RESUMO
The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.
Assuntos
Proteínas de Neoplasias , Multimerização Proteica , Molécula 1 de Interação Estromal , Humanos , Sítios de Ligação , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/química , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/química , Ligação Proteica , Domínios Proteicos , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/químicaRESUMO
The calcium release activated calcium channel is activated by the endoplasmic reticulum-resident calcium sensor protein STIM1. On activation, STIM1 C terminus changes from an inactive, tight to an active, extended conformation. A coiled-coil clamp involving the CC1 and CC3 domains is essential in controlling STIM1 activation, with CC1 as the key entity. The nuclear magnetic resonance-derived solution structure of the CC1 domain represents a three-helix bundle stabilized by interhelical contacts, which are absent in the Stormorken disease-related STIM1 R304W mutant. Two interhelical sites between the CC1α1 and CC1α2 helices are key in controlling STIM1 activation, affecting the balance between tight and extended conformations. Nuclear magnetic resonance-directed mutations within these interhelical interactions restore the physiological, store-dependent activation behavior of the gain-of-function STIM1 R304W mutant. This study reveals the functional impact of interhelical interactions within the CC1 domain for modifying the CC1-CC3 clamp strength to control the activation of STIM1.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Transtornos Plaquetários/genética , Clonagem Molecular , Dislexia/genética , Eritrócitos Anormais , Células HEK293 , Humanos , Ictiose/genética , Espectroscopia de Ressonância Magnética , Transtornos de Enxaqueca/genética , Miose/genética , Modelos Moleculares , Fadiga Muscular/genética , Mutação/genética , Conformação de Ácido Nucleico , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Baço/anormalidadesRESUMO
The initial activation step in the gating of ubiquitously expressed Orai1 calcium (Ca2+) ion channels represents the activation of the Ca2+-sensor protein STIM1 upon Ca2+ store depletion of the endoplasmic reticulum. Previous studies using constitutively active Orai1 mutants gave rise to, but did not directly test, the hypothesis that STIM1-mediated Orai1 pore opening is accompanied by a global conformational change of all Orai transmembrane domain (TM) helices within the channel complex. We prove that a local conformational change spreads omnidirectionally within the Orai1 complex. Our results demonstrate that these locally induced global, opening-permissive TM motions are indispensable for pore opening and require clearance of a series of Orai1 gating checkpoints. We discovered these gating checkpoints in the 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 with one gain-of-function point mutation in a series of possible combinations. We demonstrated that an array of loss-of-function mutations are dominant over most gain-of-function mutations within the same as well as of an adjacent Orai subunit. We further identified 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 are required to allow STIM1 coupling and Orai1 pore opening. Our results unravel novel insights in the preconditions of the unique fingerprint of CRAC channel activation, provide a valuable source for future structural resolutions, and help to understand the molecular basis of disease-causing mutations.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Ativação do Canal Iônico/genética , Proteínas de Neoplasias/química , Proteína ORAI1/química , Molécula 1 de Interação Estromal/química , Substituição de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Regulação da Expressão Gênica , Genes Reporter , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Lipossomos/química , Lipossomos/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Simulação de Dinâmica Molecular , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Técnicas de Patch-Clamp , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
The calcium release-activated calcium (CRAC) channel consists of STIM1, a Ca2+ sensor in the endoplasmic reticulum (ER), and Orai1, the Ca2+ ion channel in the plasma membrane. Ca2+ store depletion triggers conformational changes and oligomerization of STIM1 proteins and their direct interaction with Orai1. Structural alterations include the transition of STIM1 C-terminus from a folded to an extended conformation thereby exposing CAD (CRAC activation domain)/SOAR (STIM1-Orai1 activation region) for coupling to Orai1. In this study, we discovered that different point mutations of F394 in the small alpha helical segment (STIM1 α2) within the CAD/SOAR apex entail a rich plethora of effects on diverse STIM1 activation steps. An alanine substitution (STIM1 F394A) destabilized the STIM1 quiescent state, as evident from its constitutive activity. Single point mutation to hydrophilic, charged amino acids (STIM1 F394D, STIM1 F394K) impaired STIM1 homomerization and subsequent Orai1 activation. MD simulations suggest that their loss of homomerization may arise from altered formation of the CC1α1-SOAR/CAD interface and potential electrostatic interactions with lipid headgroups in the ER membrane. Consistent with these findings, we provide experimental evidence that the perturbing effects of F394D depend on the distance of the apex from the ER membrane. Taken together, our results suggest that the CAD/SOAR apex is in the immediate vicinity of the ER membrane in the STIM1 quiescent state and that different mutations therein can impact the STIM1/Orai1 activation cascade in various manners. Legend: Upon intracellular Ca2+ store depletion of the endoplasmic reticulum (ER), Ca2+ dissociates from STIM1. As a result, STIM1 adopts an elongated conformation and elicits Ca2+ influx from the extracellular matrix (EM) into the cell due to binding to and activation of Ca2+-selective Orai1 channels (left). The effects of three point mutations within the SOARα2 domain highlight the manifold roles of this region in the STIM1/Orai1 activation cascade: STIM1 F394A is active irrespective of the intracellular ER Ca2+ store level, but activates Orai1 channels to a reduced extent (middle). On the other hand, STIM1 F394D/K cannot adopt an elongated conformation upon Ca2+ store-depletion due to altered formation of the CC1α1-SOAR/CAD interface and/or electrostatic interaction of the respective side-chain charge with corresponding opposite charges on lipid headgroups in the ER membrane (right).
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Proteínas de Neoplasias/genética , Molécula 1 de Interação Estromal/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Linhagem Celular , Membrana Celular/genética , Retículo Endoplasmático/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Mutação Puntual/genéticaRESUMO
Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Moléculas de Interação Estromal/metabolismo , Animais , HumanosRESUMO
Calcium (Ca2+) is an essential second messenger required for diverse signaling processes in immune cells. Ca2+ release-activated Ca2+ (CRAC) channels represent one main Ca2+ entry pathway into the cell. They are fully reconstituted via two proteins, the stromal interaction molecule 1 (STIM1), a Ca2+ sensor in the endoplasmic reticulum, and the Ca2+ ion channel Orai in the plasma membrane. After Ca2+ store depletion, STIM1 and Orai couple to each other, allowing Ca2+ influx. CRAC-/STIM1-mediated Orai channel currents display characteristic hallmarks such as high Ca2+ selectivity, an increase in current density when switching from a Ca2+-containing solution to a divalent-free Na+ one, and fast Ca2+-dependent inactivation. Here, we discovered several constitutively active Orai1 and Orai3 mutants, containing substitutions in the TM3 and/or TM4 regions, all of which displayed a loss of the typical CRAC channel hallmarks. Restoring authentic CRAC channel activity required both the presence of STIM1 and the conserved Orai N-terminal portion. Similarly, these structural requisites were found in store-operated Orai channels. Key molecular determinants within the Orai N terminus that together with STIM1 maintained the typical CRAC channel hallmarks were distinct from those that controlled store-dependent Orai activation. In conclusion, the conserved portion of the Orai N terminus is essential for STIM1, as it fine-tunes the open Orai channel gating, thereby establishing authentic CRAC channel activity.
Assuntos
Canais de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Ativação do Canal Iônico , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Canais de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Domínios Proteicos , Molécula 1 de Interação Estromal/genéticaRESUMO
Ca2+ release-activated Ca2+ (CRAC) channels constitute the major Ca2+ entry pathway into the cell. They are fully reconstituted via intermembrane coupling of the Ca2+-selective Orai channel and the Ca2+-sensing protein STIM1. In addition to the Orai C terminus, the main coupling site for STIM1, the Orai N terminus is indispensable for Orai channel gating. Although the extended transmembrane Orai N-terminal region (Orai1 amino acids 73-91; Orai3 amino acids 48-65) is fully conserved in the Orai1 and Orai3 isoforms, Orai3 tolerates larger N-terminal truncations than Orai1 in retaining store-operated activation. In an attempt to uncover the reason for these isoform-specific structural requirements, we analyzed a series of Orai mutants and chimeras. We discovered that it was not the N termini, but the loop2 regions connecting TM2 and TM3 of Orai1 and Orai3 that featured distinct properties, which explained the different, isoform-specific behavior of Orai N-truncation mutants. Atomic force microscopy studies and MD simulations suggested that the remaining N-terminal portion in the non-functional Orai1 N-truncation mutants formed new, inhibitory interactions with the Orai1-loop2 regions, but not with Orai3-loop2. Such a loop2 swap restored activation of the N-truncation Orai1 mutants. To mimic interactions between the N terminus and loop2 in full-length Orai1 channels, we induced close proximity of the N terminus and loop2 via cysteine cross-linking, which actually caused significant inhibition of STIM1-mediated Orai currents. In aggregate, maintenance of Orai activation required not only the conserved N-terminal region but also permissive communication of the Orai N terminus and loop2 in an isoform-specific manner.
Assuntos
Canais de Cálcio/química , Proteína ORAI1/química , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
We report a new NMR-scale purification procedure for two recombinant wild type fragments of the stromal interaction molecule 1 (STIM1). This protein acts as a calcium sensor in the endoplasmic reticulum (ER) and extends into the cytosol accumulating at ER - plasma membrane (PM) junctions upon calcium store depletion ultimately leading to activation of the Orai/CRAC channel. The functionally relevant cytosolic part of STIM1 consists of three coiled coil domains, which are mainly involved in intra- and inter-molecular homomeric interactions as well as coupling to and gating of CRAC channels. The optimized one-step rapid purification procedure for two 15N,13C isotope-labeled cytosolic coiled coil fragments, which avoids the problems of previous approaches. The high yields of soluble well folded 15N,13C isotope-labeled cytosolic coiled coil fragments followed by detergent screening provide for initial NMR characterization of these domains. The longer 30.5â¯kDa fragment represents the largest STIM1 wild type fragment that has been recombinantly prepared and characterized in solution without need for mutation or refolding.
Assuntos
Proteínas de Neoplasias/química , Molécula 1 de Interação Estromal/química , Isótopos de Carbono/química , Isótopos de Carbono/isolamento & purificação , Cromatografia de Afinidade , Difusão Dinâmica da Luz , Eletroforese em Gel de Poliacrilamida , Humanos , Marcação por Isótopo , Proteínas de Neoplasias/isolamento & purificação , Isótopos de Nitrogênio/química , Isótopos de Nitrogênio/isolamento & purificação , Ressonância Magnética Nuclear Biomolecular , Domínios Proteicos , Dobramento de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Solubilidade , Molécula 1 de Interação Estromal/isolamento & purificaçãoRESUMO
Store-operated Ca(2+) entry (SOCE) is a universal Ca(2+) influx pathway that is important for the function of many cell types. SOCE occurs upon depletion of endoplasmic reticulum (ER) Ca(2+) stores and relies on a complex molecular interplay between the plasma membrane (PM) Ca(2+) channel ORAI1 and the ER Ca(2+) sensor stromal interaction molecule (STIM) 1. Patients with null mutations in ORAI1 or STIM1 genes present with severe combined immunodeficiency (SCID)-like disease. Here, we describe the molecular mechanisms by which a loss-of-function STIM1 mutation (R429C) in human patients abolishes SOCE. R429 is located in the third coiled-coil (CC3) domain of the cytoplasmic C terminus of STIM1. Mutation of R429 destabilizes the CC3 structure and alters the conformation of the STIM1 C terminus, thereby releasing a polybasic domain that promotes STIM1 recruitment to ER-PM junctions. However, the mutation also impairs cytoplasmic STIM1 oligomerization and abolishes STIM1-ORAI1 interactions. Thus, despite its constitutive localization at ER-PM junctions, mutant STIM1 fails to activate SOCE. Our results demonstrate multifunctional roles of the CC3 domain in regulating intra- and intermolecular STIM1 interactions that control (i) transition of STIM1 from a quiescent to an active conformational state, (ii) cytoplasmic STIM1 oligomerization, and (iii) STIM1-ORAI1 binding required for ORAI1 activation.
Assuntos
Síndromes de Imunodeficiência/genética , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Cálcio/química , Canais de Cálcio/metabolismo , Citoplasma/metabolismo , Dimerização , Retículo Endoplasmático/metabolismo , Transferência Ressonante de Energia de Fluorescência , Genes Recessivos , Células HEK293 , Homozigoto , Humanos , Microscopia Confocal , Proteína ORAI1 , Estrutura Terciária de Proteína , Molécula 1 de Interação EstromalRESUMO
A primary Ca2+ entry pathway in non-excitable cells is established by the Ca2+ release-activated Ca2+ channels. Their two limiting molecular components include the Ca2+-sensor protein STIM1 located in the endoplasmic reticulum and the Orai channel in the plasma membrane. STIM1 senses the luminal Ca2+ content, and store depletion induces its oligomerization into puncta-like structures, thereby triggering coupling to as well as activation of Orai channels. A C-terminal STIM1 domain is assumed to couple to both C- and N-terminal, cytosolic strands of Orai, accomplishing gating of the channel. Here we highlight the inter- and intramolecular steps of the STIM1-Orai signaling cascade together with critical sites of the pore structure that accomplishes Ca2+ permeation.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Membrana Celular/metabolismo , HumanosRESUMO
Calmodulin (CaM) binds most of its targets by wrapping around an amphipathic α-helix. The N-terminus of Orai proteins contains a conserved CaM-binding segment but the binding mechanism has been only partially characterized. Here, microscale thermophoresis (MST), surface plasmon resonance (SPR), and atomic force microscopy (AFM) were employed to study the binding equilibria, the kinetics, and the single-molecule interaction forces involved in the binding of CaM to the conserved helical segments of Orai1 and Orai3. The results consistently indicated stepwise binding of two separate target peptides to the two lobes of CaM. An unparalleled high affinity was found when two Orai peptides were dimerized or immobilized at high lateral density, thereby mimicking the close proximity of the N-termini in native Orai oligomers. The analogous experiments with smooth muscle myosin light chain kinase (smMLCK) showed only the expected 1:1 binding, confirming the validity of our methods.
Assuntos
Canais de Cálcio/química , Calmodulina/química , Proteína ORAI1/química , Humanos , Ligação ProteicaRESUMO
Ca(2+) influx via store-operated Ca(2+) release activated Ca(2+) (CRAC) channels represents a main signalling pathway for a variety of cell functions, including T-cell activation as well as mast-cell degranulation. Depletion of [Ca(2+)]ER results in activation of Ca(2+) channels within the plasmamembrane that mediate sustained Ca(2+) influx which is required for refilling Ca(2+) stores and down-stream Ca(2+) signalling. The CRAC channel is the best characterized store-operated channel (SOC) with well-defined electrophysiological properties. In recent years, the molecular components of the CRAC channel have been defined. The ER - located Ca(2+)-sensor, STIM1 and the Ca(2+)-selective ion pore, Orai1 in the membrane are sufficient to fully reconstitute CRAC currents. Stromal interaction molecule (STIM) 1 is localized in the ER, senses [Ca(2+)]ER and activates the CRAC channel upon store depletion by direct binding to Orai1 in the plasmamembrane. The identification of STIM1 and Orai1 and recently the structural resolution of both proteins by X-ray crystallography and nuclear magnetic resonance substantiated many findings from structure-function studies which has substantially improved the understanding of CRAC channel activation. Within this review, we summarize the functional and structural mechanisms of CRAC channel regulation, present a detailed overview of the STIM1/Orai1 signalling pathway where we focus on the critical domains mediating interactions and on the ion permeation pathway. We portray a mechanistic view of the steps in the dynamics of CRAC channel signalling ranging from STIM1 oligomerization over STIM1-Orai1 coupling to CRAC channel activation and permeation.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Humanos , Transporte de Íons , Proteínas de Membrana/química , Modelos Moleculares , Proteínas de Neoplasias/química , Molécula 1 de Interação EstromalRESUMO
Store-operated Ca(2+) entry, essential for the adaptive immunity, is initiated by the endoplasmic reticulum (ER) Ca(2+) sensor STIM1. Ca(2+) entry occurs through the plasma membrane resident Ca(2+) channel Orai1 that directly interacts with the C-terminal STIM1 domain, named SOAR/CAD. Depletion of the ER Ca(2+) store controls this STIM1/Orai1 interaction via transition to an extended STIM1 C-terminal conformation, exposure of the SOAR/CAD domain, and STIM1/Orai1 co-clustering. Here we developed a novel approach termed FRET-derived Interaction in a Restricted Environment (FIRE) in an attempt to dissect the interplay of coiled-coil (CC) interactions in controlling STIM1 quiescent as well as active conformation and cluster formation. We present evidence of a sequential activation mechanism in the STIM1 cytosolic domains where the interaction between CC1 and CC3 segment regulates both SOAR/CAD exposure and CC3-mediated higher-order oligomerization as well as cluster formation. These dual levels of STIM1 auto-inhibition provide efficient control over the coupling to and activation of Orai1 channels.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Cálcio/genética , Membrana Celular/genética , Células HEK293 , Humanos , Proteínas de Membrana/genética , Proteínas de Neoplasias/genética , Proteína ORAI1 , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Molécula 1 de Interação EstromalRESUMO
Stromal interaction molecule (STIM1) and ORAI1 are key components of the Ca(2+) release-activated Ca(2+) (CRAC) current having an important role in T-cell activation and mast cell degranulation. CRAC channel activation occurs via physical interaction of ORAI1 with STIM1 when endoplasmic reticulum Ca(2+) stores are depleted. Here we show, utilizing a novel STIM1-derived Förster resonance energy transfer sensor, that the ORAI1 activating small fragment (OASF) undergoes a C-terminal, intramolecular transition into an extended conformation when activating ORAI1. The C-terminal rearrangement of STIM1 does not require a functional CRAC channel, suggesting interaction with ORAI1 as sufficient for this conformational switch. Extended conformations were also engineered by mutations within the first and third coiled-coil domains in the cytosolic portion of STIM1 revealing the involvement of hydrophobic residues in the intramolecular transition. Corresponding full-length STIM1 mutants exhibited enhanced interaction with ORAI1 inducing constitutive CRAC currents, even in the absence of store depletion. We suggest that these mutant STIM1 proteins imitate a physiological activated state, which mimics the intramolecular transition that occurs in native STIM1 upon store depletion.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Conformação Proteica , Western Blotting , Cromatografia em Gel , Clonagem Molecular , Eletrofisiologia , Transferência Ressonante de Energia de Fluorescência , Humanos , Proteínas de Membrana/genética , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Proteínas de Neoplasias/genética , Proteína ORAI1 , Molécula 1 de Interação Estromal , TransfecçãoRESUMO
RATIONALE: 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. OBJECTIVE: 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. METHODS AND RESULTS: 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. CONCLUSIONS: 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.
Assuntos
Canais de Cálcio/fisiologia , Lesões das Artérias Carótidas/fisiopatologia , Leucotrieno C4/metabolismo , Músculo Liso Vascular/fisiopatologia , Neointima/fisiopatologia , Angioplastia com Balão/efeitos adversos , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Lesões das Artérias Carótidas/etiologia , Lesões das Artérias Carótidas/patologia , Citosol/metabolismo , Modelos Animais de Doenças , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Músculo Liso Vascular/patologia , Neointima/etiologia , Neointima/patologia , Proteína ORAI1 , Técnicas de Patch-Clamp , Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Molécula 1 de Interação Estromal , Trombina/metabolismo , Trombina/farmacologiaRESUMO
STIM1 and Orai1 represent the two molecular key components of the Ca(2+) release-activated Ca(2+) channels. Their activation involves STIM1 C terminus coupling to both the N terminus and the C terminus of Orai. Here we focused on the extended transmembrane Orai1 N-terminal (ETON, aa73-90) region, conserved among the Orai family forming an elongated helix of TM1 as recently shown by x-ray crystallography. To identify "hot spot" residues in the ETON binding interface for STIM1 interaction, numerous Orai1 constructs with N-terminal truncations or point mutations within the ETON region were generated. N-terminal truncations of the first four residues of the ETON region or beyond completely abolished STIM1-dependent Orai1 function. Loss of Orai1 function resulted from neither an impairment of plasma membrane targeting nor pore damage, but from a disruption of STIM1 interaction. In a complementary approach, we monitored STIM1-Orai interaction via Orai1 V102A by determining restored Ca(2+) selectivity as a consequence of STIM1 coupling. Orai1 N-terminal truncations that led to a loss of function consistently failed to restore Ca(2+) selectivity of Orai1 V102A in the presence of STIM1, demonstrating impairment of STIM1 binding. Hence, the major portion of the ETON region (aa76-90) is essential for STIM1 binding and Orai1 activation. Mutagenesis within the ETON region revealed several hydrophobic and basic hot spot residues that appear to control STIM1 coupling to Orai1 in a concerted manner. Moreover, we identified two basic residues, which protrude into the elongated pore to redound to Orai1 gating. We suggest that several hot spot residues in the ETON region contribute in aggregate to the binding of STIM1, which in turn is coupled to a conformational reorientation of the gate.
Assuntos
Canais de Cálcio/química , Canais de Cálcio/metabolismo , Ativação do Canal Iônico , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Potenciais de Ação , Sequência de Aminoácidos , Aminoácidos/metabolismo , Sítios de Ligação , Humanos , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Proteína ORAI1 , Ligação Proteica , Estrutura Terciária de Proteína , Deleção de Sequência/genética , Molécula 1 de Interação Estromal , Relação Estrutura-AtividadeRESUMO
Store-operated Ca(2+) entry describes the phenomenon that connects a depletion of internal Ca(2+) stores to an activation of plasma membrane-located Ca(2+) selective ion channels. Tremendous progress towards the underlying molecular mechanism came with the discovery of the two respective limiting components, STIM and Orai. STIM1 represents the ER-located Ca(2+) sensor and transmits the signal of store depletion to the plasma membrane. Here it couples to and activates Orai, the highly Ca(2+)-selective pore-forming subunit of Ca(2+) release-activated Ca(2+) channels. In this review, we focus on the molecular steps that these two proteins undergo from store-depletion to their coupling, the activation, and regulation of Ca(2+) currents.
Assuntos
Canais de Cálcio/fisiologia , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Cálcio/química , Canais de Cálcio/química , Canais de Cálcio/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/fisiologia , Modelos Biológicos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/fisiologia , Proteína ORAI1 , Estrutura Terciária de Proteína , Molécula 1 de Interação EstromalRESUMO
Calcium (Ca2+) entry into non-excitable cells is mainly carried by store-operated channels, which serve essential functions ranging from regulation of transcription to cell growth. The best-characterized store-operated current, initially discovered in T lymphocytes and mast cells, is the Ca2+ release-activated Ca2+ (CRAC) current. The search for the molecular components of the CRAC channel has recently identified stromal interaction molecule 1 (STIM1) as the Ca2+ sensor in the endoplasmic reticulum (ER) and Orai1 as the CRAC channel pore. ER store depletion results in formation of STIM1 puncta that trigger Ca2+ influx via Orai1 channels. This review covers the role of domains within STIM1 and Orai and enlightens their function in the STIM1/Orai coupling process. Moreover, a molecular interpretation focuses on interactions between cytosolic portions of STIM1 and Orai together with a mechanistic view on the loss of function of the SCID (severe combined immunodeficiency)-linked Orai1 R91W mutant channel. The architecture of the selectivity filter of Orai channels is finally elucidated based on permeation properties of Orai pore mutants.
Assuntos
Canais de Cálcio/metabolismo , Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Animais , Canais de Cálcio/química , Canais de Cálcio/genética , Citosol/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Ligação ProteicaRESUMO
Stromal interaction molecule 1 (STIM1) resides in the endoplasmic reticulum (ER) membrane and senses luminal calcium (Ca2+ ) concentration. STIM1 activation involves a large-scale conformational transition that exposes a STIM1 domain termed "CAD/SOAR", - which is required for activation of the calcium channel Orai. Under resting cell conditions, STIM1 assumes a quiescent state where CAD/SOAR is suspended in an intramolecular clamp formed by the coiled-coil 1 domain (CC1) and CAD/SOAR. Here, we present a structural model of the cytosolic part of the STIM1 resting state using molecular docking simulations that take into account previously reported interaction sites between the CC1α1 and CAD/SOAR domains. We corroborate and refine previously reported interdomain coiled-coil contacts. Based on our model, we provide a detailed analysis of the CC1-CAD/SOAR binding interface using molecular dynamics simulations. We find a very similar binding interface for a proposed domain-swapped configuration of STIM1, where the CAD/SOAR domain of one monomer interacts with the CC1α1 domain of another monomer of STIM1. The rich structural and dynamical information obtained from our simulations reveals novel interaction sites such as M244, I409, or E370, which are crucial for STIM1 quiescent state stability. We tested our predictions by electrophysiological and Förster resonance energy transfer experiments on corresponding single-point mutants. These experiments provide compelling support for the structural model of the STIM1 quiescent state reported here. Based on transitions observed in enhanced-sampling simulations paired with an analysis of the quiescent STIM1 conformational dynamics, our work offers a first atomistic model for CC1α1-CAD/SOAR detachment.
Assuntos
Canais de Cálcio , Cálcio , Humanos , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Células HEK293 , Simulação de Acoplamento Molecular , Proteína ORAI1/metabolismo , Domínios Proteicos , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/metabolismoRESUMO
Ca2+ release-activated Ca2+ (CRAC) channels, indispensable for the immune system and various other human body functions, consist of two transmembrane (TM) proteins, the Ca2+-sensor STIM1 in the ER membrane and the Ca2+ ion channel Orai1 in the plasma membrane. Here we employ genetic code expansion in mammalian cell lines to incorporate the photocrosslinking unnatural amino acids (UAA), p-benzoyl-L-phenylalanine (Bpa) and p-azido-L-phenylalanine (Azi), into the Orai1 TM domains at different sites. Characterization of the respective UAA-containing Orai1 mutants using Ca2+ imaging and electrophysiology reveal that exposure to UV light triggers a range of effects depending on the UAA and its site of incorporation. In particular, photoactivation at A137 using Bpa in Orai1 activates Ca2+ currents that best match the biophysical properties of CRAC channels and are capable of triggering downstream signaling pathways such as nuclear factor of activated T-cells (NFAT) translocation into the nucleus without the need for the physiological activator STIM1.