RESUMEN
Potassium ion channels are one of the most diversely and widely distributed channels, which are involved in all kinds of physiological functions in both excitable and non-excitable cells. The expression of voltage-gated potassium ion (Kv) channels is highly variable according to the state of macrophages activation. Macrophages have an important function in innate immunity against intruding pathogens. They produce a variety of inflammatory and immunoactive molecules that modulate imflammatory responses. Here we show that blockade of K+ channels by non-selective Kv channel inhibitor tetraethylammonium chloride (TEA), and 4-aminopyridine (4-AP) inhibited proinflammatory cytokines expression, cell proliferation, and reactive oxygen species (ROS) production in LPS-stimulated macrophages of Sea perch (Lateolabrax japonicas). Then we isolated four Kv channels genes (spKv1.1, spKv1.2, spKv1.5 and spKv3.1) in LPS-activated fish macrophages. These channels genes were up-regulated after LPS stimulation except spKv3.1, which remained unchanged during the test. The results of this study indicate that Kv channels could be required for modulating the immune function of fish macrophages.
Asunto(s)
Citocinas/genética , Proteínas de Peces/genética , Activación de Macrófagos/efectos de los fármacos , Perciformes/genética , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/genética , Especies Reactivas de Oxígeno/metabolismo , 4-Aminopiridina/farmacología , Secuencia de Aminoácidos , Animales , Clonación Molecular , Citocinas/inmunología , Citocinas/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Proteínas de Peces/química , Proteínas de Peces/metabolismo , Inmunidad Innata/efectos de los fármacos , Inmunidad Innata/inmunología , Lipopolisacáridos/farmacología , Perciformes/inmunología , Perciformes/metabolismo , Filogenia , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Alineación de Secuencia/veterinaria , Tetraetilamonio/farmacologíaRESUMEN
Voltage sensor domains (VSD) of voltage-dependent ion channels share a basic molecular structure with a voltage-sensing phosphatase and a voltage-gated proton channel. The VSD senses and responds to changes in the membrane potential by undergoing conformational changes associated with the movement of the charged arginines located on the S4 segment. Although several functional and structural studies have provided useful information about the conformational changes in many ion channels, a detailed and unambiguous explanation has not been published. Therefore, understanding the principle of voltage-dependent gating at an atomic level is required. In this study, we took advantage of the available spin labeling electron paramagnetic resonance spectrometry data and computational methods to investigate the structure and dynamic properties of the Up-state (activated) and Down-state (resting) conformations of the VSD by means of all-atom molecular dynamics (MD) simulations. The MD results of the Down conformation determined in bilayers with and without lipid phosphates both revealed a different shape of the aqueous crevice, in which more water molecules surround and fill the intracellular crevice in its Down state than in its Up state. The solvent accessible surface within the crevice has a complementary shape that can account for water-mediated interactions between the voltage sensor and the lipid bilayer. The results support the previously reported experimental data.
Asunto(s)
Simulación de Dinámica Molecular , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Agua/química , Enlace de Hidrógeno , Activación del Canal Iónico , Potenciales de la Membrana , Estructura Terciaria de Proteína , Marcadores de SpinRESUMEN
The potassium channel KcsA was heterologously expressed in a eukaryotic cell-free system. Both, the expression yields and functional analysis of the protein were reported. Qualitative and quantitative analyses of KcsA expression were performed by using (14)C-labeled leucine as one of the amino acids supplemented in the cell-free reaction mixture. There was a time dependent increase in the protein yield as well as the intensity of the native tetramer band in insect cell derived microsomes. Electrophysiology measurements demonstrated the functional activity of the microsomes harboring KcsA showing single-channel currents with the typical biophysical characteristics of the ion channel. The channel behavior was asymmetric and showed positive rectification with larger currents towards positive voltages. KcsA channel currents were effectively blocked by potassium selective barium (Ba(2+)). This functional demonstration of an ion channel in eukaryotic cell-free system has a large potential for future applications including drug screening, diagnostic applications and functional assessment of complex membrane proteins like GPCRs by coupling them to ion channels in cell-free systems. Furthermore, membrane proteins can be expressed directly from linear DNA templates within 90 min, eliminating the need for additional cloning steps, which makes this cell-free system fast and efficient.
Asunto(s)
Proteínas Bacterianas/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Streptomyces lividans/metabolismo , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Línea Celular , Clonación Molecular , Microsomas/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Biosíntesis de Proteínas , Multimerización de Proteína , Streptomyces lividans/química , Streptomyces lividans/genéticaRESUMEN
Marine snails of the genus Conus (â¼500 species) are tropical predators that produce venoms for capturing prey, defense and competitive interactions. These venoms contain 50-200 different peptides ("conotoxins") that generally comprise 7-40 amino acid residues (including 0-5 disulfide bridges), and that frequently contain diverse posttranslational modifications, some of which have been demonstrated to be important for folding, stability, and biological activity. Most conotoxins affect voltage- and ligand-gated ion channels, G protein-coupled receptors, and neurotransmitter transporters, generally with high affinity and specificity. Due to these features, several conotoxins are used as molecular tools, diagnostic agents, medicines, and models for drug design. Based on the signal sequence of their precursors, conotoxins have been classified into genetic superfamilies, whereas their molecular targets allow them to be classified into pharmacological families. The objective of this work was to identify and analyze partial cDNAs encoding precursors of conotoxins belonging to I superfamily from three vermivorous species of the Mexican Pacific coast: C. brunneus, C. nux and C. princeps. The precursors identified contain diverse numbers of amino acid residues (C. brunneus, 65 or 71; C. nux, 70; C. princeps, 72 or 73), and all include a highly conserved signal peptide, a C-terminal propeptide, and a mature toxin. All the latter have one of the typical Cys frameworks of the I-conotoxins (C-C-CC-CC-C-C). The prepropeptides belong to the I2-superfamily, and encode eight different hydrophilic and acidic mature toxins, rather similar among them, and some of which have similarity with I2-conotoxins targeting voltage- and voltage-and-calcium-gated potassium channels.
Asunto(s)
Conotoxinas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Caracoles/genética , Secuencia de Aminoácidos , Animales , Conotoxinas/química , Caracol Conus/genética , ADN Complementario , México , Datos de Secuencia Molecular , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Homología de Secuencia de AminoácidoRESUMEN
Human voltage-gated potassium channel Kv1.3 is an important pharmacological target for the treatment of autoimmune and metabolic diseases. Increasing clinical demands stipulate an active search for efficient and selective Kv1.3 blockers. Here we present a new, reliable, and easy-to-use analytical system designed to seek for and study Kv1.3 ligands that bind to the extracellular vestibule of the K(+)-conducting pore. It is based on Escherichia coli spheroplasts with the hybrid protein KcsA-Kv1.3 embedded into the membrane, fluorescently labeled Kv1.3 blocker agitoxin-2, and confocal laser scanning microscopy as a detection method. This system is a powerful alternative to radioligand and patch-clamp techniques. It enables one to search for Kv1.3 ligands both among individual compounds and in complex mixtures, as well as to characterize their affinity to Kv1.3 channel using the "mix and read" mode. To demonstrate the potential of the system, we performed characterization of several known Kv1.3 ligands, tested nine spider venoms for the presence of Kv1.3 ligands, and conducted guided purification of a channel blocker from scorpion venom.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Escherichia coli/genética , Canal de Potasio Kv1.3/química , Microscopía Confocal/métodos , Animales , Escherichia coli/química , Escherichia coli/metabolismo , Expresión Génica , Humanos , Canal de Potasio Kv1.3/genética , Canal de Potasio Kv1.3/metabolismo , Ligandos , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/genética , Venenos de Escorpión/metabolismo , Escorpiones , Esferoplastos/química , Esferoplastos/genética , Esferoplastos/metabolismo , Venenos de Araña/química , ArañasRESUMEN
Voltage-gated potassium (Kv) channels on cell plasma membrane play an important role in both excitable cells and non-excitable cells and Kv1 subfamily is most extensively studied channel in mammalian cells. Recently, this potassium channel was reported to control processes inside mammalian T lymphocytes such as cell proliferation and volume regulation. Little is known about Kv1 channels in fish. We have postulated the presence of such a channel in lymphocytes and speculated its potential role in immunoregulation in ï¬sh. Employing speciï¬c primers and RNA template, we cloned a segment of a novel gene from sea perch blood sample and subsequently obtained a full cDNA sequence using RACE approach. Bioinformatic analysis revealed structural and phylogenetic characteristics of a novel Kv channel gene, designated as spKv1.3, which exhibits homologous domains to the members of Kv1.3 family, but it differs notably from some other members of that family at the carboxyl terminus. Full-length of spKv1.3 cDNA is 2152 bp with a 1440 bp open reading frame encoding a protein of 480 amino acids. SpKv1.3 gene is expressed in all of the tested organs and tissues of sea perch. To assess the postulated immune function of spKv1.3, we stimulated lymphocytes with LPS and/or channel blocker 4-AP. Expression levels of messenger RNA (mRNA) of spKv1.3 under stimulation conditions were measured by quantitative RT-PCR. The results showed that LPS can motivate the up-regulation of spKv1.3 expression significantly. Interestingly, we found for the first time that 4-AP with LPS can also increase the spKv1.3 mRNA expression levels in time course. Although 4-AP could block potassium channels physically, we speculated that its effect on blockage of potassium channel may start up an alternative mechanism which feed back and evoke the spKv1.3 mRNA expression.
Asunto(s)
Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Perciformes/genética , Perciformes/inmunología , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , 4-Aminopiridina/administración & dosificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Biología Computacional , ADN Complementario/genética , Proteínas de Peces/química , Regulación de la Expresión Génica , Inmunidad Innata , Lipopolisacáridos/administración & dosificación , Linfocitos/química , Linfocitos/metabolismo , Datos de Secuencia Molecular , Filogenia , Canales de Potasio con Entrada de Voltaje/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Alineación de SecuenciaRESUMEN
We present experiments where the gating behavior of a voltage-gated ion channel is modulated by artificial ligand binding. We construct a channel-DNA chimera with the KvAP potassium channel reconstituted in an artificial membrane. The channel is functional and the single channel ion conductivity unperturbed by the presence of the DNA. However, the channel opening probability vs. bias voltage, i.e., the gating, can be shifted considerably by the electrostatic force between the charges on the DNA and the voltage sensing domain of the protein. Different hybridization states of the chimera DNA thus lead to different response curves of the channel.
Asunto(s)
ADN/metabolismo , Activación del Canal Iónico/fisiología , Membranas Artificiales , Canales de Potasio con Entrada de Voltaje/metabolismo , Aeropyrum , Secuencia de Bases , ADN/genética , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Espacio Intracelular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Hibridación de Ácido Nucleico , Canales de Potasio con Entrada de Voltaje/química , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Electricidad EstáticaRESUMEN
The slowly activating delayed rectifier K(+) channels (I(Ks)) are one of the main pharmacological targets for development of drugs against cardiovascular diseases. Cardiac I(Ks) consists of KCNQ1 plus KCNE1 subunits. Ginsenoside, one of the active ingredient of Panax ginseng, enhances cardiac I(Ks) currents. However, little is known about the molecular mechanisms of how ginsenoside interacts with channel proteins to enhance cardiac I(Ks). In the present study, we investigated ginsenoside Rg(3) (Rg(3)) effects on human I(Ks) by co-expressing human KCNQ1 plus KCNE1 subunits in Xenopus oocytes. Rg(3) enhanced I(Ks) currents in concentration- and voltage-dependent manners. The EC(50) was 15.2+/-8.7 microM. However, in oocytes expressing KCNQ1 alone, Rg(3) inhibited the currents with concentration- and voltage-dependent manners. The IC(50) was 4.8+/-0.6 microM. Since Rg(3) acts opposite ways in oocytes expressing KCNQ1 alone or KCNQ1 plus KCNE1 subunits, we examined Rg(3) effects after co-expression of different ratios of KCNE1 and KCNQ1. The increase of KCNE1/KCNQ1 ratio converted I(Ks) inhibition to I(Ks) activations. One to ten ratio of KCNE1 and KCNQ1 subunit is required for Rg(3) activation of I(Ks). Mutations of K318 and V319 into K318Y and V319Y of KCNQ1 channel abolished Rg(3) effects on KCNQ1 or KCNQ1 plus KCNE1 channel currents. The docked modeling revealed that K318 residue plays a key role in stabilization between Rg(3) and KCNQ1 plus KCNE1 or KCNQ1 subunit. These results indicate that Rg(3)-induced activation of I(Ks) requires co-assembly of KCNQ1 and KCNE1 subunits and achieves this through interaction with residues K318 and V319 of KCNQ1 subunit.
Asunto(s)
Ginsenósidos/farmacología , Activación del Canal Iónico/efectos de los fármacos , Canal de Potasio KCNQ1/agonistas , Canal de Potasio KCNQ1/metabolismo , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Subunidades de Proteína/metabolismo , Potenciales de Acción/efectos de los fármacos , Animales , Secuencia de Bases , Dominio Catalítico , Relación Dosis-Respuesta a Droga , Humanos , Activación del Canal Iónico/genética , Activación del Canal Iónico/fisiología , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/genética , Lisina/metabolismo , Mutación , Oocitos/metabolismo , Panax/química , Canales de Potasio con Entrada de Voltaje/genética , Subunidades de Proteína/química , Subunidades de Proteína/genética , Valina/metabolismo , Xenopus laevisRESUMEN
Ephedrine (Eph) is an alkaloid extracted from the Chinese traditional medicine plant Ephedra Sinica or Ma huang, which has been known for effects on the central nervous system, cardiovascular system, and smooth muscles. However, the corresponding molecular mechanism of these effects remains unknown. In this study, we investigated the influences of Eph on heart rate, QTc interval in vivo, and the slowly activated K channels (IKs) that were composed of both KCNQ1 and KCNE1 subunits in vitro. Results demonstrated that Eph, but not pseudoephedrine, could increase the heart rate and shorten QTc interval of BALB/c mouse. Besides, Eph markedly activated cardiac IKs currents with EC50 = 50 nM and shifted G-V curves to left. But pseudoephedrine had no effects on Iks currents. The onset and offset time constants of IKs currents activated by Eph at 1 M were tauon = 49 seconds and tauoff = 400 seconds. A pair of binding sites of Eph on KCNQ1/KCNE1 channel was also shown to occur at F296 and Y299 in the S5-S6 P-loop of the KCNQ1 channel. As both amino acids are highly conserved in the KCNQ family, Eph can possibly activate other members of the KCNQ family. The mechanism of Iks activated by Eph may provide a clue for drug design in the future.
Asunto(s)
Efedrina/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Canales de Potasio con Entrada de Voltaje/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Humanos , Canal de Potasio KCNQ1/química , Canal de Potasio KCNQ1/metabolismo , Síndrome de QT Prolongado/metabolismo , Síndrome de QT Prolongado/fisiopatología , Masculino , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/fisiologíaRESUMEN
Two venom peptides, CPY-Pl1 (EU000528) and CPY-Fe1 (EU000529), characterized from the vermivorous marine snails Conus planorbis and Conus ferrugineus, define a new class of conopeptides, the conopeptide Y (CPY) family. The peptides have no disulfide cross-links and are 30 amino acids long; the high content of tyrosine is unprecedented for any native gene product. The CPY peptides were chemically synthesized and shown to be biologically active upon injection into both mice and Caenorhabditis elegans; activity on mammalian Kv1 channel isoforms was demonstrated using an oocyte heterologous expression system, and selectivity for Kv1.6 was found. NMR spectroscopy revealed that the peptides were unstructured in aqueous solution; however, a helical region including residues 12-18 for one peptide, CPY-Pl1, formed in trifluoroethanol buffer. Clones obtained from cDNA of both species encoded prepropeptide precursors that shared a unique signal sequence, indicating that these peptides are encoded by a novel gene family. This is the first report of tyrosine-rich bioactive peptides in Conus venom.
Asunto(s)
Péptidos/química , Canales de Potasio con Entrada de Voltaje/química , Tirosina/química , Secuencia de Aminoácidos , Animales , Caracol Conus , ADN Complementario/metabolismo , Canal de Potasio Kv1.6/química , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Venenos de Moluscos/metabolismo , Oocitos/metabolismo , Homología de Secuencia de Aminoácido , Fracciones Subcelulares , Trifluoroetanol/químicaRESUMEN
A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.
Asunto(s)
Cnidarios/genética , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Clonación Molecular , Cnidarios/química , ADN Complementario/genética , Combinación de Medicamentos , Venenos Elapídicos/metabolismo , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Péptidos/análisis , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/química , Unión Proteica , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Alineación de Secuencia , Sinaptosomas/metabolismoRESUMEN
The voltage-gated potassium channels (Kv) are partially responsible for the contraction/relaxation of blood vessels in response to changes in the Po(2) level. The present study determined the expression of Kvbeta1 and four oxygen-sensitive Kvalpha subunits (Kv1.2, Kv1.5, Kv2.1, and Kv9.3) in the ductus arteriosus (DA), the aorta (Ao), and the pulmonary artery (PA) in porcine neonates immediately after birth. We cloned three Kvbeta1 transcript variants (Kvbeta1.2, Kvbeta1.3, and Kvbeta1.4), Kv1.2, Kv1.5, and Kv9.3 from piglets. Three Kvbeta1 transcripts, Kv1.2, Kv1.5, and Kv9.3, encode predicted proteins of 401, 408, 202, 499, 600, and 491 residues. These Kv showed a high degree of sequence conservation with the corresponding Kv in human. Northern and quantitative real-time PCR (qr-PCR) analyses showed that Kvbeta1.2 expression was high in the DA and Ao but low in the PA. Kv1.5 expression was high in the Ao and PA but low in the DA. Expression of Kvbeta1.3, Kvbeta1.4, Kv1.2, Kv2.1, and Kv9.3 was low in these blood vessels. The inactivation property of Kvbeta1.2 against Kv1.5 was confirmed using Xenopus laevis oocytes. Our findings suggest that the molecular basis for the differential electrophysiological characteristics including opposing response to oxygen in the DA and the PA are partially due to diversity in expression of Kv1.5 and Kvbeta1.2 subunits. The high expression of Kvbeta1.2 and relatively low expression of Kv1.5 in the DA might be partially responsible for the ductal closure after birth.
Asunto(s)
Conducto Arterial/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Secuencia de Bases , Northern Blotting , Bovinos , Clonación Molecular , ADN Complementario , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Homología de Secuencia de AminoácidoRESUMEN
DPL2 (DPP10) found at chromosome 2q14.1 is a member of the dipeptidyl peptidase IV (DPIV) gene family. Here we characterize a novel short DPL2 isoform (DPL2-s), a 789-amino acid protein, that differs from the previously described long DPL2 isoform (DPL2-l) at the N-terminal cytoplasmic domain by 13 amino acids. The two DPL2 isoforms use alternate first exons. DPL2 mRNA was expressed mainly in the brain and pancreas. Multiple forms of recombinant DPL2-s protein were observed in 293T cells, having mobilities 96 kDa, 100 kDa, and approximately 250 kDa which may represent soluble DPL2, transmembrane DPL2 and multimeric DPL2 respectively. DPL2 is glycosylated as a band shift is observed following PNGase F deglycosylation. DPL2-s was expressed primarily on the cell surface of transfected 293T and PC12 cells. DPL2-s exhibits high sequence homology with other DPIV peptidases, but lacks a catalytic serine residue and lacks dipeptidyl peptidase activity. Substitutions of Gly(644)-->Ser, Lys(643)Gly(644)-->TrpSer, or Asp(561)Lys(643)Gly(644)-->TyrTrpSer in the catalytic motif did not confer dipeptidyl peptidase activity upon DPL2-s. Thus, although DPL2 is similar in structure and sequence to the other dipeptidyl peptidases, it lacks vital residues required to confer dipeptidyl peptidase activity and has instead evolved features that enable it to act as an important component of voltage-gated potassium channels.
Asunto(s)
Encéfalo/enzimología , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/química , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Empalme Alternativo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia de Bases , Línea Celular , Membrana Celular/enzimología , Clonación Molecular , Citoplasma/enzimología , ADN Complementario/genética , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/metabolismo , Expresión Génica , Glicosilación , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Páncreas/enzimología , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio con Entrada de Voltaje/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Voltage-gated ion channels open and close in response to voltage changes across electrically excitable cell membranes. Voltage-gated potassium (Kv) channels are homotetramers with each subunit constructed from six transmembrane segments, S1-S6 (ref. 2). The voltage-sensing domain (segments S1-S4) contains charged arginine residues on S4 that move across the membrane electric field, modulating channel open probability. Understanding the physical movements of this voltage sensor is of fundamental importance and is the subject of controversy. Recently, the crystal structure of the KvAP channel motivated an unconventional 'paddle model' of S4 charge movement, indicating that the segments S3b and S4 might move as a unit through the lipid bilayer with a large (15-20-A) transmembrane displacement. Here we show that the voltage-sensor segments do not undergo significant transmembrane translation. We tested the movement of these segments in functional Shaker K+ channels by using luminescence resonance energy transfer to measure distances between the voltage sensors and a pore-bound scorpion toxin. Our results are consistent with a 2-A vertical displacement of S4, not the large excursion predicted by the paddle model. This small movement supports an alternative model in which the protein shapes the electric field profile, focusing it across a narrow region of S4 (ref. 6).
Asunto(s)
Transferencia de Energía , Activación del Canal Iónico , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Mediciones Luminiscentes , Movimiento , Oocitos/metabolismo , Potasio/metabolismo , Canales de Potasio/química , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de Potasio con Entrada de Voltaje/genética , Canales de Potasio de la Superfamilia Shaker , Xenopus laevisRESUMEN
Voltage-dependent potassium channels (Kv) in leukocytes are involved in the immune response. In bone marrow-derived macrophages (BMDM), proliferation and activation induce delayed rectifier K+ currents, generated by Kv1.3, via transcriptional, translational, and posttranslational controls. Furthermore, modulatory Kv beta subunits coassociate with Kv alpha subunits, increasing channel diversity and function. In this study we have identified Kv beta subunits in mouse BMDM, studied their regulation during proliferation and activation, and analyzed K+ current parameters influenced by these proteins. BMDM express all isoforms of Kv beta1 (Kv beta1.1, Kv beta1.2, and Kv beta1.3) and Kv beta2 (Kv beta2.1), but not Kv beta4, the alternatively spliced murine Kv beta3 variant. M-CSF-dependent proliferation induced all Kv beta isoforms. However, LPS- and TNF-alpha-induced activation differentially regulated these subunits. Although LPS increased Kv beta1.3, reduced Kv beta1.2, and maintained Kv beta1.1 mRNA levels constant, TNF-alpha up-regulated Kv beta1.1, down-regulated Kv beta1.2, and left Kv beta1.3 expression unchanged. Moreover, in contrast to TNF-alpha, M-CSF- and LPS- up-regulated Kv beta2.1. K+ currents from M-CSF- and LPS-stimulated BMDM exhibited faster inactivation, whereas TNF-alpha increased tau values. Although in M-CSF-stimulated cells the half-inactivation voltage shifted to more positive potentials, the incubation with LPS and TNF-alpha resulted in a hyperpolarizing displacement similar to that in resting BMDM. Furthermore, activation time constants of K+ currents and the kinetics of the tail currents were different depending upon the mode of activation. Our results indicate that differential Kv beta expression modifies the electrical properties of Kv in BMDM, dependent upon proliferation and the mode of activation. This could determine physiologically appropriate surface channel complexes, allowing for greater flexibility in the precise regulation of the immune response.
Asunto(s)
Activación de Macrófagos , Macrófagos/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Animales , Secuencia de Bases , Línea Celular , Proliferación Celular , Células Cultivadas , ADN Complementario/genética , Electrofisiología , Regulación de la Expresión Génica , Humanos , Lipopolisacáridos/farmacología , Activación de Macrófagos/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Canales de Potasio con Entrada de Voltaje/química , Canales de Potasio con Entrada de Voltaje/genética , Subunidades de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
A-type K(+) channels belonging to the Shal subfamily are found in various receptor and neuronal cells. Although their kinetics and cell surface expression are regulated by auxiliary subunits, little is known about the proteins that may interact with Kv4 during development. A yeast two-hybrid screening of a cDNA library made from the sensory epithelium of embryonic chick cochlea revealed a novel association of Kv4.2 with a protein containing a pentraxin domain (PPTX). Sequence analysis shows that PPTX is a member of the long pentraxin family, is 53% identical to mouse PTX3, and has a signal peptide at the N terminus. Studies with chick cochlear tissues reveal that Kv4.2 coprecipitates PPTX and that both proteins are colocalized to the sensory and ganglion cells. A yeast two-hybrid assay demonstrated that the last 22 amino acids of the PPTX C terminus interact with the N terminus of Kv4.2. Chinese hamster ovary cells transfected with recombinant PPTX reveal secretory products in both non-truncated and truncated forms. Among the secreted variants are several blocked by Brefeldin A, suggesting export via a classical pathway. PPTX is soluble in the presence of sodium carbonate, suggesting localization to the cytosolic side of the plasmalemma. Immunohistochemical studies show that Kv4.2 and PPTX colocalize in the region of the plasmalemma of Chinese hamster ovary cells; however, both are locked in the endoplasmic reticulum of COS-7 cells, suggesting that PPTX does not act as a shuttle protein. Reverse transcription-PCR demonstrates that PPTX mRNA is found in tissues that include brain, eye, heart, and blood vessels.
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Proteína C-Reactiva/química , Regulación de la Expresión Génica , Proteínas del Tejido Nervioso/química , Canales de Potasio/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Western Blotting , Células CHO , Células COS , Carbonatos/farmacología , Embrión de Pollo , Cóclea/metabolismo , Cricetinae , ADN Complementario/metabolismo , Biblioteca de Genes , Inmunohistoquímica , Inmunoprecipitación , Ratones , Microscopía Fluorescente , Modelos Biológicos , Datos de Secuencia Molecular , Péptidos/química , Plásmidos/metabolismo , Potasio/química , Canales de Potasio con Entrada de Voltaje/química , Unión Proteica , Estructura Terciaria de Proteína , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Potasio Shal , Transducción de Señal , Distribución Tisular , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
Genomic microarray analysis of genes specifically expressed in a pure cell isolate from a heterocellular organ identified the likely K efflux channel associated with the gastric H-K-ATPase. The function of this channel is to supply K to the luminal surface of the pump to allow H for K exchange. KCNQ1-KCNE2 was the most highly expressed and significantly enriched member of the large variety of K channels expressed in the gastric epithelium. The function of this K channel in acid secretion was then shown by inhibition of secretion in isolated gastric glands with specific KCNQ inhibitors and by colocalization of the channel with the H-K-ATPase in the secretory canaliculus of the parietal cell. KCNQ1-KCNE2 appears to be the K efflux channel that is essential for gastric acid secretion.
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Adenosina Trifosfatasas/química , Epitelio/metabolismo , Mucosa Gástrica/metabolismo , Canal de Potasio KCNQ1/biosíntesis , Canales de Potasio con Entrada de Voltaje/biosíntesis , Canales de Potasio/química , Naranja de Acridina/farmacología , Aminopirina/química , Animales , Separación Celular , Cartilla de ADN/química , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Ácido Gástrico/química , Inmunohistoquímica , Masculino , Microscopía Confocal , Análisis de Secuencia por Matrices de Oligonucleótidos , Oligonucleótidos/química , Potasio/química , Canales de Potasio con Entrada de Voltaje/química , ARN Complementario/metabolismo , ARN Mensajero/metabolismo , Conejos , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Putative interactions between the Human Ether-a-go-go Related Gene (HERG), QT interval prolongation and Torsades de Pointes (TdP) are now integral components of any discussion on drug safety. HERG encodes for the inwardly rectifying potassium channel (I(Kr)), which is essential to the maintenance of normal cardiac function. HERG channel mutations are responsible for one form of familial long QT syndrome, a potentially deadly inherited cardiac disorder associated with TdP. Moreover, drug-induced (acquired) QT interval prolongation has been associated with an increase in the incidence of sudden unexplained deaths, with HERG inhibition implicated as the underlying cause. Subsequently, a number of non-cardiovascular drugs which induce QT interval prolongation and/or TdP have been withdrawn. However, a definitive link between HERG, QT interval prolongation and arrhythmogenesis has not been established. Nevertheless, this area is subject to ever increasing regulatory scrutiny. Here we review the relationship between HERG, long QT syndrome and TdP, together with a summary of the associated regulatory issues, and developments in pre-clinical screening.
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Diseño de Fármacos , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Síndrome de QT Prolongado/inducido químicamente , Síndrome de QT Prolongado/genética , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio con Entrada de Voltaje/genética , Potenciales de Acción/efectos de los fármacos , Animales , Ensayos Clínicos como Asunto , Evaluación Preclínica de Medicamentos , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go , Corazón/fisiología , Humanos , Síndrome de QT Prolongado/metabolismo , Modelos Moleculares , Mutación , Canales de Potasio con Entrada de Voltaje/química , Torsades de Pointes/inducido químicamenteRESUMEN
Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1-S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5-S6, equivalent to M1-M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4-S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.