RESUMEN
The genetic architecture of common epilepsies is largely unknown. HCNs are excellent epilepsy candidate genes because of their fundamental neurophysiological roles. Screening in subjects with febrile seizures and genetic epilepsy with febrile seizures plus revealed that 2.4% carried a common triple proline deletion (delPPP) in HCN2 that was seen in only 0.2% of blood bank controls. Currents generated by mutant HCN2 channels were approximately 35% larger than those of controls; an effect revealed using automated electrophysiology and an appropriately powered sample size. This is the first association of HCN2 and familial epilepsy, demonstrating gain of function of HCN2 current as a potential contributor to polygenic epilepsy.
Asunto(s)
Canales Iónicos/genética , Convulsiones Febriles/genética , Eliminación de Secuencia/genética , Animales , Biofisica/métodos , AMP Cíclico/farmacología , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Análisis Mutacional de ADN/métodos , Estimulación Eléctrica/métodos , Frecuencia de los Genes , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Oocitos , Técnicas de Placa-Clamp/métodos , Canales de Potasio/genética , Prolina/genética , Transfección/métodos , XenopusRESUMEN
PURPOSE: Studies in genetic absence epileptic rats from Strasbourg (GAERS) indicate that enhancement of gamma aminobutyric acid (GABA(A)) receptor activity is a critical mechanism in the aggravation of seizures by carbamazepine (CBZ). We examined whether structural analogs of CBZ, oxcarbazepine (OXC), and its active metabolite, monohydroxy derivative (MHD), also potentiate GABA(A) receptor current and aggravate seizures. METHODS: In vitro studies in Xenopus oocytes compared the three drugs' effect on GABA(A) receptor currents. In vivo studies compared seizure activity in GAERS after intraperitoneal drug administration. RESULTS: OXC potentiated GABA(A) receptor current and aggravated seizures in GAERS, similarly to the effect of CBZ. Conversely, MHD showed only a minor potentiation of GABA(A) receptor current and did not aggravate seizures. DISCUSSION: A hydroxyl group at the C-10 position on the CBZ tricyclic structure in MHD reduces GABA(A) receptor potentiation and seizure aggravation. Reports of the aggravation of absence seizures in patients taking OXC may result from circulating unmetabolized OXC rather than MHD.
Asunto(s)
Anticonvulsivantes/farmacología , Carbamazepina/análogos & derivados , Epilepsia Tipo Ausencia/inducido químicamente , Receptores de GABA-A/efectos de los fármacos , Animales , Anticonvulsivantes/efectos adversos , Encéfalo/efectos de los fármacos , Encéfalo/fisiopatología , Carbamazepina/efectos adversos , Carbamazepina/farmacología , Electroencefalografía , Epilepsia Tipo Ausencia/diagnóstico , Epilepsia Tipo Ausencia/fisiopatología , Femenino , Oxcarbazepina , RatasRESUMEN
Mutations in the GABA(A) receptor gamma2 subunit are associated with childhood absence epilepsy and febrile seizures. To understand better the molecular basis of absence epilepsy in man, we developed a mouse model harboring a gamma2 subunit point mutation (R43Q) found in a large Australian family. Mice heterozygous for the mutation demonstrated behavioral arrest associated with 6-to 7-Hz spike-and-wave discharges, which are blocked by ethosuximide, a first-line treatment for absence epilepsy in man. Seizures in the mouse showed an abrupt onset at around age 20 days corresponding to the childhood nature of this disease. Reduced cell surface expression of gamma2(R43Q) was seen in heterozygous mice in the absence of any change in alpha1 subunit surface expression, ruling out a dominant-negative effect. GABA(A)-mediated synaptic currents recorded from cortical pyramidal neurons revealed a small but significant reduction that was not seen in the reticular or ventrobasal thalamic nuclei. We hypothesize that a subtle reduction in cortical inhibition underlies childhood absence epilepsy seen in humans harboring the R43Q mutation.
Asunto(s)
Epilepsia Tipo Ausencia/fisiopatología , Animales , Modelos Animales de Enfermedad , Epilepsia Tipo Ausencia/tratamiento farmacológico , Epilepsia Tipo Ausencia/genética , Epilepsia Tipo Ausencia/metabolismo , Etosuximida/farmacología , Predisposición Genética a la Enfermedad , Ratones , Fenotipo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores de GABA-A/genética , Receptores de GABA-A/metabolismoRESUMEN
Carbamazepine (CBZ) aggravates many generalized seizures types, particularly absence seizures, but the mechanisms underlying this are poorly understood. GABA signaling within the reticular nucleus (Rt) and the ventrobasal complex (VB) of the thalamus is critical to the neurophysiology of absence seizures. The hypothesis that CBZ aggravates absence seizures by acting at the VB thalamus via a GABA(A) receptor-mediated mechanism was investigated in a genetic rat model, generalized absence epilepsy rats from Strasbourg (GAERS). Seizure activity was quantified by a 90-min electroencephalogram recording postdrug injection. Intracerebroventricular injections of CBZ (15 microg in 4 microl) resulted in seizure aggravation versus vehicle treatment, with a mean increase in seizure time of 40%. This indicates that CBZ acts directly, rather than via a metabolite, on the brain to aggravate seizures. Seizure aggravation also occurred following bilateral microinjection of CBZ (0.75 microg in 0.2 microl) into the VB (53%) but not following injection into the Rt (-9%). However, seizure aggravation was blocked when the GABA(A) receptor antagonist, bicuculline (BIC, 0.04 microg in 0.2 microl), was coinjected with CBZ into the VB. Injection of BIC alone (versus vehicle) into the VB also blocked seizure aggravation following systemic administration of CBZ (15 mg/kg i.p.). In vitro studies in Xenopus oocytes expressing recombinant GABA(A) receptors demonstrated that CBZ produced a dose-dependent potentiation of the GABA current at a physiological relevant concentration range (1-100 microM). These data demonstrate that CBZ acts at the VB thalamus to aggravate absence seizures in GAERS and that activation of GABA(A) receptors is critical to this effect.
Asunto(s)
Anticonvulsivantes/efectos adversos , Carbamazepina/efectos adversos , Epilepsia Tipo Ausencia/etiología , Animales , Encéfalo/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Antagonistas de Receptores de GABA-A , Ratas , Ratas Wistar , Tálamo/efectos de los fármacos , XenopusRESUMEN
The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.
Asunto(s)
Adenosina Trifosfato/química , Macrófagos/metabolismo , Mycobacterium/metabolismo , Polimorfismo Genético , Receptores Purinérgicos P2/genética , Serina/genética , Treonina/genética , Alelos , Animales , Bario/farmacología , Diferenciación Celular , Línea Celular , Codón de Terminación , Electrofisiología , Etidio/farmacología , Femenino , Citometría de Flujo , Frecuencia de los Genes , Proteínas Fluorescentes Verdes/metabolismo , Heterocigoto , Homocigoto , Humanos , Interferón gamma/metabolismo , Linfocitos/citología , Linfocitos/metabolismo , Linfocitos/microbiología , Macrófagos/microbiología , Masculino , Microscopía Fluorescente , Monocitos/citología , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Mycobacterium bovis/metabolismo , Oocitos/metabolismo , Polimorfismo de Nucleótido Simple , Receptores Purinérgicos P2X7 , Serina/química , Treonina/química , Transfección , Xenopus laevisRESUMEN
The P2X(7) receptor is a ligand-gated channel that is highly expressed on mononuclear cells of the immune system and that mediates ATP-induced apoptosis. Wide variations in the function of the P2X receptor have been observed, explained in part by (7)loss-of-function polymorphisms that change Glu(496) to Ala (E496A) and Ile(568) to Asn (I568N). In this study, a third polymorphism, which substitutes an uncharged glutamine for the highly positively charged Arg(307) (R307Q), has been found in heterozygous dosage in 12 of 420 subjects studied. P2X(7) function was measured by ATP-induced fluxes of Rb(+), Ba(2+), and ethidium(+) into peripheral blood monocytes or various lymphocyte subsets and was either absent or markedly decreased. Transfection experiments showed that P2X(7) carrying the R307Q mutation lacked either channel or pore function despite robust protein synthesis and surface expression of the receptor. The monoclonal antibody (clone L4) that binds to the extracellular domain of wild type P2X(7) and blocks P2X(7) function failed to bind to the R307Q mutant receptor. Differentiation of monocytes to macrophages up-regulated P2X(7) function in cells heterozygous for the R307Q to a value 10-40% of that for wild type macrophages. However, macrophages from a subject who was double heterozygous for R307Q/I568N remained totally non-functional for P2X(7), and lymphocytes from the same subject also lacked ATP-stimulated phospholipase D activity. These data identify a third loss-of-function polymorphism affecting the human P2X(7) receptor, and since the affected Arg(307) is homologous to those amino acids essential for ATP binding to P2X(1) and P2X(2), it is likely that this polymorphism abolishes the binding of ATP to the extracellular domain of P2X(7).
Asunto(s)
Polimorfismo de Nucleótido Simple , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2/metabolismo , Adenosina Trifosfato/metabolismo , Sustitución de Aminoácidos , Animales , Bario/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Cartilla de ADN/genética , Femenino , Heterocigoto , Humanos , Técnicas In Vitro , Transporte Iónico , Leucocitos/metabolismo , Macrófagos/metabolismo , Oocitos/metabolismo , Fosfolipasa D/metabolismo , Receptores Purinérgicos P2/química , Receptores Purinérgicos P2X7 , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Xenopus laevisRESUMEN
We have recently identified and cloned the cDNA of a new member of the glucose transporter family that has been designated GLUT12. GLUT12 possesses the structural features critical to facilitative transport of glucose but the key to understanding the possible physiological roles of this novel protein requires analysis of functional glucose transport. In the current study, we have utilized the Xenopus laevis oocyte expression system to assay transport of the glucose analog 2-deoxy-D-glucose and characterize the glucose transport properties and hexose affinities of GLUT12. Our results demonstrate that GLUT12 facilitates transport of glucose with an apparent preferential substrate affinity for glucose over other hexoses assayed. The results are significant to understanding the potential role and importance of GLUT12 in insulin-sensitive tissues and also cells with high glucose utilization such as cancer cells.
Asunto(s)
Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Animales , Transporte Biológico , Glucosa/metabolismo , Proteínas Facilitadoras del Transporte de la Glucosa , Transportador de Glucosa de Tipo 4 , Microscopía Confocal , Proteínas de Transporte de Monosacáridos/análisis , Proteínas de Transporte de Monosacáridos/fisiología , Oocitos/metabolismo , Especificidad por Sustrato , Xenopus laevisRESUMEN
Fatty acids and other negatively charged single-chain lipids increase large-conductance Ca(2+)-activated K(+) (BK(Ca)) channel activity, whereas sphingosine and other positively charged single-chain lipids suppress activity. Because these molecules are effective on both inside-out and outside-out patches and because they can flip across the bilayer, the location of their site of action is unclear. To identify the site of action of charged lipids on this channel, we used two compounds that are unlikely to flip across the lipid bilayer. Palmitoyl coenzyme A (PCoA) was used to identify the site of action of negatively charged lipids, and a positively charged myristoylated pentapeptide (myr-KPRPK) was used to investigate the site of action of positively charged lipids. The effect of these compounds on channel activity was studied in excised patches using patch-clamp techniques. In "normal" ionic strength solutions and in experiments where high-ionic strength solutions were used to shield membrane surface charge, PCoA increased channel activity only when applied to outside-out patches, suggesting that the site of action of negatively charged lipids is located on the outer surface of the membrane. A decrease in activity, similar to that of other positively charged lipids, was observed only when myr-KPRPK was applied to outside-out patches, suggesting that positively charged lipids suppress activity by also acting on the outer membrane surface. Some channel blockade effects of myr-KPRPK and KPRPK are also described. The sidedness of action suggests that modulation of channel activity by single-chain lipids can occur by their interaction with the channel protein.
Asunto(s)
Aniones/metabolismo , Arterias/metabolismo , Membrana Celular/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos , Músculo Liso Vascular/metabolismo , Miocitos del Músculo Liso/metabolismo , Canales de Potasio Calcio-Activados/metabolismo , Aminas/farmacología , Aniones/farmacología , Arterias/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Ácidos Grasos/farmacología , Lípidos/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/efectos de los fármacos , Palmitoil Coenzima A/metabolismo , Palmitoil Coenzima A/farmacología , Fragmentos de Péptidos/metabolismo , Fragmentos de Péptidos/farmacología , Canales de Potasio Calcio-Activados/efectos de los fármacos , Compuestos de Amonio Cuaternario/farmacología , Compuestos de TrimetilamonioRESUMEN
To determine the mechanism of fatty acid modulation of rabbit pulmonary artery large-conductance Ca2+ -activated K+ (BK(Ca)) channel activity, we studied effects of fatty acids and other lipids on channel activity in excised patches with patch-clamp techniques. The structural features of the fatty acid required to increase BK(Ca) channel activity (or average number of open channels, NP(o)) were identified to be the negatively charged head group and a sufficiently long (C > 8) carbon chain. Positively charged lipids like sphingosine, which have a sufficiently long alkyl chain (C >or= 8), produced a decrease in NP(o). Neutral and short-chain lipids did not alter NP(o). Screening of membrane surface charge with high-ionic-strength bathing solutions (330 mM K+ or 130 mM K+, 300 mM Na+) did not alter the modulation of the BK(Ca) channel NP(o) by fatty acids and other charged lipids, indicating that channel modulation is unlikely to be due to an alteration of the membrane electric field or the attraction of local counterions to the channel. Fatty acids and other negatively charged lipids were able to modulate BK(Ca) channel activity in bathing solutions containing 0 mM Ca2+, 20 mM EGTA, suggesting that calcium is not required for this modulation. Together, these results indicate that modulation of BK(Ca) channels by fatty acids and other charged lipids most likely occurs by their direct interaction with the channel protein itself or with some other channel-associated component.
Asunto(s)
Ácidos Grasos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canales de Potasio Calcio-Activados/química , Canales de Potasio Calcio-Activados/metabolismo , Aminas/farmacología , Animales , Ácido Araquidónico/farmacología , Membrana Celular/enzimología , Electroquímica , Activación del Canal Iónico/fisiología , Canales de Potasio de Gran Conductancia Activados por el Calcio , Músculo Liso Vascular/fisiología , Ácido Mirístico/farmacología , Ácido Oléico/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Quinasas/metabolismo , Conejos , Esfingosina/farmacología , Relación Estructura-ActividadRESUMEN
The mechanism by which dietary fatty acids confer protection against cardiac arrhythmias and sudden cardiac death is not resolved. Here, we study the effects of several known cardio-protective and arrhythmogenic fatty acids on the slowly activating delayed rectifier potassium current (IKs), which is responsible for the repolarization phase of the cardiac action potential. cRNAs encoding either or both of the two subunits, KvLQT1 and hminK, that together produce IKs, were injected into Xenopus oocytes, and the effects of various fatty acids were determined. Docosahexaenoic acid (DHA) significantly augmented IKs as did the short-chained fully saturated lauric acid, and to a lesser extent the cis-unsaturated oleic acid. Eicosapentaenoic acid (EPA) was without significant effect on current magnitude, although it reduced the rate of activation. These results suggest that not all "antiarrhythmic" fatty acids target the same channel. To examine the role of hminK in this response, KvLQT1 was expressed alone. In this case, DHA, lauric acid, and oleic acid did not augment current, suggesting that hminK confers fatty acid sensitivity to IKs.