RESUMEN
The PI3K (phosphoinositide 3-kinase) family of lipid kinases regulate cell motility in diverse organisms and cell types. In mammals, the main PI3K enzyme activated by chemokine receptor signalling is the class IB isoform, p110gamma. Studies of p110gamma-knockout mice have shown an essential function for this isoform in chemotaxis of neutrophils and macrophages both in vitro and in vivo. However, the roles of p110gamma and other PI3K enzymes and regulatory subunits in lymphocyte motility have been more difficult to discern. Recent studies of adoptively transferred, fluorescently labelled lymphocytes have revealed complex and unexpected functions for PI3K in lymphocyte migration in vivo. In this review we highlight cell-type-specific roles for PI3K catalytic and regulatory subunits in the homing and basal motility of lymphocytes in the intact lymph node.
Asunto(s)
Quimiotaxis de Leucocito , Linfocitos/citología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Linfocitos/enzimología , Tejido Linfoide/citología , Ratones , Ratones NoqueadosRESUMEN
The small-conductance calcium-activated K(+) channel SK3 (SKCa3/KCNN3) regulates electrical excitability and neurotransmitter release in monoaminergic neurons, and has been implicated in schizophrenia, ataxia and anorexia nervosa. We have identified a novel SK3 transcript, SK3-1B that utilizes an alternative first exon (exon 1B), but is otherwise identical to SK3. SK3-1B, mRNA is widely distributed in human tissues and is present at 20-60% of SK3 in the brain. The SK3-1B protein lacks the N-terminus and first transmembrane segment, and begins eight residues upstream of the second transmembrane segment. When expressed alone, SK3-1B did not produce functional channels, but selectively suppressed endogenous SK3 currents in the pheochromocytoma cell line, PC12, in a dominant-negative fashion. This dominant inhibitory effect extended to other members of the SK subfamily, but not to voltage-gated K(+) channels, and appears to be due to intracellular trapping of endogenous SK channels. The effect of SK3-1B expression is very similar to that produced by expression of the rare SK3 truncation allele, SK3-Delta, found in a patient with schizophrenia. Regulation of SK3 and SK3-1B levels may provide a potent mechanism to titrate neuronal firing rates and neurotransmitter release in monoaminergic neurons, and alterations in the relative abundance of these proteins could contribute to abnormal neuronal excitability, and to the pathogenesis of schizophrenia.
Asunto(s)
Calcio/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/genética , Canales de Potasio/metabolismo , Esquizofrenia/genética , Animales , Secuencia de Bases , Química Encefálica/genética , Genes Dominantes , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos/metabolismo , Isomerismo , Células Jurkat , Proteínas Luminiscentes/genética , Potenciales de la Membrana/fisiología , Datos de Secuencia Molecular , Neuronas/fisiología , Células PC12 , Canales de Potasio/química , Estructura Terciaria de Proteína , Ratas , Esquizofrenia/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el CalcioRESUMEN
Adoptive transfer experimental autoimmune encephalomyelitis (AT-EAE), a disease resembling multiple sclerosis, is induced in rats by myelin basic protein (MBP)-activated CD4(+) T lymphocytes. By patch-clamp analysis, encephalitogenic rat T cells stimulated repeatedly in vitro expressed a unique channel phenotype ("chronically activated") with large numbers of Kv1.3 voltage-gated channels (approximately 1500 per cell) and small numbers of IKCa1 Ca(2+)-activated K(+) channels (approximately 50-120 per cell). In contrast, resting T cells displayed 0-10 Kv1.3 and 10-20 IKCa1 channels per cell ("quiescent" phenotype), whereas T cells stimulated once or twice expressed approximately 200 Kv1.3 and approximately 350 IKCa1 channels per cell ("acutely activated" phenotype). Consistent with their channel phenotype, [(3)H]thymidine incorporation by MBP-stimulated chronically activated T cells was suppressed by the peptide ShK, a blocker of Kv1.3 and IKCa1, and by an analog (ShK-Dap(22)) engineered to be highly specific for Kv1.3, but not by a selective IKCa1 blocker (TRAM-34). The combination of ShK-Dap(22) and TRAM-34 enhanced the suppression of MBP-stimulated T cell proliferation. Based on these in vitro results, we assessed the efficacy of K(+) channel blockers in AT-EAE. Specific and simultaneous blockade of the T cell channels by ShK or by a combination of ShK-Dap(22) plus TRAM-34 prevented lethal AT-EAE. Blockade of Kv1.3 alone with ShK-Dap(22), but not of IKCa1 with TRAM-34, was also effective. When administered after the onset of symptoms, ShK or the combination of ShK-Dap(22) plus TRAM-34 greatly ameliorated the clinical course of both moderate and severe AT-EAE. We conclude that selective targeting of Kv1.3, alone or with IKCa1, may provide an effective new mode of therapy for multiple sclerosis.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Encefalomielitis Autoinmune Experimental/prevención & control , Esclerosis Múltiple/prevención & control , Bloqueadores de los Canales de Potasio , Canales de Potasio con Entrada de Voltaje , Canales de Potasio/metabolismo , Animales , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacocinética , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Venenos de Cnidarios/administración & dosificación , Venenos de Cnidarios/farmacocinética , Venenos de Cnidarios/farmacología , Modelos Animales de Enfermedad , Encefalomielitis Autoinmune Experimental/metabolismo , Femenino , Cobayas , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Marcaje Isotópico , Canal de Potasio Kv1.3 , Esclerosis Múltiple/metabolismo , Fenotipo , Bloqueadores de los Canales de Potasio/administración & dosificación , Bloqueadores de los Canales de Potasio/farmacocinética , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/administración & dosificación , Pirazoles/farmacocinética , Pirazoles/farmacología , Ratas , Ratas Endogámicas Lew , Timidina/metabolismo , Tritio/metabolismoRESUMEN
Apamin-sensitive small conductance calcium-activated potassium channels (SKCa1-3) mediate the slow afterhyperpolarization in neurons, but the molecular identity of the channel has not been defined because of the lack of specific inhibitors. Here we describe the structure-based design of a selective inhibitor of SKCa2. Leiurotoxin I (Lei) and PO5, peptide toxins that share the RXCQ motif, potently blocked human SKCa2 and SKCa3 but not SKCa1, whereas maurotoxin, Pi1, Tskappa, and PO1 were ineffective. Lei blocked these channels more potently than PO5 because of the presence of Ala(1), Phe(2), and Met(7). By replacing Met(7) in the RXCQ motif of Lei with the shorter, unnatural, positively charged diaminobutanoic acid (Dab), we generated Lei-Dab(7), a selective SKCa2 inhibitor (K(d) = 3.8 nm) that interacts with residues in the external vestibule of the channel. SKCa3 was rendered sensitive to Lei-Dab(7) by replacing His(521) with the corresponding SKCa2 residue (Asn(367)). Intracerebroventricular injection of Lei-Dab(7) into mice resulted in no gross central nervous system toxicity at concentrations that specifically blocked SKCa2 homotetramers. Lei-Dab(7) will be a useful tool to investigate the functional role of SKCa2 in mammalian tissues.
Asunto(s)
Péptidos/química , Bloqueadores de los Canales de Potasio , Canales de Potasio Calcio-Activados/antagonistas & inhibidores , Canales de Potasio/química , Venenos de Escorpión/farmacología , Alanina/farmacología , Secuencia de Aminoácidos , Animales , Arginina/química , Células COS , Línea Celular , Clonación Molecular , Relación Dosis-Respuesta a Droga , Electrofisiología , Humanos , Cinética , Metionina/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Neurotoxinas/farmacología , Células PC12 , Péptidos/farmacología , Fenilalanina/farmacología , Unión Proteica , Ratas , Venenos de Escorpión/química , Homología de Secuencia de Aminoácido , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , Treonina/química , Transfección , Valina/química , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The discovery of a diverse and unique set of ion channels in T lymphocytes has led to a rapidly growing body of knowledge about their functional roles in the immune system. Here we review the biophysical and molecular characterization of K+, Ca2+, and Cl- channels in T lymphocytes. Potent and specific blockers, especially of K+ channels, have provided molecular tools to elucidate the involvement of voltage- and calcium-activated potassium channels in T-cell activation and cell-volume regulation. Their unique and differential expression makes lymphocyte K+ channels excellent pharmaceutical targets for modulating immune system function. This review surveys recent progress at the biophysical, molecular, and functional roles of the ion channels found in T lymphocytes.
Asunto(s)
Canales Iónicos/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Fenómenos Biofísicos , Biofisica , Señalización del Calcio , Electrofisiología , Femenino , Humanos , Canales Iónicos/química , Canales Iónicos/genética , Canales Iónicos/fisiología , Intercambio Materno-Fetal , Modelos Biológicos , Datos de Secuencia Molecular , Canales de Potasio/genética , Canales de Potasio/inmunología , Canales de Potasio/fisiología , Embarazo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/fisiologíaRESUMEN
The small conductance calcium-activated K+ channel gene SKCa3/KCNN3 maps to 1q21, a region strongly linked to schizophrenia. Recently, a 4-base pair deletion in SKCa3 was reported in a patient with schizophrenia, which truncates the protein at the end of the N-terminal cytoplasmic region (SKCa3Delta). We generated a green fluorescent protein-SKCa3 N-terminal construct (SKCa3-1/285) that is identical to SKCa3Delta except for the last two residues. Using confocal microscopy we demonstrate that SKCa3-1/285 localizes rapidly and exclusively to the nucleus of mammalian cells like several other pathogenic polyglutamine-containing proteins. This nuclear targeting is mediated in part by two polybasic sequences present at the C-terminal end of SKCa3-1/285. In contrast, full-length SKCa3, SKCa2, and IKCa1 polypeptides are all excluded from the nucleus and express as functional channels. When overexpressed in human Jurkat T cells, SKCa3-1/285 can suppress endogenous SKCa2 currents but not voltage-gated K+ currents. This dominant-negative suppression is most likely mediated through the co-assembly of SKCa3-1/285 with native subunits and the formation of non-functional tetramers. The nuclear localization of SKCa3-1/285 may alter neuronal architecture, and its ability to dominantly suppress endogenous small conductance K(Ca) currents may affect patterns of neuronal firing. Together, these two effects may play a part in the pathogenesis of schizophrenia and other neuropsychiatric disorders.
Asunto(s)
Núcleo Celular/metabolismo , Canales de Potasio Calcio-Activados , Canales de Potasio/química , Esquizofrenia/genética , Esquizofrenia/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Línea Celular , ADN Complementario/metabolismo , Electrofisiología , Eliminación de Gen , Genes Dominantes , Proteínas Fluorescentes Verdes , Humanos , Células Jurkat , Proteínas Luminiscentes/metabolismo , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Ratas , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Canales de Potasio de Pequeña Conductancia Activados por el Calcio , TransfecciónRESUMEN
Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr(250) or Val(275) in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(250) and Val(275) line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobic interactions with the pi-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside.
Asunto(s)
Canales de Calcio/metabolismo , Calcio/metabolismo , Clotrimazol/metabolismo , Canales de Potasio/metabolismo , Pirazoles/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Canales de Calcio/química , Citoplasma/metabolismo , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Modelos Moleculares , Datos de Secuencia Molecular , Canales de Potasio/química , Conformación Proteica , Pirazoles/antagonistas & inhibidores , Homología de Secuencia de AminoácidoRESUMEN
UNLABELLED: Transesophageal echocardiography (TEE) may improve intraoperative decision-making and patient outcome if it is performed and interpreted correctly. After revising our TEE examination to fulfill the published guidelines for basic TEE practitioners, we prospectively evaluated the ability of our cardiac anesthesiologists (all very experienced with TEE) to record and interpret this revised examination. Educational aids and regular TEE performance feedback were provided to the anesthesiologists. Their interpretations were compared with the independently determined results of experts. Compared with their own historical controls (42% recording rate), all anesthesiologists showed significant improvement in their ability to record a basic intraoperative TEE examination resulting in 81% (P < 0.0001) of all required images being recorded: 88% before cardiopulmonary bypass, 77% immediately after bypass, and 64% after chest closure. Seventy-nine percent of the images recorded at baseline were correctly interpreted, 6% were incorrectly interpreted, and 15% were not evaluated. Our attempt to assess compliance with published guidelines for basic intraoperative TEE resulted in a marked improvement in our intraoperative TEE practice. Most, but not all, standard cross-sections are recorded or interpreted correctly, even by highly experienced and motivated practitioners. IMPLICATIONS: Experienced cardiac anesthesiologists can obtain and correctly interpret most basic intraoperative transesophageal echocardiograms.
Asunto(s)
Anestesiología , Competencia Clínica , Ecocardiografía Transesofágica , Cardiopatías/diagnóstico por imagen , Ecocardiografía Transesofágica/normas , Evaluación Educacional , Adhesión a Directriz , Humanos , Periodo Intraoperatorio , Guías de Práctica Clínica como Asunto , Estudios ProspectivosRESUMEN
UNLABELLED: We tested the hypothesis that sevoflurane is a safer and more effective anesthetic than halothane during the induction and maintenance of anesthesia for infants and children with congenital heart disease undergoing cardiac surgery. With a background of fentanyl (5 microg/kg bolus, then 5 microg. kg(-1). h(-1)), the two inhaled anesthetics were directly compared in a randomized, double-blinded, open-label study involving 180 infants and children. Primary outcome variables included severe hypotension, bradycardia, and oxygen desaturation, defined as a 30% decrease in the resting mean arterial blood pressure or heart rate, or a 20% decrease in the resting arterial oxygen saturation, for at least 30 s. There were no differences in the incidence of these variables; however, patients receiving halothane experienced twice as many episodes of severe hypotension as those who received sevoflurane (P = 0.03). These recurrences of hypotension occurred despite an increased incidence of vasopressor use in the halothane-treated patients than in the sevoflurane-treated patients. Multivariate stepwise logistic regression demonstrated that patients less than 1 yr old were at increased risk for hypotension compared with older children (P = 0.0004), and patients with preoperative cyanosis were at increased risk for developing severe desaturation (P = 0.049). Sevoflurane may have hemodynamic advantages over halothane in infants and children with congenital heart disease. IMPLICATIONS: In infants and children with congenital heart disease, anesthesia with sevoflurane may result in fewer episodes of severe hypotension and less emergent drug use than anesthesia with halothane.
Asunto(s)
Anestésicos por Inhalación , Cardiopatías Congénitas/cirugía , Éteres Metílicos , Anestésicos por Inhalación/efectos adversos , Anestésicos Intravenosos , Procedimientos Quirúrgicos Cardíacos , Niño , Preescolar , Método Doble Ciego , Fentanilo , Halotano/efectos adversos , Humanos , Lactante , Recién Nacido , Modelos Logísticos , Éteres Metílicos/efectos adversos , Estudios Prospectivos , Factores de Riesgo , SevofluranoRESUMEN
To maintain Ca(2+) entry during T lymphocyte activation, a balancing efflux of cations is necessary. Using three approaches, we demonstrate that this cation efflux is mediated by Ca(2+)-activated K(+) (K(Ca)) channels, hSKCa2 in the human leukemic T cell line Jurkat and hIKCa1 in mitogen-activated human T cells. First, several recently developed, selective and potent pharmacological inhibitors of K(Ca) channels but not K(V) channels reduce Ca(2+) entry in Jurkat and in mitogen-activated human T cells. Second, dominant-negative suppression of the native K(Ca) channel in Jurkat T cells by overexpression of a truncated fragment of the cloned hSKCa2 channel decreases Ca(2+) influx. Finally, introduction of the hIKCa1 channel into Jurkat T cells maintains rapid Ca(2+) entry despite pharmacological inhibition of the native small conductance K(Ca) channel. Thus, K(Ca) channels play a vital role in T cell Ca(2+) signaling.
Asunto(s)
Señalización del Calcio , Calcio/metabolismo , Canales de Potasio/metabolismo , Linfocitos T/metabolismo , Animales , Células COS , Humanos , Células JurkatRESUMEN
Although the crucial role of Ca(2+) influx in lymphocyte activation has been well documented, little is known about the properties or expression levels of Ca(2+) channels in normal human T lymphocytes. The use of Na(+) as the permeant ion in divalent-free solution permitted Ca(2+) release-activated Ca(2+) (CRAC) channel activation, kinetic properties, and functional expression levels to be investigated with single channel resolution in resting and phytohemagglutinin (PHA)-activated human T cells. Passive Ca(2+) store depletion resulted in the opening of 41-pS CRAC channels characterized by high open probabilities, voltage-dependent block by extracellular Ca(2+) in the micromolar range, selective Ca(2+) permeation in the millimolar range, and inactivation that depended upon intracellular Mg(2+) ions. The number of CRAC channels per cell increased greatly from approximately 15 in resting T cells to approximately 140 in activated T cells. Treatment with the phorbol ester PMA also increased CRAC channel expression to approximately 60 channels per cell, whereas the immunosuppressive drug cyclosporin A (1 microM) suppressed the PHA-induced increase in functional channel expression. Capacitative Ca(2+) influx induced by thapsigargin was also significantly enhanced in activated T cells. We conclude that a surprisingly low number of CRAC channels are sufficient to mediate Ca(2+) influx in human resting T cells, and that the expression of CRAC channels increases approximately 10-fold during activation, resulting in enhanced Ca(2+) signaling.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/inmunología , Activación del Canal Iónico/fisiología , Linfocitos T/fisiología , Calcio/metabolismo , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Humanos , Células Jurkat , Cinética , Activación de Linfocitos/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Fitohemaglutininas/farmacología , Sodio/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunologíaRESUMEN
We used whole cell recording to evaluate functional expression of the intermediate conductance Ca(2+)-activated K(+) channel, IKCa1, in response to various mitogenic stimuli. One to two days following engagement of T-cell receptors to trigger both PKC- and Ca(2+)-dependent events, IKCa1 expression increased from an average of 8 to 300-800 channels/cell. Selective stimulation of the PKC pathway resulted in equivalent up-regulation, whereas a calcium ionophore was relatively ineffective. Enhancement in IKCa1 mRNA levels paralleled the increased channel number. The genomic organization of IKCa1, SKCa2, and SKCa3 were defined, and IK(Ca) and SK(Ca) genes were found to have a remarkably similar intron-exon structure. Mitogens enhanced IKCa1 promoter activity proportional to the increase in IKCa1 mRNA, suggesting that transcriptional mechanisms underlie channel up-regulation. Mutation of motifs for AP1 and Ikaros-2 in the promoter abolished this induction. Selective Kv1.3 inhibitors ShK-Dap(22), margatoxin, and correolide suppressed mitogenesis of resting T-cells but not preactivated T-cells with up-regulated IKCa1 channel expression. Selectively blocking IKCa1 channels with clotrimazole or TRAM-34 suppressed mitogenesis of preactivated lymphocytes, whereas resting T-cells were less sensitive. Thus, Kv1.3 channels are essential for activation of quiescent cells, but signaling through the PKC pathway enhances expression of IKCa1 channels that are required for continued proliferation.
Asunto(s)
Canales de Calcio/biosíntesis , Activación de Linfocitos , Canales de Potasio , Linfocitos T/metabolismo , Regulación hacia Arriba , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/genética , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Activación de Linfocitos/efectos de los fármacos , Mitógenos/farmacología , Modelos Biológicos , Datos de Secuencia Molecular , Fitohemaglutininas/farmacología , Regiones Promotoras Genéticas , Pirazoles/farmacología , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunología , Acetato de Tetradecanoilforbol/farmacología , Transcripción GenéticaRESUMEN
The antimycotic clotrimazole, a potent inhibitor of the intermediate-conductance calcium-activated K(+) channel, IKCa1, is in clinical trials for the treatment of sickle cell disease and diarrhea and is effective in ameliorating the symptoms of rheumatoid arthritis. However, inhibition of cytochrome P450 enzymes by clotrimazole limits its therapeutic value. We have used a rational design strategy to develop a clotrimazole analog that selectively inhibits IKCa1 without blocking cytochrome P450 enzymes. A screen of 83 triarylmethanes revealed the pharmacophore for channel block to be different from that required for cytochrome P450 inhibition. The "IKCa1-pharmacophore" consists of a (2-halogenophenyl)diphenylmethane moiety substituted by an unsubstituted polar pi-electron-rich heterocycle (pyrazole or tetrazole) or a -C≡N group, whereas cytochrome P450 inhibition absolutely requires the imidazole ring. A series of pyrazoles, acetonitriles, and tetrazoles were synthesized and found to selectively block IKCa1. TRAM-34 (1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole) inhibits the cloned and the native IKCa1 channel in human T lymphocytes with a K(d) of 20-25 nM and is 200- to 1,500-fold selective over other ion channels. Using TRAM-34, we show that blocking IKCa1 in human lymphocytes, in the absence of P450-inhibition, results in suppression of mitogen-stimulated [(3)H]thymidine incorporation of preactivated lymphocytes with EC(50)-values of 100 nM-1 microM depending on the donor. Combinations of TRAM-34 and cyclosporin A are more effective in suppressing lymphocyte mitogenesis than either compound alone. Our studies suggest that TRAM-34 and related compounds may hold therapeutic promise as immunosuppressants.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/efectos de los fármacos , Inmunosupresores/farmacología , Canales de Potasio , Pirazoles/farmacología , Clotrimazol/química , Ciclosporina/farmacología , Sistema Enzimático del Citocromo P-450/efectos de los fármacos , Diseño de Fármacos , Interacciones Farmacológicas , Conductividad Eléctrica , Humanos , Canales de Potasio de Conductancia Intermedia Activados por el Calcio , Activación del Canal Iónico , Ionomicina/farmacología , Activación de Linfocitos/efectos de los fármacos , Relación Estructura-Actividad , Linfocitos T/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
Dysregulation of calcium signaling has been causally implicated in brain aging and Alzheimer's disease. Mutations in the presenilin genes (PS1, PS2), the leading cause of autosomal dominant familial Alzheimer's disease (FAD), cause highly specific alterations in intracellular calcium signaling pathways that may contribute to the neurodegenerative and pathological lesions of the disease. To elucidate the cellular mechanisms underlying these disturbances, we studied calcium signaling in fibroblasts isolated from mutant PS1 knockin mice. Mutant PS1 knockin cells exhibited a marked potentiation in the amplitude of calcium transients evoked by agonist stimulation. These cells also showed significant impairments in capacitative calcium entry (CCE, also known as store-operated calcium entry), an important cellular signaling pathway wherein depletion of intracellular calcium stores triggers influx of extracellular calcium into the cytosol. Notably, deficits in CCE were evident after agonist stimulation, but not if intracellular calcium stores were completely depleted with thapsigargin. Treatment with ionomycin and thapsigargin revealed that calcium levels within the ER were significantly increased in mutant PS1 knockin cells. Collectively, our findings suggest that the overfilling of calcium stores represents the fundamental cellular defect underlying the alterations in calcium signaling conferred by presenilin mutations.
Asunto(s)
Señalización del Calcio , Proteínas de la Membrana/metabolismo , Enfermedad de Alzheimer/etiología , Animales , Bombesina/farmacología , Bradiquinina/farmacología , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteínas de la Membrana/genética , Ratones , Ratones Mutantes , Fosfatidilinositoles/metabolismo , Presenilina-1RESUMEN
Perillyl alcohol (POH) inhibits isoprenylation and has shown anticancer and chemopreventive properties in rodent models. The mechanism that underlies the anticancer activity of POH and other isoprenylation inhibitors is unknown but has been postulated to involve decreased levels of isoprenylated Ras and Ras-related proteins. Previously we demonstrated that POH effectively inhibits human T cell proliferation in vitro and can prevent acute and chronic rejection in a rat cardiac transplant model. In this report, we investigate the effects of POH on T lymphocytes at the single-cell level. POH disrupts the polarized shape and motility of antigen-specific murine 1E5 T cells. Using an optical trap to position anti-CD3-coated beads in contact with 1E5 T cells, we demonstrate that POH inhibits their TCR-mediated calcium response. Furthermore, we show that POH preferentially induces apoptosis in PHA-activated human T cells as well as in 1E5 T cells.
Asunto(s)
Transferasas Alquil y Aril/antagonistas & inhibidores , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Movimiento Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Monoterpenos , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/fisiología , Linfocitos T/efectos de los fármacos , Terpenos/farmacología , Adulto , Animales , Polaridad Celular/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Humanos , Ratones , Linfocitos T/metabolismo , Linfocitos T/fisiologíaRESUMEN
In Th1 and Th2 lymphocytes, activation begins with identical stimuli but results in the production of different cytokines. The expression of some cytokine genes is differentially induced according to the amplitude and pattern of Ca2+ signaling. Using fura- 2 Ca2+ imaging of murine Th1 and Th2 clones, we observed that the Ca2+ rise elicited following store depletion with thapsigargin is significantly lower in Th2 cells than in Th1 cells. Maximal Ca2+ influx rates and whole-cell Ca2+ currents showed that both Th1 and Th2 cells express indistinguishable Ca2+-release-activated Ca2+ channels. Therefore, we investigated other mechanisms controlling the concentration of intracellular Ca2+, including K+ channels and Ca2+ clearance from the cytosol. Whole-cell recording demonstrated that there is no distinction in the amplitudes of voltage-gated K+ currents in the two cell types. Ca2+-activated K+ (KCa) currents, however, were significantly smaller in Th2 cells than in Th1 cells. Pharmacological equalization of Ca2+-activated K+ currents in the two cell types reduced but did not completely eliminate the difference between Th1 and Th2 Ca2+ responses, suggesting divergence in an additional Ca2+ regulatory mechanism. Therefore, we analyzed Ca2+ clearance from the cytosol of both cell types and found that Th2 cells extrude Ca2+ more quickly than Th1 cells. The combination of a faster Ca2+ clearance mechanism and smaller Ca2+-activated K+ currents in Th2 cells accounts for the lower Ca2+ response of Th2 cells compared with Th1 cells.
Asunto(s)
Calcio/metabolismo , Canales de Potasio/metabolismo , Células TH1/metabolismo , Células Th2/metabolismo , Animales , Canales de Calcio/metabolismo , Canales de Calcio/fisiología , Señalización del Calcio/inmunología , Células Clonales , Citosol/inmunología , Citosol/metabolismo , Activación del Canal Iónico/inmunología , Potenciales de la Membrana/inmunología , Ratones , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Células TH1/fisiología , Células Th2/fisiologíaRESUMEN
We have used a structure-based design strategy to transform the polypeptide toxin charybdotoxin, which blocks several voltage-gated and Ca(2+)-activated K(+) channels, into a selective inhibitor. As a model system, we chose two channels in T-lymphocytes, the voltage-gated channel Kv1.3 and the Ca(2+)-activated channel IKCa1. Homology models of both channels were generated based on the crystal structure of the bacterial channel KcsA. Initial docking of charybdotoxin was undertaken with both models, and the accuracy of these docking configurations was tested by mutant cycle analyses, establishing that charybdotoxin has a similar docking configuration in the external vestibules of IKCa1 and Kv1.3. Comparison of the refined models revealed a unique cluster of negatively charged residues in the turret of Kv1.3, not present in IKCa1. To exploit this difference, three novel charybdotoxin analogs were designed by introducing negatively charged residues in place of charybdotoxin Lys(32), which lies in close proximity to this cluster. These analogs block IKCa1 with approximately 20-fold higher affinity than Kv1.3. The other charybdotoxin-sensitive Kv channels, Kv1.2 and Kv1. 6, contain the negative cluster and are predictably insensitive to the charybdotoxin position 32 analogs, whereas the maxi-K(Ca) channel, hSlo, lacking the cluster, is sensitive to the analogs. This provides strong evidence for topological similarity of the external vestibules of diverse K(+) channels and demonstrates the feasibility of using structure-based strategies to design selective inhibitors for mammalian K(+) channels. The availability of potent and selective inhibitors of IKCa1 will help to elucidate the role of this channel in T-lymphocytes during the immune response as well as in erythrocytes and colonic epithelia.