Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 28
Filtrar
Más filtros

Banco de datos
Tipo del documento
Intervalo de año de publicación
1.
Circulation ; 139(18): 2157-2169, 2019 04 30.
Artículo en Inglés | MEDLINE | ID: mdl-30764634

RESUMEN

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.


Asunto(s)
Fibrilación Atrial , Bloqueo Atrioventricular , Bradicardia , Canales de Potasio Rectificados Internamente Asociados a la Proteína G , Enfermedades Genéticas Congénitas , Mutación Missense , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Fibrilación Atrial/genética , Fibrilación Atrial/metabolismo , Fibrilación Atrial/patología , Fibrilación Atrial/fisiopatología , Bloqueo Atrioventricular/genética , Bloqueo Atrioventricular/metabolismo , Bloqueo Atrioventricular/patología , Bloqueo Atrioventricular/fisiopatología , Benzopiranos/farmacología , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/patología , Bradicardia/fisiopatología , Técnicas Electrofisiológicas Cardíacas , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Enfermedades Genéticas Congénitas/patología , Enfermedades Genéticas Congénitas/fisiopatología , Humanos , Masculino , Xenopus laevis , Pez Cebra
2.
Mol Pharmacol ; 93(6): 592-600, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29650538

RESUMEN

Many compounds inhibit tetrameric and pseudo-tetrameric cation channels by associating with the central cavity located in the middle of the membrane plane. They traverse the ion conduction pathway from the intracellular side and through access to the cavity. Previously, we reported that the bacteriostatic agent, proflavine, preferentially blocked a subset of inward rectifier K+ (Kir) channels. However, the development of the inhibition of Kir1.1 by the compound was obviously different from that operating in Kir3.2 as a pore blocker. To gain mechanistic insights into the compound-channel interaction, we analyzed its chemical specificity, subunit selectivity, and voltage dependency using 13 different combinations of Kir-channel family members and 11 proflavine derivatives. The Kir-channel family members were classified into three groups: 1) Kir2.2, Kir3.x, Kir4.2, and Kir6.2Δ36, which exhibited Kir3.2-type inhibition (slow onset and recovery, irreversible, and voltage-dependent blockage); 2) Kir1.1 and Kir4.1/Kir5.1 (prompt onset and recovery, reversible, and voltage-independent blockage); and 3) Kir2.1, Kir2.3, Kir4.1, and Kir7.1 (no response). The degree of current inhibition depended on the combination of compounds and channels. Chimera between proflavine-sensitive Kir1.1 and -insensitive Kir4.1 revealed that the extracellular portion of Kir1.1 is crucial for the recognition of the proflavine derivative acrinol. In conclusion, preferential blockage of Kir-channel family members by proflavine derivatives is based on multiple modes of action. This raises the possibility of designing subunit-specific inhibitors.


Asunto(s)
Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Canales de Potasio de Rectificación Interna/metabolismo , Proflavina/farmacología , Animales , Humanos , Ratones , Ratas
3.
Physiol Rev ; 90(1): 291-366, 2010 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-20086079

RESUMEN

Inwardly rectifying K(+) (Kir) channels allow K(+) to move more easily into rather than out of the cell. They have diverse physiological functions depending on their type and their location. There are seven Kir channel subfamilies that can be classified into four functional groups: classical Kir channels (Kir2.x) are constitutively active, G protein-gated Kir channels (Kir3.x) are regulated by G protein-coupled receptors, ATP-sensitive K(+) channels (Kir6.x) are tightly linked to cellular metabolism, and K(+) transport channels (Kir1.x, Kir4.x, Kir5.x, and Kir7.x). Inward rectification results from pore block by intracellular substances such as Mg(2+) and polyamines. Kir channel activity can be modulated by ions, phospholipids, and binding proteins. The basic building block of a Kir channel is made up of two transmembrane helices with cytoplasmic NH(2) and COOH termini and an extracellular loop which folds back to form the pore-lining ion selectivity filter. In vivo, functional Kir channels are composed of four such subunits which are either homo- or heterotetramers. Gene targeting and genetic analysis have linked Kir channel dysfunction to diverse pathologies. The crystal structure of different Kir channels is opening the way to understanding the structure-function relationships of this simple but diverse ion channel family.


Asunto(s)
Canales de Potasio de Rectificación Interna/química , Canales de Potasio de Rectificación Interna/fisiología , Animales , Membrana Celular/química , Membrana Celular/fisiología , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Humanos , Canales KATP , Ratones , Ratones Noqueados , Fenómenos Farmacológicos/fisiología
4.
Biochim Biophys Acta ; 1838(2): 521-31, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24028827

RESUMEN

A variety of extracellular stimuli regulate cellular responses via membrane receptors. A well-known group of seven-transmembrane domain-containing proteins referred to as G protein-coupled receptors, directly couple with the intracellular GTP-binding proteins (G proteins) across cell membranes and trigger various cellular responses by regulating the activity of several enzymes as well as ion channels. Many specific populations of ion channels are directly controlled by G proteins; however, indirect modulation of some channels by G protein-dependent phosphorylation events and lipid metabolism is also observed. G protein-mediated diverse modifications affect the ion channel activities and spatio-temporally regulate membrane potentials as well as of intracellular Ca(2+) concentrations in both excitatory and non-excitatory cells. This article is part of a Special Issue entitled: Reciprocal influences between cell cytoskeleton and membrane channels, receptors and transporters. Guest Editor: Jean Claude Hervé.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Canales Iónicos/metabolismo , Animales , Humanos , Transducción de Señal
5.
Cell Physiol Biochem ; 36(5): 1847-61, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26184980

RESUMEN

BACKGROUND/AIMS: KCNQ channels transport K+ ions and participate in various cellular functions. The channels directly assemble with auxiliary proteins such as a ubiquitous Ca2+- sensor protein, calmodulin (CaM), to configure the physiological properties in a tissue-specific manner. Although many CaM-like Ca2+-sensor proteins have been identified in eukaryotes, how KCNQ channels selectively interact with CaM and how the homologues modulate the functionality of the channels remain unclear. METHODS: We developed protocols to evaluate the interaction between the green fluorescent protein-tagged C-terminus of KCNQ1 (KCNQ1cL) and Ca2+-sensors by detecting its fluorescence in size exclusion chromatography and electrophoresed gels. The effects of Ca2+-sensor proteins on KCNQ1 activity was measured by two electrode voltage clamp technique of Xenopus oocytes. RESULTS: When co-expressed CaM and KCNQ1cL, they assemble in a 4:4 stoichiometry, forming a hetero-octamer. Among nine CaM homologues tested, Calml3 was found to form a hetero-octamer with KCNQ1cL and to associate with the full-length KCNQ1 in a competitive manner with CaM. When co-expressed in oocytes, Calml3 rendered KCNQ1 channels resistant to the voltage-dependent depletion of phosphatidylinositol 4,5-bisphosphate by voltage-sensitive phosphatase. CONCLUSION: Since Calml3 is closely related to CaM and is prominently expressed in epithelial cells, Calml3 may be a constituent of epithelial KCNQ1 channels and underscores the molecular diversity of endogenous KCNQ1 channels.


Asunto(s)
Calmodulina/fisiología , Canal de Potasio KCNQ1/fisiología , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Electroforesis en Gel de Poliacrilamida , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Humanos , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/genética , Datos de Secuencia Molecular , Unión Proteica , Homología de Secuencia de Aminoácido , Espectrometría de Fluorescencia , Xenopus laevis
6.
Cell Tissue Res ; 359(2): 627-634, 2015 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-25380566

RESUMEN

Brain ependymal cells, which form an epithelial layer covering the cerebral ventricles, have been shown to play a role in the regulation of cerebrospinal and interstitial fluids. The machinery underlying this, however, remains largely unknown. Here, we report the specific localization of an inwardly rectifying K(+) channel, Kir4.1, on the ependymal cell membrane suggesting involvement of the channel in this function. Immunohistochemical study with confocal microscopy identified Kir4.1 labeling on the lateral but not apical membrane of ependymal cells. Ultrastructural analysis revealed that Kir4.1-immunogold particles were specifically localized and clustered on adjacent membranes at puncta adherens type junctions, whereas an aquaporin water channel, AQP4, that was also detected on the lateral membrane only occurred at components other than adherens junctions. Therefore, in ependymal cells, Kir4.1 and AQP4 are partitioned into distinct membrane compartments that might respectively transport either K(+) or water. Kir4.1 was also expressed in a specialized form of ependymal cell, namely the tanycyte, being abundant in tanycyte processes wrapping neuropils and blood vessels. These specific localizations suggest that Kir4.1 mediates intercellular K(+) exchange between ependymal cells and also K(+)-buffering transport via tanycytes that can interconnect neurons and vessels/ventricles. We propose that ependymal cells and tanycytes differentially operate Kir4.1 and AQP4 actively to control the property of fluids at local areas in the brain.


Asunto(s)
Compartimento Celular , Membrana Celular/metabolismo , Epéndimo/citología , Canales de Potasio de Rectificación Interna/metabolismo , Animales , Acuaporina 4/metabolismo , Membrana Celular/ultraestructura , Epéndimo/metabolismo , Epéndimo/ultraestructura , Células Ependimogliales/citología , Células Ependimogliales/metabolismo , Masculino , Transporte de Proteínas , Ratas Wistar , Fracciones Subcelulares/metabolismo
7.
J Physiol ; 592(6): 1237-48, 2014 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-24421355

RESUMEN

Partial agonists are used clinically to avoid overstimulation of receptor-mediated signalling, as they produce a submaximal response even at 100% receptor occupancy. The submaximal efficacy of partial agonists is due to conformational change of the agonist-receptor complex, which reduces effector activation. In addition to signalling activators, several regulators help control intracellular signal transductions. However, it remains unclear whether these signalling regulators contribute to partial agonism. Here we show that regulator of G-protein signalling (RGS) 4 is a determinant for partial agonism of the M2 muscarinic receptor (M2R). In rat atrial myocytes, pilocarpine evoked smaller G-protein-gated K(+) inwardly rectifying (KG) currents than those evoked by ACh. In a Xenopus oocyte expression system, pilocarpine acted as a partial agonist in the presence of RGS4 as it did in atrial myocytes, while it acted like a full agonist in the absence of RGS4. Functional couplings within the agonist-receptor complex/G-protein/RGS4 system controlled the efficacy of pilocarpine relative to ACh. The pilocarpine-M2R complex suppressed G-protein-mediated activation of KG currents via RGS4. Our results demonstrate that partial agonism of M2R is regulated by the RGS4-mediated inhibition of G-protein signalling. This finding helps us to understand the molecular components and mechanism underlying the partial agonism of M2R-mediated physiological responses.


Asunto(s)
Potasio/metabolismo , Proteínas RGS/metabolismo , Receptor Muscarínico M2/agonistas , Acetilcolina/farmacología , Animales , Membrana Celular/metabolismo , Dopamina/farmacología , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Masculino , Potenciales de la Membrana , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Pilocarpina/farmacología , Dominios y Motivos de Interacción de Proteínas , Proteínas RGS/química , Proteínas RGS/genética , Ratas , Ratas Wistar , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Xenopus laevis
8.
Biophys J ; 105(6): 1515-25, 2013 Sep 17.
Artículo en Inglés | MEDLINE | ID: mdl-24048003

RESUMEN

Acetylcholine (ACh) rapidly increases cardiac K(+) currents (IKACh) by activating muscarinic K(+) (KACh) channels followed by a gradual amplitude decrease within seconds. This phenomenon is called short-term desensitization and its precise mechanism and physiological role are still unclear. We constructed a mathematical model for IKACh to examine the conditions required to reconstitute short-term desensitization. Two conditions were crucial: two distinct muscarinic receptors (m2Rs) with different affinities for ACh, which conferred an IKACh response over a wide range of ACh concentrations, and two distinct KACh channels with different affinities for the G-protein ßγ subunits, which contributed to reconstitution of the temporal behavior of IKACh. Under these conditions, the model quantitatively reproduced several unique properties of short-term desensitization observed in myocytes: 1), the peak and quasi-steady states with 0.01-100 µM [ACh]; 2), effects of ACh preperfusion; and 3), recovery from short-term desensitization. In the presence of 10 µM ACh, the IKACh model conferred recurring spontaneous firing after asystole of 8.9 s and 10.7 s for the Demir and Kurata sinoatrial node models, respectively. Therefore, two different populations of KACh channels and m2Rs may participate in short-term desensitization of IKACh in native myocytes, and may be responsible for vagal escape at nodal cells.


Asunto(s)
Acetilcolina/metabolismo , Fenómenos Electrofisiológicos , Corazón/fisiología , Modelos Biológicos , Potasio/metabolismo , Potenciales de Acción , Proteínas de Unión al GTP/metabolismo , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Células Musculares/citología , Células Musculares/metabolismo , Receptores Muscarínicos/metabolismo , Nodo Sinoatrial/citología , Nodo Sinoatrial/metabolismo , Nodo Sinoatrial/fisiología , Factores de Tiempo , Estimulación del Nervio Vago
9.
J Biol Chem ; 286(48): 41801-41811, 2011 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-21982822

RESUMEN

Ion channels gate at membrane-embedded domains by changing their conformation along the ion conduction pathway. Inward rectifier K(+) (Kir) channels possess a unique extramembrane cytoplasmic domain that extends this pathway. However, the relevance and contribution of this domain to ion permeation remain unclear. By qualitative x-ray crystallographic analysis, we found that the pore in the cytoplasmic domain of Kir3.2 binds cations in a valency-dependent manner and does not allow the displacement of Mg(2+) by monovalent cations or spermine. Electrophysiological analyses revealed that the cytoplasmic pore of Kir3.2 selectively binds positively charged molecules and has a higher affinity for Mg(2+) when it has a low probability of being open. The selective blocking of chemical modification of the side chain of pore-facing residues by Mg(2+) indicates that the mode of binding of Mg(2+) is likely to be similar to that observed in the crystal structure. These results indicate that the Kir3.2 crystal structure has a closed conformation with a negative electrostatic field potential at the cytoplasmic pore, the potential of which may be controlled by conformational changes in the cytoplasmic domain to regulate ion diffusion along the pore.


Asunto(s)
Cationes Bivalentes/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Magnesio/química , Animales , Cationes Bivalentes/metabolismo , Cristalografía por Rayos X , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Células HEK293 , Humanos , Magnesio/metabolismo , Ratones , Estructura Terciaria de Proteína
10.
Biochem Biophys Res Commun ; 418(1): 161-6, 2012 Feb 03.
Artículo en Inglés | MEDLINE | ID: mdl-22244872

RESUMEN

Human ether-a-go-go-related gene (hERG) channels play a critical role in cardiac action potential repolarization. The unintended block of hERG channels by compounds can prolong the cardiac action potential duration and induce arrhythmia. Several compounds not only block hERG channels but also enhance channel activation after the application of a depolarizing voltage step. This is referred to as facilitation. In this study, we tried to extract the property of compounds that induce hERG channel facilitation. We first examined the facilitation effects of structurally diverse hERG channel blockers in Xenopus oocytes. Ten of 13 assayed compounds allowed facilitation, suggesting that it is an effect common to most hERG channel blockers. We constructed a pharmacophore model for hERG channel facilitation. The model consisted of one positively ionizable feature and three hydrophobic features. Verification experiments suggest that the model well describes the structure-activity relationship for facilitation. Comparison of the pharmacophore for facilitation with that for hERG channel block showed that the spatial arrangement of features is clearly different. It is therefore conceivable that two different interactions of a compound with hERG channels exert two pharmacological effects, block and facilitation.


Asunto(s)
Canales de Potasio Éter-A-Go-Go/fisiología , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/farmacología , Relación Estructura-Actividad Cuantitativa , Animales , Atenolol/química , Atenolol/farmacología , Clorfeniramina/química , Clorfeniramina/farmacología , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Fluoxetina/química , Fluoxetina/farmacología , Haloperidol/química , Haloperidol/farmacología , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Imipramina/química , Imipramina/farmacología , Metoprolol/química , Metoprolol/farmacología , Nortriptilina/química , Nortriptilina/farmacología , Prometazina/química , Prometazina/farmacología , Propranolol/química , Propranolol/farmacología , Sotalol/química , Sotalol/farmacología , Terfenadina/química , Terfenadina/farmacología , Verapamilo/química , Verapamilo/farmacología , Xenopus laevis
11.
J Biol Chem ; 285(49): 38517-23, 2010 Dec 03.
Artículo en Inglés | MEDLINE | ID: mdl-20880843

RESUMEN

Inward rectifier K(+) (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP(2)), but G protein-gated Kir (K(G)) channels further require either G protein ßγ subunits (Gßγ) or intracellular Na(+) for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of K(G) channel subunit Kir3.2 obtained in the presence and the absence of Na(+). The Na(+)-free Kir3.2, but not the Na(+)-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gßγ- and Na(+)-dependent activation, lowered PIP(2) sensitivity. The conservation of these residues within the K(G) channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP(2) sensitivity.


Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Fosfatidilinositol 4,5-Difosfato/química , Sodio/química , Animales , Cristalografía por Rayos X , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Ratones , Fosfatidilinositol 4,5-Difosfato/genética , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sodio/metabolismo , Relación Estructura-Actividad
12.
Biochem Biophys Res Commun ; 407(2): 366-71, 2011 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-21396912

RESUMEN

The gate at the pore-forming domain of potassium channels is allosterically controlled by a stimulus-sensing domain. Using Cd²(+) as a probe, we examined the structural elements responsible for gating in an inward-rectifier K(+) channel (Kir3.2). One of four endogenous cysteines facing the cytoplasm contributes to a high-affinity site for inhibition by internal Cd²(+). Crystal structure of its cytoplasmic domain in complex with Cd²(+) reveals that octahedral coordination geometry supports the high-affinity binding. This mode of action causes the tethering of the N-terminus to CD loop in the stimulus-sensing domain, suggesting that their conformational changes participate in gating and Cd²(+) inhibits Kir3.2 by trapping the conformation in the closed state like "inverse agonist".


Asunto(s)
Cadmio/farmacología , Agonismo Inverso de Drogas , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/agonistas , Animales , Cisteína/química , Cisteína/genética , Citoplasma/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Células HEK293 , Humanos , Activación del Canal Iónico/efectos de los fármacos , Ratones , Estabilidad Proteica/efectos de los fármacos , Estructura Terciaria de Proteína/efectos de los fármacos , Porcinos , Difracción de Rayos X
13.
Biochem Biophys Res Commun ; 415(1): 141-6, 2011 Nov 11.
Artículo en Inglés | MEDLINE | ID: mdl-22020101

RESUMEN

Nifekalant and azimilide, Class III antiarrhythmic agents, block the human ether-à-go-go-related gene K(+) (hERG) channel. However, when a depolarizing membrane potential is applied, they also increase the current at low potentials by shifting its activation curve towards hyperpolarizing voltages. This phenomenon is called 'facilitation'. In this study, we tried to address the mechanism underlying the facilitation by analyzing the effects of various compounds on hERG expressed in Xenopus oocytes. Like nifekalant, amiodarone, quinidine and carvedilol, but not by dofetilide, caused the current facilitation of hERG, suggesting that the facilitation is a common effect to a subset of hERG blockers. As the concentration of each compound was increased, the total hERG current was suppressed progressively, while the current at low potentials was augmented. Activation curves of the remaining hERG current in the facilitation condition could be described as the sum of two Boltzmann functions reflecting two populations of hERG currents having different activation curves. The voltage shift in the activation curve from control was constant for each compound even at different concentrations; -31 mV in amiodarone, -27 mV in nifekalant, -17 mV in quinidine and -12 mV in carvedilol. Therefore, the facilitation is based on the appearance of hERG whose voltage-dependence for the activation is shifted towards hyperpolarizing voltages.


Asunto(s)
Antiarrítmicos/farmacología , Canales de Potasio Éter-A-Go-Go/agonistas , Amiodarona/farmacología , Animales , Canal de Potasio ERG1 , Humanos , Hidantoínas , Imidazolidinas/farmacología , Potenciales de la Membrana/efectos de los fármacos , Piperazinas/farmacología , Pirimidinonas/farmacología , Xenopus laevis
14.
Neuron ; 47(1): 71-84, 2005 Jul 07.
Artículo en Inglés | MEDLINE | ID: mdl-15996549

RESUMEN

Partial agonists produce submaximal activation of ligand-gated ion channels. To address the question of partial agonist action at the NR1 subunit of the NMDA receptor, we performed crystallographic and electrophysiological studies with 1-aminocyclopropane-1-carboxylic acid (ACPC), 1-aminocyclobutane-1-carboxylic acid (ACBC), and 1-aminocyclopentane-1-carboxylic acid (cycloleucine), three compounds with incrementally larger carbocyclic rings. Whereas ACPC and ACBC partially activate the NMDA receptor by 80% and 42%, respectively, their cocrystal structures of the NR1 ligand binding core show the same degree of domain closure as found in the complex with glycine, a full agonist, illustrating that the NR1 subunit provides a new paradigm for partial agonist action that is distinct from that of the evolutionarily related GluR2, AMPA-sensitive receptor. Cycloleucine behaves as an antagonist and stabilizes an open-cleft conformation. The NR1-cycloleucine complex forms a dimer that is similar to the GluR2 dimer, thereby suggesting a conserved mode of subunit-subunit interaction in AMPA and NMDA receptors.


Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Receptores de N-Metil-D-Aspartato/agonistas , Aminoácidos Cíclicos/farmacología , Animales , Unión Competitiva/efectos de los fármacos , Cristalografía por Rayos X , Cicloleucina/farmacología , Electrofisiología , Femenino , Cinética , Ligandos , Modelos Moleculares , Mutación , Oocitos/metabolismo , Técnicas de Placa-Clamp , Conformación Proteica , Ratas , Receptores AMPA/metabolismo , Receptores de Glicina/efectos de los fármacos , Receptores de Glicina/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/genética , Xenopus
15.
Mol Pharmacol ; 75(6): 1287-95, 2009 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-19264848

RESUMEN

Drug interaction with target proteins including ion channels is essential for pharmacological control of various cellular functions, but the majority of its molecular mechanisms is still elusive. We recently found that a series of antidepressants preferentially block astroglial K(+)-buffering inwardly rectifying potassium channel (Kir) 4.1 channels over Kir1.1 channels. Here, using electrophysiological analyses of drug action on mutated Kir4.1 channel as well as computational analyses of three-dimensional (3D) arrangements of the ligands (i.e., bidirectional analyses), we examined the underlying mechanism for the antidepressant-Kir4.1 channel interaction. First, the effects of the selective serotonin reuptake inhibitor fluoxetine and the tricyclic antidepressant nortriptyline on chimeric and site-directed mutants of Kir4.1 expressed in Xenopus laevis oocytes were examined using the two-electrode voltage-clamp technique. Two amino acids, Thr128 and Glu158, on transmembrane domain 2 were critical for the drug inhibition of the current. The closed and open conformation models of the Kir4.1 pore suggested that both residues faced the central cavity, and they were positioned within a geometrical range capable of interacting with the drugs. Second, to represent molecular properties of active ligands in geometric terms, a 3D quantitative structure-activity relationship model of antidepressants was generated, which suggested that they share common features bearing a hydrogen bond acceptor and a positively charged moiety. 3D structures and physicochemical features of receptor and ligand were fitted together. Our results strongly suggest that antidepressants interact with Kir4.1 channel pore residues by hydrogen bond and ionic interactions, which account for their preferential inhibitory action on Kir4.1 current. This study may represent a possible general approach for the understanding of the mechanism of ligand-protein interactions.


Asunto(s)
Antidepresivos/farmacología , Astrocitos/metabolismo , Canales de Potasio de Rectificación Interna/antagonistas & inhibidores , Animales , Antidepresivos/química , Sitios de Unión , Línea Celular , Femenino , Fluoxetina/química , Fluoxetina/farmacología , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Ligandos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mutación , Nortriptilina/química , Nortriptilina/farmacología , Oocitos/fisiología , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/genética , Conformación Proteica , Relación Estructura-Actividad Cuantitativa , Ratas , Xenopus laevis
16.
Brain Res ; 1178: 44-51, 2007 Oct 31.
Artículo en Inglés | MEDLINE | ID: mdl-17920044

RESUMEN

The inwardly rectifying K+ (Kir) channel Kir4.1 is responsible for astroglial K+ buffering. We recently found that tricyclic antidepressants (TCAs) inhibit Kir4.1 channel currents, which suggests that astroglial Kir currents might be involved in the pharmacological action of antidepressants. We therefore further examined the effects of the currently most popular antidepressants, selective serotonin reuptake inhibitors (SSRIs), and other related agents on Kir4.1 channels heterologously expressed in HEK293T cells. The whole-cell patch clamp technique was used. Fluoxetine, the typical SSRI, inhibited Kir4.1 channel currents in a concentration-dependent manner with an IC50 value of 15.2 microM. The inhibitory effect of fluoxetine was reversible and essentially voltage-independent. Fluoxetine had little or no effect upon Kir1.1 (ROMK1) or Kir2.1 (IRK1) channel currents. Other SSRIs, sertraline and fluvoxamine, also inhibited Kir4.1 channel currents whereas the tetracyclic (mianserin) or the 5-HT1A receptor-related (buspirone) antidepressants did not. This study shows that SSRIs such as fluoxetine and sertraline preferentially block astroglial Kir4.1 rather than Kir1.1 or Kir2.1 channels in the brain, which may be implicated in their therapeutic and/or adverse actions.


Asunto(s)
Astrocitos/metabolismo , Bloqueadores de los Canales de Potasio , Canales de Potasio de Rectificación Interna/metabolismo , Inhibidores Selectivos de la Recaptación de Serotonina/farmacología , Antidepresivos Tricíclicos/farmacología , Astrocitos/efectos de los fármacos , Células Cultivadas , Interpretación Estadística de Datos , Electrofisiología , Fluoxetina/farmacología , Humanos , Técnicas de Placa-Clamp , Canales de Potasio de Rectificación Interna/efectos de los fármacos , Transfección
17.
Front Mol Neurosci ; 10: 408, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29358904

RESUMEN

Inwardly rectifying potassium (Kir) 4.1 channels in astrocytes regulate neuronal excitability by mediating spatial potassium buffering. Although dysfunction of astrocytic Kir4.1 channels is implicated in the development of epileptic seizures, the functional mechanisms of Kir4.1 channels in modulating epileptogenesis remain unknown. We herein evaluated the effects of Kir4.1 inhibition (blockade and knockdown) on expression of brain-derived neurotrophic factor (BDNF), a key modulator of epileptogenesis, in the primary cultures of mouse astrocytes. For blockade of Kir4.1 channels, we tested several antidepressant agents which reportedly bound to and blocked Kir4.1 channels in a subunit-specific manner. Treatment of astrocytes with fluoxetine enhanced BDNF mRNA expression in a concentration-dependent manner and increased the BDNF protein level. Other antidepressants (e.g., sertraline and imipramine) also increased the expression of BDNF mRNA with relative potencies similar to those for inhibition of Kir4.1 channels. In addition, suppression of Kir4.1 expression by the transfection of small interfering RNA (siRNA) targeting Kir4.1 significantly increased the mRNA and protein levels of BDNF. The BDNF induction by Kir4.1 siRNA transfection was suppressed by the MEK1/2 inhibitor U0126, but not by the p38 MAPK inhibitor SB202190 or the JNK inhibitor SP600125. The present results demonstrated that inhibition of Kir4.1 channels facilitates BDNF expression in astrocytes primarily by activating the Ras/Raf/MEK/ERK pathway, which may be linked to the development of epilepsy and other neuropsychiatric disorders.

18.
Neuropharmacology ; 109: 18-28, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27236080

RESUMEN

The overexpression of Kir3.2, a subunit of the G protein-gated inwardly rectifying K(+) channel, is implicated in some of the neurological phenotypes of Down syndrome (DS). Chemical compounds that block Kir3.2 are expected to improve the symptoms of DS. The purpose of this study is to develop a cell-based screening system to identify Kir3.2 blockers and then investigate the mode of action of the blocker. Chemical screening was carried out using a K(+) transporter-deficient yeast strain that expressed a constitutively active Kir3.2 mutant. The mode of action of an effective blocker was electrophysiologically analyzed using Kir channels expressed in Xenopus oocytes. Proflavine was identified to inhibit the growth of Kir3.2-transformant cells and Kir3.2 activity in a concentration-dependent manner. The current inhibition was strong when membrane potentials (Vm) was above equilibrium potential of K(+) (EK). When Vm was below EK, the blockage apparently depended on the difference between Vm and [K(+)]. Furthermore, the inhibition became stronger by lowering extracellular [K(+)]. These results indicated that the yeast strain serves as a screening system to isolate Kir3.2 blockers and proflavine is a prototype of a pore blocker of Kir3.2.


Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/antagonistas & inhibidores , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/fisiología , Inhibidores de Crecimiento/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Proflavina/farmacología , Animales , Proliferación Celular/efectos de los fármacos , Proliferación Celular/fisiología , Relación Dosis-Respuesta a Droga , Femenino , Inhibidores de Crecimiento/química , Ratones , Bloqueadores de los Canales de Potasio/química , Proflavina/química , Xenopus laevis
19.
PLoS One ; 8(11): e79844, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-24244570

RESUMEN

The cytoplasmic domain of inward rectifier K(+) (Kir) channels associates with cytoplasmic ligands and undergoes conformational change to control the gate present in its transmembrane domain. Ligand-operated activation appears to cause dilation of the pore at the cytoplasmic domain. However, it is still unclear how the cytoplasmic domain supports pore dilation and how alterations to this domain affect channel activity. In the present study, we focused on 2 spatially adjacent residues, i.e., Glu236 and Met313, of the G protein-gated Kir channel subunit Kir3.2. In the closed state, these pore-facing residues are present on adjacent ßD and ßH strands, respectively. We mutated both residues, expressed them with the m2-muscarinic receptor in Xenopus oocytes, and measured the acetylcholine-dependent K(+) currents. The dose-response curves of the Glu236 mutants tended to be shifted to the right. In comparison, the slopes of the concentration-dependent curves were reduced and the single-channel properties were altered in the Met313 mutants. The introduction of arginine at position 236 conferred constitutive activity and caused a leftward shift in the conductance-voltage relationship. The crystal structure of the cytoplasmic domain of the mutant showed that the arginine contacts the main chains of the ßH and ßI strands of the adjacent subunit. Because the ßH strand forms a ß sheet with the ßI and ßD strands, the immobilization of the pore-forming ß sheet appears to confer unique properties to the mutant. These results suggest that the G protein association triggers pore dilation at the cytoplasmic domain in functional channels, and the pore-constituting structural elements contribute differently to these conformational changes.


Asunto(s)
Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Ácido Glutámico/química , Metionina/química , Potasio/metabolismo , Subunidades de Proteína/química , Acetilcolina/metabolismo , Acetilcolina/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Sustitución de Aminoácidos , Animales , Cationes Monovalentes , Relación Dosis-Respuesta a Droga , Femenino , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Expresión Génica , Ácido Glutámico/metabolismo , Activación del Canal Iónico , Transporte Iónico , Metionina/metabolismo , Ratones , Modelos Moleculares , Oocitos/citología , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Potasio/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptor Muscarínico M2/genética , Receptor Muscarínico M2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevis
20.
Philos Trans A Math Phys Eng Sci ; 368(1921): 2983-3000, 2010 Jun 28.
Artículo en Inglés | MEDLINE | ID: mdl-20478917

RESUMEN

The first model of G-protein-K(ACh) channel interaction was developed 14 years ago and then expanded to include both the receptor-G-protein cycle and G-protein-K(ACh) channel interaction. The G-protein-K(ACh) channel interaction used the Monod-Wyman-Changeux allosteric model with the idea that one K(ACh) channel is composed of four subunits, each of which binds one active G-protein subunit (G(betagamma)). The receptor-G-protein cycle used a previous model to account for the steady-state relationship between K(ACh) current and intracellular guanosine-5-triphosphate at various extracellular concentrations of acetylcholine (ACh). However, simulations of the activation and deactivation of K(ACh) current upon ACh application or removal were much slower than experimental results. This clearly indicates some essential elements were absent from the model. We recently found that regulators of G-protein signalling are involved in the control of K(ACh) channel activity. They are responsible for the voltage-dependent relaxation behaviour of K(ACh) channels. Based on this finding, we have improved the receptor-G-protein cycle model to reproduce the relaxation behaviour. With this modification, the activation and deactivation of K(ACh) current in the model are much faster and now fall within physiological ranges.


Asunto(s)
Proteínas de Unión al GTP/metabolismo , Atrios Cardíacos/citología , Atrios Cardíacos/metabolismo , Modelos Biológicos , Canales de Potasio/metabolismo , Regulación Alostérica , Animales , Simulación por Computador , Conductividad Eléctrica , Guanosina Trifosfato/metabolismo , Cinética , Masculino , Ratas , Ratas Endogámicas WKY
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA