RESUMEN
Store-operated calcium entry (SOCE) plays a crucial role in maintaining cellular calcium homeostasis. This mechanism involves proteins, such as stromal interaction molecule 1 (STIM1) and ORAI1. Mutations in the genes encoding these proteins, especially STIM1, can lead to various diseases, including CRAC channelopathies associated with severe combined immunodeficiency. Herein, we describe a novel homozygous mutation, NM_003156 c.792-3C > G, in STIM1 in a patient with a clinical profile of CRAC channelopathy, including immune system deficiencies and muscle weakness. Functional analyses revealed three distinct spliced forms in the patient cells: wild-type, exon 7 skipping, and intronic retention. Calcium influx analysis revealed impaired SOCE in the patient cells, indicating a loss of STIM1 function. We developed an antisense oligonucleotide treatment that improves STIM1 splicing and highlighted its potential as a therapeutic approach. Our findings provide insights into the complex effects of STIM1 mutations and shed light on the multifaceted clinical presentation of the patient.
Asunto(s)
Calcio , Mutación , Proteínas de Neoplasias , Molécula de Interacción Estromal 1 , Humanos , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Calcio/metabolismo , Canalopatías/genética , Masculino , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Femenino , Inmunodeficiencia Combinada Grave/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismoRESUMEN
T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Señalización del Calcio , Humanos , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/genética , Células Jurkat , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismo , Técnicas de Inactivación de Genes , Modelos Biológicos , Isoformas de Proteínas , Transporte de Proteínas/genética , Proliferación Celular/genética , Supervivencia Celular/genéticaRESUMEN
Stormorken syndrome is a multiorgan hereditary disease caused by dysfunction of the endoplasmic reticulum (ER) Ca2+ sensor protein STIM1, which forms the Ca2+ release-activated Ca2+ (CRAC) channel together with the plasma membrane channel Orai1. ER Ca2+ store depletion activates STIM1 by releasing the intramolecular "clamp" formed between the coiled coil 1 (CC1) and CC3 domains of the protein, enabling the C terminus to extend and interact with Orai1. The most frequently occurring mutation in patients with Stormorken syndrome is R304W, which destabilizes and extends the STIM1 C terminus independently of ER Ca2+ store depletion, causing constitutive binding to Orai1 and CRAC channel activation. We found that in cis deletion of one amino acid residue, Glu296 (which we called E296del) reversed the pathological effects of R304W. Homozygous Stim1 E296del+R304W mice were viable and phenotypically indistinguishable from wild-type mice. NMR spectroscopy, molecular dynamics simulations, and cellular experiments revealed that although the R304W mutation prevented CC1 from interacting with CC3, the additional deletion of Glu296 opposed this effect by enabling CC1-CC3 binding and restoring the CC domain interactions within STIM1 that are critical for proper CRAC channel function. Our results provide insight into the activation mechanism of STIM1 by clarifying the molecular basis of mutation-elicited protein dysfunction and pathophysiology.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Proteínas de la Membrana , Ratones , Animales , Proteínas de la Membrana/metabolismo , Canales de Calcio/metabolismo , Aminoácidos/metabolismo , Mutación , Retículo Endoplásmico/metabolismo , Molécula de Interacción Estromal 1/genética , Canales de Calcio Activados por la Liberación de Calcio/genética , Proteína ORAI1/metabolismo , Calcio/metabolismoRESUMEN
Ca2+ release-activated Ca2+ (CRAC) channels are activated by direct physical interactions between Orai1, the channel protein, and STIM1, the endoplasmic reticulum Ca2+ sensor. A hallmark of CRAC channels is fast Ca2+-dependent inactivation (CDI) which provides negative feedback to limit Ca2+ entry through CRAC channels. Although STIM1 is thought to be essential for CDI, its molecular mechanism remains largely unknown. Here, we examined a poorly understood gain-of-function (GOF) human Orai1 disease mutation, L138F, that causes tubular aggregate myopathy. Through pairwise mutational analysis, we determine that large amino acid substitutions at either L138 or the neighboring T92 locus located on the pore helix evoke highly Ca2+-selective currents in the absence of STIM1. We find that the GOF phenotype of the L138 pathogenic mutation arises due to steric clash between L138 and T92. Surprisingly, strongly activating L138 and T92 mutations showed CDI in the absence of STIM1, contradicting prevailing views that STIM1 is required for CDI. CDI of constitutively open T92W and L138F mutants showed enhanced intracellular Ca2+ sensitivity, which was normalized by re-adding STIM1 to the cells. Truncation of the Orai1 C-terminus reduced T92W CDI, indicating a key role for the Orai1 C-terminus for CDI. Overall, these results identify the molecular basis of a disease phenotype with broad implications for activation and inactivation of Orai1 channels.
Asunto(s)
Canales de Calcio , Canales de Calcio Activados por la Liberación de Calcio , Humanos , Canales de Calcio/metabolismo , Proteína ORAI1/genética , Mutación , Canales de Calcio Activados por la Liberación de Calcio/genética , Mutación con Ganancia de Función , Molécula de Interacción Estromal 1/genética , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
SignificanceCalcium release-activated calcium (CRAC) channels play key roles in the regulation of cellular signaling, transcription, and migration. Here, we describe the design, chemical synthesis, and characterization of photoswitchable channel inhibitors that can be switched on and off depending on the wavelength of light used. We use the compounds to induce light-dependent modulation of channel activity and downstream gene expression in human immune cells. We further expand the usage of the compounds to control seeding of cancer cells in target tissue and regulation of response to noxious stimuli in vivo in mice.
Asunto(s)
Canales de Calcio , Canales de Calcio Activados por la Liberación de Calcio , Animales , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/fisiología , Ratones , Molécula de Interacción Estromal 1/metabolismoRESUMEN
CRAC channel regulator 2 A (CRACR2A) is a large Rab GTPase that is expressed abundantly in T cells and acts as a signal transmitter between T cell receptor stimulation and activation of the Ca2+-NFAT and JNK-AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies, however, to date no patient with damaging variants in CRACR2A has been identified. In this study, we describe a patient harboring biallelic variants in CRACR2A [paternal allele c.834 gaG> gaT (p.E278D) and maternal alelle c.430 Aga > Gga (p.R144G) c.898 Gag> Tag (p.E300*)], the gene encoding CRACR2A. The 33-year-old patient of East-Asian origin exhibited late onset combined immunodeficiency characterised by recurrent chest infections, panhypogammaglobulinemia and CD4+ T cell lymphopenia. In vitro exposure of patient B cells to a T-dependent stimulus resulted in normal generation of antibody-secreting cells, however the patient's T cells showed pronounced reduction in CRACR2A protein levels and reduced proximal TCR signaling, including dampened SOCE and reduced JNK phosphorylation, that contributed to a defect in proliferation and cytokine production. Expression of individual allelic mutants in CRACR2A-deleted T cells showed that the CRACR2AE278D mutant did not affect JNK phosphorylation, but impaired SOCE which resulted in reduced cytokine production. The truncated double mutant CRACR2AR144G/E300* showed a pronounced defect in JNK phosphorylation as well as SOCE and strong impairment in cytokine production. Thus, we have identified variants in CRACR2A that led to late-stage combined immunodeficiency characterized by loss of function in T cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Citocinas/biosíntesis , Mutación , Enfermedades de Inmunodeficiencia Primaria/genética , Enfermedades de Inmunodeficiencia Primaria/fisiopatología , Receptores de Antígenos de Linfocitos T/metabolismo , Adulto , Pueblo Asiatico , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Citocinas/genética , Variación Genética , Humanos , Enfermedades de Inmunodeficiencia Primaria/inmunología , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunologíaRESUMEN
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).
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/genética , Proteínas de Neoplasias/genética , Molécula de Interacción Estromal 1/genética , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular , Membrana Celular/genética , Retículo Endoplásmico/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , Mutación Puntual/genéticaRESUMEN
Ca2+ ion channels are critical in a variety of physiological events, including cell growth, differentiation, gene transcription and apoptosis. One such essential entry pathway for calcium into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel. It consists of the Ca2+ sensing protein, stromal interaction molecule 1 (STIM1) located in the endoplasmic reticulum (ER) and a Ca2+ ion channel Orai in the plasma membrane. The Orai channel family includes three homologues Orai1, Orai2 and Orai3. While Orai1 is the "classical" Ca2+ ion channel within the CRAC channel complex and plays a universal role in the human body, there is increasing evidence that Orai2 and Orai3 are important in specific physiological and pathophysiological processes. This makes them an attractive target in drug discovery, but requires a detailed understanding of the three Orai channels and, in particular, their differences. Orai channel activation is initiated via Ca2+ store depletion, which is sensed by STIM1 proteins, and induces their conformational change and oligomerization. Upon STIM1 coupling, Orai channels activate to allow Ca2+ permeation into the cell. While this activation mechanism is comparable among the isoforms, they differ by a number of functional and structural properties due to non-conserved regions in their sequences. In this review, we summarize the knowledge as well as open questions in our current understanding of the three isoforms in terms of their structure/function relationship, downstream signaling and physiology as well as pathophysiology.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Señalización del Calcio , Calcio , Retículo Endoplásmico , Animales , Calcio/química , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/química , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Retículo Endoplásmico/química , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Humanos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Relación Estructura-ActividadRESUMEN
The population of regulatory T cells (Tregs) is critical for immunological self-tolerance and homeostasis. Proper ion regulation contributes to Treg lineage identity, regulation, and effector function. Identified ion channels include Ca2+ release-activated Ca2+, transient receptor potential, P2X, volume-regulated anion and K+ channels Kv1.3 and KCa3.1. Ion channel modulation represents a promising therapeutic approach for the treatment of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. This review summarizes studies with gene-targeted mice and pharmacological modulators affecting Treg number and function. Furthermore, participation of ion channels is illustrated and the power of future research possibilities is discussed.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Calcio/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Moduladores del Transporte de Membrana/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Linfocitos T Reguladores/efectos de los fármacos , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Calcio/inmunología , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Señalización del Calcio , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/inmunología , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/inmunología , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Moduladores del Transporte de Membrana/química , Ratones , Esclerosis Múltiple/genética , Esclerosis Múltiple/inmunología , Esclerosis Múltiple/metabolismo , Receptores Purinérgicos P2X/genética , Receptores Purinérgicos P2X/inmunología , Receptores Purinérgicos P2X7/genética , Receptores Purinérgicos P2X7/inmunología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/patología , Canales de Potencial de Receptor Transitorio/genética , Canales de Potencial de Receptor Transitorio/inmunologíaRESUMEN
Mast cells (MCs) are tissue resident immune cells that play important roles in the pathogenesis of allergic disorders. These responses are mediated via the cross-linking of cell surface high affinity IgE receptor (FcϵRI) by antigen resulting in calcium (Ca2+) mobilization, followed by degranulation and release of proinflammatory mediators. In addition to FcϵRI, cutaneous MCs express Mas-related G protein-coupled receptor X2 (MRGPRX2; mouse ortholog MrgprB2). Activation of MRGPRX2/B2 by the neuropeptide substance P (SP) is implicated in neurogenic inflammation, chronic urticaria, mastocytosis and atopic dermatitis. Although Ca2+ entry is required for MRGPRX2/B2-mediated MC responses, the possibility that calcium release-activated calcium (CRAC/Orai) channels participate in these responses has not been tested. Lentiviral shRNA-mediated silencing of Orai1, Orai2 or Orai3 in a human MC line (LAD2 cells) resulted in partial inhibition of SP-induced Ca2+ mobilization, degranulation and cytokine/chemokine generation (TNF-α, IL-8, and CCL-3). Synta66, which blocks homo and hetero-dimerization of Orai channels, caused a more robust inhibition of SP-induced responses than knockdown of individual Orai channels. Synta66 also blocked SP-induced extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation and abrogated cytokine/chemokine production. It also inhibited SP-induced Ca2+ mobilization and degranulation in primary human skin MCs and mouse peritoneal MCs. Furthermore, Synta66 attenuated both SP-induced cutaneous vascular permeability and leukocyte recruitment in mouse peritoneum. These findings demonstrate that Orai channels contribute to MRGPRX2/B2-mediated MC activation and suggest that their inhibition could provide a novel approach for the modulation of SP-induced MC/MRGPRX2-mediated disorders.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Mastocitos/inmunología , Mastocitos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animales , Calcio/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Activados por la Liberación de Calcio/antagonistas & inhibidores , Canales de Calcio Activados por la Liberación de Calcio/genética , Señalización del Calcio , Permeabilidad Capilar/efectos de los fármacos , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Noqueados , Familia de MultigenesRESUMEN
Calcium (Ca2+) signals initiate egg activation across the animal kingdom and in at least some plants. These signals are crucial for the success of development and, in the case of mammals, health of the offspring. The mechanisms associated with fertilization that trigger these signals and the molecules that regulate their characteristic patterns vary widely. With few exceptions, a major contributor to fertilization-induced elevation in cytoplasmic Ca2+ is release from endoplasmic reticulum stores through the IP3 receptor. In some cases, Ca2+ influx from the extracellular space and/or release from alternative intracellular stores contribute to the rise in cytoplasmic Ca2+. Following the Ca2+ rise, the reuptake of Ca2+ into intracellular stores or efflux of Ca2+ out of the egg drive the return of cytoplasmic Ca2+ back to baseline levels. The molecular mediators of these Ca2+ fluxes in different organisms include Ca2+ release channels, uptake channels, exchangers and pumps. The functions of these mediators are regulated by their particular activating mechanisms but also by alterations in their expression and spatial organization. We discuss here the molecular basis for modulation of Ca2+ signalling at fertilization, highlighting differences across several animal phyla, and we mention key areas where questions remain.
Asunto(s)
Señalización del Calcio/genética , Calcio/metabolismo , Fertilización/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Canales de Calcio Activados por la Liberación de Calcio/genética , Retículo Endoplásmico/genética , HumanosRESUMEN
Calcium (Ca2+) signals play fundamental roles in immune cell function. The main sources of Ca2+ influx in mammalian lymphocytes following antigen receptor stimulation are Ca2+ release-activated Ca2+ (CRAC) channels. These are formed by ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the endoplasmic reticulum (ER). Human loss-of-function (LOF) mutations in ORAI1 and STIM1 that abolish Ca2+ influx cause a unique disease syndrome called CRAC channelopathy that is characterized by immunodeficiency autoimmunity and non-immunological symptoms. Studies in mice lacking Stim and Orai genes have illuminated many cellular and molecular mechanisms by which these molecules control lymphocyte function. CRAC channels are required for the differentiation and function of several T lymphocyte subsets that provide immunity to infection, mediate inflammation and prevent autoimmunity. This review examines new insights into how CRAC channels control T cell-mediated immunity.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Señalización del Calcio , Linfocitos T , Animales , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/inmunología , Señalización del Calcio/inmunología , Humanos , Inmunidad Celular/genética , Inmunidad Celular/inmunología , Proteína ORAI1/genética , Proteína ORAI1/inmunología , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/inmunología , Linfocitos T/inmunologíaRESUMEN
BACKGROUND: Calcium (Ca2+) ion is a major intracellular signaling messenger, controlling a diverse array of cellular functions like gene expression, secretion, cell growth, proliferation, and apoptosis. The major mechanism controlling this Ca2+ homeostasis is store-operated Ca2+ release-activated Ca2+ (CRAC) channels. CRAC channels are integral membrane protein majorly constituted via two proteins, the stromal interaction molecule (STIM) and ORAI. Following Ca2+ depletion in the Endoplasmic reticulum (ER) store, STIM1 interacts with ORAI1 and leads to the opening of the CRAC channel gate and consequently allows the influx of Ca2+ ions. A plethora of studies report that aberrant CRAC channel activity due to Loss- or gain-of-function mutations in ORAI1 and STIM1 disturbs this Ca2+ homeostasis and causes several autoimmune disorders. Hence, it clearly indicates that the therapeutic target of CRAC channels provides the space for a new approach to treat autoimmune disorders. OBJECTIVE: This review aims to provide the key structural and mechanical insights of STIM1, ORAI1 and other molecular modulators involved in CRAC channel regulation. RESULTS AND CONCLUSION: Understanding the structure and function of the protein is the foremost step towards improving the effective target specificity by limiting their potential side effects. Herein, the review mainly focusses on the structural underpinnings of the CRAC channel gating mechanism along with its biophysical properties that would provide the solid foundation to aid the development of novel targeted drugs for an autoimmune disorder. Finally, the immune deficiencies caused due to mutations in CRAC channel and currently used pharmacological blockers with their limitation are briefly summarized.
Asunto(s)
Enfermedades Autoinmunes/tratamiento farmacológico , Canales de Calcio Activados por la Liberación de Calcio/química , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Proteína ORAI1/química , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/química , Molécula de Interacción Estromal 1/metabolismo , Enfermedades Autoinmunes/genética , Enfermedades Autoinmunes/patología , Canales de Calcio Activados por la Liberación de Calcio/antagonistas & inhibidores , Canales de Calcio Activados por la Liberación de Calcio/genética , Señalización del Calcio/fisiología , Humanos , Proteína ORAI1/genética , Molécula de Interacción Estromal 1/genéticaRESUMEN
Ca2+ release activated Ca2+ (CRAC) channels represent a primary pathway for Ca2+ to enter non-excitable cells. The two key players in this process are the stromal interaction molecule (STIM), a Ca2+ sensor embedded in the membrane of the endoplasmic reticulum, and Orai, a highly Ca2+ selective ion channel located in the plasma membrane. Upon depletion of the internal Ca2+ stores, STIM is activated, oligomerizes, couples to and activates Orai. This review provides an overview of novel findings about the CRAC channel activation mechanisms, structure and gating. In addition, it highlights, among diverse STIM and Orai mutants, also the disease-related mutants and their implications.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio , Membrana Celular , Animales , Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/sangre , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio/fisiología , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Humanos , Mutación , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
Approximately one-fourth of patients with essential thrombocythemia or primary myelofibrosis carry a somatic mutation of the calreticulin gene (CALR), the gene encoding for calreticulin. A 52-bp deletion (type I mutation) and a 5-bp insertion (type II mutation) are the most frequent genetic lesions. The mechanism(s) by which a CALR mutation leads to a myeloproliferative phenotype has been clarified only in part. We studied the interaction between calreticulin and store-operated calcium (Ca2+) entry (SOCE) machinery in megakaryocytes (Mks) from healthy individuals and from patients with CALR-mutated myeloproliferative neoplasms (MPNs). In Mks from healthy subjects, binding of recombinant human thrombopoietin to c-Mpl induced the activation of signal transducer and activator of transcription 5, AKT, and extracellular signal-regulated kinase 1/2, determining inositol triphosphate-dependent Ca2+ release from the endoplasmic reticulum (ER). This resulted in the dissociation of the ER protein 57 (ERp57)-mediated complex between calreticulin and stromal interaction molecule 1 (STIM1), a protein of the SOCE machinery that leads to Ca2+ mobilization. In Mks from patients with CALR-mutated MPNs, defective interactions between mutant calreticulin, ERp57, and STIM1 activated SOCE and generated spontaneous cytosolic Ca2+ flows. In turn, this resulted in abnormal Mk proliferation that was reverted using a specific SOCE inhibitor. In summary, the abnormal SOCE regulation of Ca2+ flows in Mks contributes to the pathophysiology of CALR-mutated MPNs. In perspective, SOCE may represent a new therapeutic target to counteract Mk proliferation and its clinical consequences in MPNs.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Calreticulina/genética , Calreticulina/metabolismo , Megacariocitos/patología , Mutación , Trastornos Mieloproliferativos/patología , Canales de Calcio Activados por la Liberación de Calcio/genética , Estudios de Casos y Controles , Humanos , Megacariocitos/metabolismo , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína Disulfuro Isomerasas/genética , Proteína Disulfuro Isomerasas/metabolismo , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismoRESUMEN
The ORAI family of ion channel-forming proteins in mammals includes three members, ORAI1, ORAI2 and ORAI3, encoded by homologous genes. Of these proteins the ORAI1 one received major attention as plasma membrane constituent of store-operated calcium entry (SOCE) in non-excitable cells. The functional significance of two other proteins, ORAI2 and ORAI3, is much less defined, although both of them participate to various extends in cell-specific modulation of SOCE as well as in supporting some of the store-independent calcium entry mechanisms. Calcium signaling becomes remodeled in cancer to promote cancer hallmarks - enhanced proliferation, resistance to apoptosis, motility and metastasizing. Although such remodeling commonly involves rearrangements of the whole molecular Ca2+-handling toolkit of the cell (Ca2+ pumps and transporters, Ca2+-binding and storage proteins, Ca2+ entry and release channels, Ca2+-dependent effectors), Ca2+ entry through Orai-based channels is especially important, as its dysregulation may contribute to several cancer hallmarks. The latter depend on the type of Ca2+-permeable channel formed by ORAI-proteins, spatiotemporal characteristics of Ca2+ signal that this channel contributes to, and the type Ca2+-dependent effector(s) targeted by this signal, all of which may be cancer-specific. By participating in global Ca2+ entry, ORAI-based SOCE may also contribute to cytosolic Ca2+ overload of cancer cells thereby playing pro-apoptotic, antineoplastic roles which can potentially be exploited for cancer treatment. This mini review examines various aspects of ORAI proteins in malignant transformation.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Calcio/metabolismo , Neoplasias/etiología , Neoplasias/metabolismo , Animales , Canales de Calcio Activados por la Liberación de Calcio/genética , Señalización del Calcio , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Astrocytes are the major glial subtype in the brain and mediate numerous functions ranging from metabolic support to gliotransmitter release through signaling mechanisms controlled by Ca2+ Despite intense interest, the Ca2+ influx pathways in astrocytes remain obscure, hindering mechanistic insights into how Ca2+ signaling is coupled to downstream astrocyte-mediated effector functions. Here, we identified store-operated Ca2+ release-activated Ca2+ (CRAC) channels encoded by Orai1 and STIM1 as a major route of Ca2+ entry for driving sustained and oscillatory Ca2+ signals in astrocytes after stimulation of metabotropic purinergic and protease-activated receptors. Using synaptopHluorin as an optical reporter, we showed that the opening of astrocyte CRAC channels stimulated vesicular exocytosis to mediate the release of gliotransmitters, including ATP. Furthermore, slice electrophysiological recordings showed that activation of astrocytes by protease-activated receptors stimulated interneurons in the CA1 hippocampus to increase inhibitory postsynaptic currents on CA1 pyramidal cells. These results reveal a central role for CRAC channels as regulators of astrocyte Ca2+ signaling, gliotransmitter release, and astrocyte-mediated tonic inhibition of CA1 pyramidal neurons.
Asunto(s)
Astrocitos/fisiología , Señalización del Calcio/fisiología , Calcio/metabolismo , Neuronas GABAérgicas/fisiología , Proteína ORAI1/metabolismo , Molécula de Interacción Estromal 1/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Región CA1 Hipocampal/citología , Región CA1 Hipocampal/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Células Cultivadas , Exocitosis/fisiología , Femenino , Neuronas GABAérgicas/citología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Proteína ORAI1/genética , Células Piramidales/citología , Células Piramidales/fisiología , Molécula de Interacción Estromal 1/genética , Transmisión Sináptica/fisiologíaRESUMEN
Inositol 1,4,5-trisphosphate receptors (IP3R) are the most widely expressed intracellular Ca2+ release channels. Their activation by IP3 and Ca2+ allows Ca2+ to pass rapidly from the ER lumen to the cytosol. The resulting increase in cytosolic [Ca2+] may directly regulate cytosolic effectors or fuel Ca2+ uptake by other organelles, while the decrease in ER luminal [Ca2+] stimulates store-operated Ca2+ entry (SOCE). We are close to understanding the structural basis of both IP3R activation, and the interactions between the ER Ca2+-sensor, STIM, and the plasma membrane Ca2+ channel, Orai, that lead to SOCE. IP3Rs are the usual means through which extracellular stimuli, through ER Ca2+ release, stimulate SOCE. Here, we review evidence that the IP3Rs most likely to respond to IP3 are optimally placed to allow regulation of SOCE. We also consider evidence that IP3Rs may regulate SOCE downstream of their ability to deplete ER Ca2+ stores. Finally, we review evidence that IP3Rs in the plasma membrane can also directly mediate Ca2+ entry in some cells.
Asunto(s)
Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Señalización del Calcio , Calcio/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Moléculas de Interacción Estromal/metabolismo , Animales , Canales de Calcio Activados por la Liberación de Calcio/genética , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Moléculas de Interacción Estromal/genéticaRESUMEN
Basal forebrain neurons control cerebral blood flow (CBF) by releasing acetylcholine (Ach), which binds to endothelial muscarinic receptors to induce nitric (NO) release and vasodilation in intraparenchymal arterioles. Nevertheless, the mechanism whereby Ach stimulates human brain microvascular endothelial cells to produce NO is still unknown. Herein, we sought to assess whether Ach stimulates NO production in a Ca2+ -dependent manner in hCMEC/D3 cells, a widespread model of human brain microvascular endothelial cells. Ach induced a dose-dependent increase in intracellular Ca2+ concentration ([Ca2+ ]i ) that was prevented by the genetic blockade of M5 muscarinic receptors (M5-mAchRs), which was the only mAchR isoform coupled to phospholipase Cß (PLCß) present in hCMEC/D3 cells. A comprehensive real-time polymerase chain reaction analysis revealed the expression of the transcripts encoding for type 3 inositol-1,4,5-trisphosphate receptors (InsP3 R3), two-pore channels 1 and 2 (TPC1-2), Stim2, Orai1-3. Pharmacological manipulation showed that the Ca2+ response to Ach was mediated by InsP3 R3, TPC1-2, and store-operated Ca2+ entry (SOCE). Ach-induced NO release, in turn, was inhibited in cells deficient of M5-mAchRs. Likewise, Ach failed to increase NO levels in the presence of l-NAME, a selective NOS inhibitor, or BAPTA, a membrane-permeant intracellular Ca2+ buffer. Moreover, the pharmacological blockade of the Ca2+ response to Ach also inhibited the accompanying NO production. These data demonstrate for the first time that synaptically released Ach may trigger NO release in human brain microvascular endothelial cells by stimulating a Ca2+ signal via M5-mAchRs.
Asunto(s)
Acetilcolina/farmacología , Señalización del Calcio/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Microvasos/efectos de los fármacos , Agonistas Muscarínicos/farmacología , Acoplamiento Neurovascular/efectos de los fármacos , Óxido Nítrico/metabolismo , Prosencéfalo/irrigación sanguínea , Receptor Muscarínico M5/agonistas , Canales de Calcio/genética , Canales de Calcio/metabolismo , Canales de Calcio Activados por la Liberación de Calcio/genética , Canales de Calcio Activados por la Liberación de Calcio/metabolismo , Células Cultivadas , Células Endoteliales/metabolismo , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Microvasos/metabolismo , Receptor Muscarínico M5/genética , Receptor Muscarínico M5/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismo , Transmisión SinápticaRESUMEN
Calcium (Ca2+) is an important ion in physiology and is found both outside and inside cells. The intracellular concentration of Ca2+ is tightly regulated as it is an intracellular signal molecule and can affect a variety of cellular processes. In immune cells Ca2+ has been shown to regulate e.g. gene transcription, cytokine secretion, proliferation and migration. Ca2+ can enter the cytoplasm either from intracellular stores or from outside the cells when Ca2+ permeable ion channels in the plasma membrane open. The Ca2+ release-activated (CRAC) channel is the most prominent Ca2+ ion channel in the plasma membrane. It is formed by ORAI1-3 and the channel is opened by the endoplasmic reticulum Ca2+ sensor proteins stromal interaction molecules (STIM) 1 and 2. Another group of Ca2+ channels in the plasma membrane are the voltage-gated Ca2+ (CaV) channels. We examined if a change in immunological tolerance is accompanied by altered ORAI, STIM and CaV gene expression in peripheral blood mononuclear cells (PBMCs) in pregnant women and in type 1 diabetic individuals. Our results show that in pregnancy and type 1 diabetes ORAI1-3 are up-regulated whereas STIM1 and 2 are down-regulated in pregnancy but only STIM2 in type 1 diabetes. Expression of L-, P/Q-, R- and T-type voltage-gated Ca2+ channels was detected in the PBMCs where the CaV2.3 gene was up-regulated in pregnancy and type 1 diabetes whereas the CaV 2.1 and CaV3.2 genes were up-regulated only in pregnancy and the CaV1.3 gene in type 1 diabetes. The results are consistent with that expression of ORAI, STIM and CaV genes correlate with a shift in immunological status of the individual in health, as during pregnancy, and in the autoimmune disease type 1 diabetes. Whether the changes are in general protective or in type 1 diabetes include some pathogenic components remains to be clarified.