiparallel interactions between equivalent α-helices. The CC2:CC2′ helix pair clamps two identical acidic Orai1 C-terminal helices at opposite ends of a hydrophobic/basic STIM–Orai association pocket. STIM1 mutants disrupting CC1:CC1′ interactions attenuate, while variants promoting CC1 stability spontaneously activate Orai1 currents. CC2 mutations cause remarkable variability in Orai1 activation because of a dual function in binding Orai1 and autoinhibiting STIM1 oligomerization via interactions with CC3. We conclude that SOCE is activated through dynamic interplay between STIM1 and Orai1 helices. When endoplasmic reticulum calcium levels are low, STIM1 binds to and opens Orai1 channels in the plasma membrane to replenish calcium stores. Stathopulos et al.present solution structures of the STIM1 coiled-coil domain in the presence and absence of Orai1, revealing the structural basis for this interaction.

Orai1

Coiled coil

Antiparallel (mathematics)

Helix (gastropod)

10.1038/ncomms3963

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Citations (192)

Shedding Light on Cardiac Excitation: In Vitro and In Silico Analysis of Native Ca²⁺ Channel Activation in Guinea Pig Cardiomyocytes Using Organic Photovoltaic Devices

IEEE Transactions on Biomedical Engineering (2024)

Theresa Rienmüller Niroj Shrestha Mathias Polz Sara Stoppacher Daniel Ziesel

Objective: This study aims to explore the potential of organic electrolytic photocapacitors (OEPCs), an innovative photovoltaic device, in mediating the activation of native voltage-gated Cav1.2 channels ( I Ca,L) in Guinea pig ventricular cardiomyocytes. Methods: Whole-cell patch-clamp recordings were employed to examine light-triggered OEPC mediated I Ca,L activation, integrating the channel's kinetic properties into a multicompartment cell model to take intracellular ion concentrations into account. A multidomain model was additionally incorporated to evaluate effects of OEPC-mediated stimulation. The final model combines external stimulation, multicompartmental cell simulation, and a patch-clamp amplifier equivalent circuit to assess the impact on achievable intracellular voltage changes. Results: Light pulses activated I Ca,L, with amplitudes similar to voltage-clamp activation and high sensitivity to the L-type Ca ²⁺ channel blocker, nifedipine. Light-triggered I Ca,L inactivation exhibited kinetic parameters comparable to voltage-induced inactivation. Conclusion: OEPC-mediated activation of I Ca,L demonstrates their potential for nongenetic optical modulation of cellular physiology potentially paving the way for the development of innovative therapies in cardiovascular health. The integrated model proves the light-mediated activation of I Ca,L and advances the understanding of the interplay between the patch-clamp amplifier and external stimulation devices. Significance: Treating cardiac conduction disorders by minimal-invasive means without genetic modifications could advance therapeutic approaches increasing patients' quality of life compared with conventional methods employing electronic devices.

10.1109/tbme.2024.3358240

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Citations (1)