RESUMO
TWIK-related acid-sensitive K(+) (TASK) channels contribute to setting the resting potential of mammalian neurons and have recently been defined as molecular targets for extracellular protons and volatile anesthetics. We have isolated a novel member of this subfamily, hTASK-5, from a human genomic library and mapped it to chromosomal region 20q12-20q13. hTASK-5 did not functionally express in Xenopus oocytes, whereas chimeric TASK-5/TASK-3 constructs containing the region between M1 and M3 of TASK-3 produced K(+) selective currents. To better correlate TASK subunits with native K(+) currents in neurons the precise cellular distribution of all TASK family members was elucidated in rat brain. A comprehensive in situ hybridization analysis revealed that both TASK-1 and TASK-3 transcripts are most strongly expressed in many neurons likely to be cholinergic, serotonergic, or noradrenergic. In contrast, TASK-5 expression is found in olfactory bulb mitral cells and Purkinje cells, but predominantly associated with the central auditory pathway. Thus, TASK-5 K(+) channels, possibly in conjunction with auxiliary proteins, may play a role in the transmission of temporal information in the auditory system.
Assuntos
Vias Auditivas/metabolismo , Encéfalo/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso , Neurônios/metabolismo , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Sequência de Aminoácidos/genética , Animais , Vias Auditivas/citologia , Sequência de Bases/genética , Encéfalo/citologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Feminino , Humanos , Hibridização In Situ , Potenciais da Membrana/genética , Dados de Sequência Molecular , Neurônios/citologia , Oócitos/citologia , Oócitos/metabolismo , Canais de Potássio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , XenopusRESUMO
BACKGROUND: K(+) channels have important functions in the kidney, such as maintenance of the membrane potential, volume regulation, recirculation, and secretion of potassium ions. The aim of this study was to obtain more information on the localization and possible functional role of the inwardly rectifying K(+) channel, Kir7.1. METHODS: Kir7.1 cDNA (1114 bp) was isolated from guinea pig kidney (gpKir7.1), and its tissue distribution was analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR). In addition, a genomic DNA fragment (6153 bp) was isolated from a genomic library. cRNA was expressed in Xenopus laevis oocytes for functional studies. Immunohistochemistry and RT-PCR were used to localize Kir7.1 in guinea pig and human kidney. RESULTS: The expression of gpKir7.1 in Xenopus laevis oocytes revealed inwardly rectifying K(+) currents. The reversal potential was strongly dependent on the extracellular K(+) concentration, shifting from -14 mV at 96 mmol/L K(+) to -90 mV at 1 mmol/L K(+). gpKir7.1 showed a low affinity for Ba(2+). Significant expression of gpKir7.1 was found in brain, kidney, and lung, but not in heart, skeletal muscle, liver, or spleen. Immunocytochemical detection in guinea pig identified the gpKir7.1 protein in the basolateral membrane of epithelial cells of the proximal tubule. RT-PCR analysis identified strong gpKir7.1 expression in the proximal tubule and weak expression in glomeruli and thick ascending limb. In isolated human tubule fragments, RT-PCR showed expression in proximal tubule and thick ascending limb. CONCLUSION: Our results suggest that Kir7.1 may contribute to basolateral K(+) recycling in the proximal tubule and in the thick ascending limb.
Assuntos
Túbulos Renais Proximais/química , Alça do Néfron/química , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/análise , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , Feminino , Expressão Gênica/fisiologia , Cobaias , Humanos , Túbulos Renais Proximais/metabolismo , Alça do Néfron/metabolismo , Masculino , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Potássio/metabolismo , Canais de Potássio/metabolismo , RNA Mensageiro/análise , Transfecção , Xenopus laevisRESUMO
BACKGROUND: The renal K(+) channel ROMK (Kir1.1) controls salt reabsorption in the kidney. Loss-of-function mutations in this channel cause hyperprostaglandin E syndrome/antenatal Bartter syndrome (HPS/aBS), which is characterized by severe renal salt and fluid wasting. METHODS: We investigated 10 HPS/aBS patients for mutations in the ROMK gene by single-strand conformation polymorphism analysis (SSCA) and direct sequencing. To assess the functional consequences, Ba(2+)-sensitive K(+) currents were measured in five mutants of the core region as well as one mutant with truncated C-terminus, using the two-electrode voltage-clamp technique after an injection of mutant cRNA into Xenopus oocytes. RESULTS: Three novel ROMK mutations were identified together with six mutations described previously. The mutations were categorized into three groups: (1) amino acid exchanges in the core region (M1-H5-M2), (2) truncation at the cytosolic C-terminus, and (3) deletions of putative promoter elements. While the core mutations W99C, N124K, and I142T led to significantly reduced macroscopic K(+) currents (1 to 8% of wild-type currents), the A103V and P110L variants retained substantial K(+) conductivity (23 and 35% of wild-type currents, respectively). Coexpression of A103V and P110L, resembling the compound heterozygous state of the affected individual, further reduced macroscopic currents to 9% of the wild-type currents. All mutants in the core region exerted a dominant-negative effect on wild-type ROMK1. The C-terminal frameshift (fs) mutation (H354fs) did not change current amplitudes compared with ROMK1 wild type, suggesting that a mechanism other than alteration of the electrophysiological properties may responsible for loss of channel activity. CONCLUSIONS: Analysis of ROMK mutants linked to HPS/aBS revealed a spectrum of mechanisms accounting for loss of channel function. Further characterization of the molecular defects might be helpful for the development of new therapeutic approaches.
Assuntos
Síndrome de Bartter/genética , Mutação , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Prostaglandinas E/metabolismo , Animais , Síndrome de Bartter/metabolismo , Sequência de Bases , Pré-Escolar , Códon sem Sentido , Primers do DNA/genética , Éxons , Feminino , Ligação Genética , Humanos , Técnicas In Vitro , Lactente , Masculino , Mutação de Sentido Incorreto , Oócitos/metabolismo , Linhagem , Canais de Potássio/química , Canais de Potássio/metabolismo , Deleção de Sequência , Síndrome , Transfecção , XenopusRESUMO
We have identified several cDNAs for the human Kir5.1 subunit of inwardly rectifying K(+) channels. Alternative splicing of exon 3 and the usage of two alternative polyadenylation sites contribute to cDNA diversity. The hKir5.1 gene KCNJ16 is assigned to chromosomal region 17q23.1-24.2, and is separated by only 34 kb from the hKir2.1 gene (KCNJ2). In the brain, Kir5.1 mRNA is restricted to the evolutionary older parts of the hindbrain, midbrain and diencephalon and overlaps with Kir2.1 in the superior/inferior colliculus and the pontine region. In the kidney Kir5.1 and Kir2.1 are colocalized in the proximal tubule. When expressed in Xenopus oocytes, Kir5.1 is efficiently targeted to the cell surface and forms electrically silent channels together with Kir2.1, thus negatively controlling Kir2.1 channel activity in native cells.
Assuntos
Ligação Genética , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Subunidades Proteicas , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Células Cultivadas , Cromossomos Humanos Par 17/genética , Humanos , Rim/metabolismo , Dados de Sequência Molecular , Oócitos/citologia , Oócitos/metabolismo , Especificidade de Órgãos , Técnicas de Patch-Clamp , Canais de Potássio/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Transfecção , XenopusRESUMO
Two cDNAs encoding novel K(+) channels, THIK-1 and THIK-2 (tandem pore domain halothane inhibited K(+) channel), were isolated from rat brain. The proteins of 405 and 430 amino acids were 58% identical to each other. Homology analysis showed that the novel channels form a separate subfamily among tandem pore domain K(+) channels. The genes of the human orthologs were identified as human genomic data base entries. They possess one intron each and were assigned to chromosomal region 14q24.1-14q24.3 (human (h) THIK-1) and 2p22-2p21 (hTHIK-2). In rat (r), THIK-1 (rTHIK-1) is expressed ubiquitously; rTHIK-2 expression was found in several tissues including brain and kidney. In situ hybridization of brain slices showed that rTHIK-2 is strongly expressed in most brain regions, whereas rTHIK-1 expression is more restricted. Heterologous expression of rTHIK-1 in Xenopus oocytes revealed a K(+) channel displaying weak inward rectification in symmetrical K(+) solution. The current was enhanced by arachidonic acid and inhibited by halothane. rTHIK-2 did not functionally express. Confocal microscopy of oocytes injected with green fluorescent protein-tagged rTHIK-1 or rTHIK-2 showed that both channel subunits are targeted to the outer membrane. However, coinjection of rTHIK-2 did not affect the currents induced by rTHIK-1, indicating that the two channel subunits do not form heteromers.
Assuntos
Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/química , Canais de Potássio/genética , Sequência de Aminoácidos , Anestésicos Inalatórios/farmacologia , Animais , Ânions , Ácido Araquidônico/farmacologia , Sequência de Bases , Encéfalo/metabolismo , Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 2 , Clonagem Molecular , DNA Complementar/metabolismo , Eletrofisiologia , Mucosa Gástrica/metabolismo , Proteínas de Fluorescência Verde , Halotano/farmacologia , Humanos , Concentração de Íons de Hidrogênio , Hibridização In Situ , Íntrons , Rim/metabolismo , Fígado/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Filogenia , Potássio/metabolismo , Canais de Potássio/biossíntese , Ratos , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Xenopus/metabolismoRESUMO
Group III metabotropic glutamate receptors (mGluRs) are highly enriched in the presynaptic terminals of glutamatergic synapses where they mediate feedback inhibition of neurotransmitter release. Here, we used the yeast two-hybrid system to identify a direct interaction of the C-terminal tail region of mGluR7 with the rat homologue of the protein kinase C substrate PICK1. This interaction is specifically mediated by the very C-terminal amino acids of the receptor and can be reconstituted in human embryonic kidney 293 cells by transfection of full-length mGluR7 and PICK1 cDNAs. Quantitative beta-galactosidase assays revealed that among the different group III mGluRs, mGluR7 is the major PICK1 binding partner although other subfamily members can also interact with PICK1. These data indicate that PDZ domain-containing proteins might contribute to the presynaptic localization of group III mGluRs.
Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Receptores de Glutamato Metabotrópico/química , Receptores de Glutamato Metabotrópico/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Caenorhabditis elegans , Proteínas de Ciclo Celular , Linhagem Celular , Clonagem Molecular , Proteínas do Citoesqueleto , Humanos , Rim , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
G protein-coupled receptors regulate gene expression by cellular signaling cascades that target transcription factors and their recognition by specific DNA sequences. In the central nervous system, heteromeric metabotropic gamma-aminobutyric acid type B (GABA(B)) receptors through adenylyl cyclase regulate cAMP levels, which may control transcription factor binding to the cAMP response element. Using yeast-two hybrid screens of rat brain libraries, we now demonstrate that GABA(B) receptors are engaged in a direct and specific interaction with the activating transcription factor 4 (ATF-4), a member of the cAMP response element-binding protein /ATF family. As confirmed by pull-down assays, ATF-4 associates via its conserved basic leucine zipper domain with the C termini of both GABA(B) receptor (GABA(B)R) 1 and GABA(B)R2 at a site which serves to assemble these receptor subunits in heterodimeric complexes. Confocal fluorescence microscopy shows that GABA(B)R and ATF-4 are strongly coclustered in the soma and at the dendritic membrane surface of both cultured hippocampal neurons as well as retinal amacrine cells in vivo. In oocyte coexpression assays short term signaling of GABA(B)Rs via G proteins was only marginally affected by the presence of the transcription factor, but ATF-4 was moderately stimulated in response to receptor activation in in vivo reporter assays. Thus, inhibitory metabotropic GABA(B)Rs may regulate activity-dependent gene expression via a direct interaction with ATF-4.
Assuntos
Receptores de GABA-B/metabolismo , Fatores de Transcrição/metabolismo , Fator 4 Ativador da Transcrição , Sequência de Aminoácidos , Animais , Western Blotting , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Clonagem Molecular , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Eletrofisiologia , Escherichia coli/metabolismo , Regulação da Expressão Gênica , Biblioteca Gênica , Genes Reporter , Glutationa Transferase/metabolismo , Imuno-Histoquímica , Luciferases/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Neurônios/metabolismo , Oócitos/metabolismo , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Retina/metabolismo , Vírus da Floresta de Semliki/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Transcrição Gênica , Técnicas do Sistema de Duplo-Híbrido , Xenopus laevisRESUMO
Tandem pore domain acid-sensitive K(+) channel 3 (TASK-3) is a new member of the tandem pore domain potassium channel family. A cDNA encoding a 365- amino acid polypeptide with four putative transmembrane segments and two pore regions was isolated from guinea pig brain. An orthologous sequence was cloned from a human genomic library. Although TASK-3 is 62% identical to TASK-1, the cytosolic C-terminal sequence is only weakly conserved. Analysis of the gene structure identified an intron within the conserved GYG motif of the first pore region. Reverse transcriptase-polymerase chain reaction analysis showed strong expression in brain but very weak mRNA levels in other tissues. Cell-attached patch-clamp recordings of TASK-3 expressed in HEK293 cells showed that the single channel current-voltage relation was inwardly rectifying, and open probability increased markedly with depolarization. Removal of external divalent cations increased the mean single channel current measured at -100 mV from -2.3 to -5.8 pA. Expression of TASK-3 in Xenopus oocytes revealed an outwardly rectifying K(+) current that was strongly decreased in the presence of lower extracellular pH. Substitution of the histidine residue His-98 by asparagine or tyrosine abolished pH sensitivity. This histidine, which is located at the outer part of the pore adjacent to the selectivity filter, may be an essential component of the extracellular pH sensor.
Assuntos
Histidina/metabolismo , Concentração de Íons de Hidrogênio , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Complementar , Cobaias , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Canais de Potássio/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , XenopusRESUMO
In epithelial Kir7.1 channels a non-conserved methionine in the outer pore region adjacent to the G-Y-G selectivity filter (position +2) was found to determine unique properties for permeant and blocking ions characteristic of a K(+) channel in a single-occupancy state. The monovalent cation permeability sequence of Kir7.1 channels expressed in Xenopus oocytes was Tl(+)>K(+)>Rb(+)NH(4)(+)>Cs(+)>Na(+)>Li(+), but the macroscopic conductance for Rb(+) was approximately 8-fold larger than for the smaller K(+) ions, and decreased approximately 40-fold with the conserved arginine at the +2 position (Kir7.1M125R). Moreover, in Kir7.1 Rb(+) restored the typical permeation properties of other multi-ion channels indicating that a stable coordination of permeant ions at the +2 position defines the initial step in the conduction pathway of Kir channels.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/química , Canais de Potássio/metabolismo , Animais , Sítios de Ligação/genética , Feminino , Humanos , Técnicas In Vitro , Potenciais da Membrana , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Permeabilidade , Potássio/metabolismo , Canais de Potássio/genética , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
Several signaling proteins clustered at the postsynaptic density specialization in neurons harbor a conserved C-terminal PDZ domain recognition sequence (X-S/T-X-V/I) that mediates binding to members of the PSD-95/SAP90 protein family. This motif is also present in the C termini of some inwardly rectifying K(+) (Kir) channels. Constitutively active Kir2 channels as well as G protein-gated Kir3 channels, which are fundamental for neuronal excitability, were analyzed as candidates for binding to PSD-95/SAP90 family members. Therefore C termini of Kir2.1(+), Kir2.3(+), Kir2.4(-), Kir3.1(-), Kir3.2(+), Kir3.3(+) and Kir3.4(-) subunits (+, motif present; -, motif absent) were used as baits in the yeast two-hybrid assay to screen for in vivo interaction with PDZ domains 1-3 of PSD-95/SAP90. In contrast to Kir2.1 and Kir2.3, all Kir3 fragments failed to bind PSD-95 in this assay, which was supported by the lack of coimmunoprecipitation and colocalization of the entire proteins in mammalian cells. A detailed analysis of interaction domains demonstrated that the C-terminal motif in Kir3 channels is insufficient for binding PDZ domains. Kir2.1 and Kir2.3 subunits on the other hand coprecipitate with PSD-95. When coexpressed in a bicistronic internal ribosome entry site expression vector in HEK-293 cells macroscopic and elementary current analysis revealed that PSD-95 suppressed the activity of Kir2.3 channels by >50%. This inhibitory action of PSD-95, which predominantly affects the single-channel conductance, is likely attributable to a molecular association with additional internal interaction sites in the Kir2.3 protein.
Assuntos
Proteínas do Tecido Nervoso/metabolismo , Neurônios/química , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Sinapses/química , Sequência de Aminoácidos , Animais , Células COS , Proteína 4 Homóloga a Disks-Large , Eletrofisiologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Guanilato Quinases , Peptídeos e Proteínas de Sinalização Intracelular , Potenciais da Membrana/fisiologia , Proteínas de Membrana , Dados de Sequência Molecular , Núcleosídeo-Fosfato Quinase/metabolismo , Canais de Potássio/genética , Testes de Precipitina , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Associadas SAP90-PSD95 , Sinapses/enzimologia , Técnicas do Sistema de Duplo-HíbridoRESUMO
Signaling via cytosolic and receptor tyrosine kinases is associated with cell growth and differentiation but also targets onto transmitter receptors and ion channels. Here, regulation by tyrosine kinase (TK) activity was investigated for inwardly rectifying K+ (Kir2.1) channels that control membrane excitability in many central neurons. In mammalian tsA-201 cells, the membrane-permeable protein tyrosine phosphatase inhibitor, perorthovanadate (100 microM), suppressed currents through recombinant Kir2.1 channels by 60 +/- 20%. Coapplication of the TK inhibitor genistein (100 microM) completely abolished this effect. Native Kir2.1 channels in rat basophilic leukocytes were affected by manipulation of the TK and protein tyrosine phosphatase activity in a qualitatively similar manner. Site mutation of a tyrosine consensus residue for TK phosphorylation in the C-terminal domain of Kir2.1 generated channel properties indistinguishable from wild-type Kir2.1 channels. However, Kir2.1Y242F channels were no longer suppressed following exposure to perorthovanadate, indicating that the channel is a direct substrate for TKs. After coexpression of nerve growth factor receptor with Kir2.1 channels in tsA-201 cells and Xenopus oocytes, the activity of Kir2.1 was rapidly suppressed by applied nerve growth factor (0.5 microgram/ml) by 31 +/- 10 and 21 +/- 15%, respectively. Acute inhibition was also evoked by epidermal growth factor and insulin via endogenous insulin receptors, indicating that Kir2.1 channels may serve as a general target for neurotrophic growth factors in the brain.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Proteínas Tirosina Quinases/metabolismo , Tirosina , Sequência de Aminoácidos , Animais , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/fisiologia , Sequência Consenso , Fator de Crescimento Epidérmico/farmacologia , Genisteína/farmacologia , Insulina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Mutagênese Sítio-Dirigida , Fatores de Crescimento Neural/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Fosforilação , Bloqueadores dos Canais de Potássio , Ratos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Transfecção , Células Tumorais Cultivadas , Vanadatos/farmacologia , Xenopus laevisRESUMO
Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Canais de Potássio/fisiologia , Potássio/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Animais Recém-Nascidos , Northern Blotting , Encéfalo/metabolismo , Plexo Corióideo/citologia , Plexo Corióideo/metabolismo , Clonagem Molecular , Células Epiteliais/metabolismo , Humanos , Hibridização In Situ , Dados de Sequência Molecular , Canais de Potássio/metabolismo , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Xenopus laevisRESUMO
Loss of function mutations in kidney Kir1.1 (renal outer medullary potassium channel, KCNJ1) inwardly rectifying potassium channels can be found in patients suffering from hyperprostaglandin E syndrome (HPS), the antenatal form of Bartter syndrome. A novel mutation found in a sporadic case substitutes an asparagine by a positively charged lysine residue at amino acid position 124 in the extracellular M1-H5 linker region. When heterologously expressed in Xenopus oocytes and mammalian cells, current amplitudes from mutant Kir1.1a[N124K] channels were reduced by a factor of approximately 12 as compared with wild type. A lysine at the equivalent position is present in only one of the known Kir subunits, the newly identified Kir1.3, which is also poorly expressed in the recombinant system. When the lysine residue in guinea pig Kir1.3 (gpKir1.3) isolated from a genomic library was changed to an asparagine (reverse HPS mutation), mutant channels yielded macroscopic currents with amplitudes increased 6-fold. From single channel analysis it became apparent that the decrease in mutant Kir1.1 channels and the increase in mutant gpKir1.3 macroscopic currents were mainly due to the number of expressed functional channels. Coexpression experiments revealed a dominant-negative effect of Kir1.1a[N124K] and gpKir1.3 on macroscopic current amplitudes when coexpressed with wild type Kir1.1a and gpKir[K110N], respectively. Thus we postulate that in Kir1.3 channels the extracellular positively charged lysine is of crucial functional importance. The HPS phenotype in man can be explained by the lower expression of functional channels by the Kir1. 1a[N124K] mutant.
Assuntos
Síndrome de Bartter/genética , Medula Renal/metabolismo , Mutação Puntual , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Síndrome de Bartter/embriologia , Células COS , Clonagem Molecular , Feminino , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/fisiologia , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples , Canais de Potássio/química , Prostaglandinas E/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Síndrome , Transfecção , Xenopus laevisRESUMO
Members of the Kir2 subfamily of inwardly rectifying K+ channels characterized by their strong current rectification are widely expressed both in the periphery and in the CNS in mammals. We have cloned from rat brain a fourth subfamily member, designated Kir2.4 (IRK4), which shares 53-63% similarity to Kir2.1, Kir2.2, or Kir2.3 on the amino acid level. In situ hybridization analysis identifies Kir2.4 as the most restricted of all Kir subunits in the brain. Kir2. 4 transcripts are expressed predominantly in motoneurons of cranial nerve motor nuclei within the general somatic and special visceral motor cell column and thus are uniquely related to a functional system. Heterologous expression of Kir2.4 in Xenopus oocytes and mammalian cells gives rise to low-conductance channels (15 pS), with an affinity to the channel blockers Ba2+ (Ki = 390 microM) and Cs+ (Ki = 8.06 mM) 30-50-fold lower than in other Kir channels. Low Ba2+ sensitivity allows dissection of Kir2.4 currents from other Kir conductances in hypoglossal motoneurons (HMs) in rat brainstem slices. The finding that Ba2+-mediated block of Kir2.4 in HMs evokes tonic activity and increases the frequency of induced spike discharge indicates that Kir2.4 channels are of major importance in controlling excitability of motoneurons in situ.
Assuntos
Nervo Hipoglosso/química , Neurônios Motores/química , Canais de Potássio/metabolismo , Animais , Bário/farmacologia , Northern Blotting , Química Encefálica/fisiologia , Tronco Encefálico/química , Tronco Encefálico/fisiologia , Césio/farmacologia , Clonagem Molecular , DNA Complementar , Eletrofisiologia , Nervo Hipoglosso/citologia , Nervo Hipoglosso/fisiologia , Ativação do Canal Iônico/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Dados de Sequência Molecular , Neurônios Motores/fisiologia , Oócitos/fisiologia , Canais de Potássio/química , Canais de Potássio/fisiologia , Ratos , Ratos Wistar , Homologia de Sequência de Aminoácidos , Tetrodotoxina/farmacologia , XenopusRESUMO
1. A slow hyperpolarization-activated inwardly rectifying K+ current (IK(SHA)) with novel characteristics was identified from the mouse embryonic hippocampus x neuroblastoma cell line HN9.10e. 2. The non-inactivating current activated negative to a membrane potential of -80 mV with slow and complex activation kinetics (tau act approximately 1-7 s) and a characteristic delay of 1-10 s (-80 to -140 mV) that was linearly dependent on the membrane potential. 3. Tail currents and instantaneous open channel currents determined through fast voltage ramps reversed at the K+ equilibrium potential (EK) indicating that primarily K+, but not Na+, permeated the channels. 4. IK(SHA) was unaffected by altering the intracellular Ca2+ concentration between approximately 0 and 10 microM, but was susceptible to block by 5 mM extracellular Ca2+, Ba2+ (Ki = 0.42 mM), and Cs+ (Ki = 2.77 mM) 5. In cells stably transformed with M2 muscarinic receptors, IK(SHA) was rapidly, but reversibly, suppressed by application of micromolar concentrations of muscarine. 6. At the single channel level K(SHA) channel openings were observed with the characteristic delay upon membrane hyperpolarization. Analysis of unitary currents revealed an inwardly rectifying I-V profile and a channel slope conductance of 7 pS. Channel activity persisted in the inside-out configuration for many minutes. 7. It is concluded that IK(SHA) in HN9.10e cells represents a novel K+ current, which is activated upon membrane hyperpolarization. It is functionally different from both classic inwardly rectifying IKir currents and other cationic hyperpolarization-activated IH currents that have been previously described in neuronal or glial cells.
Assuntos
Hipocampo/metabolismo , Canais de Potássio/metabolismo , Animais , Cátions/metabolismo , Linhagem Celular , Estimulação Elétrica , Eletrofisiologia , Hipocampo/citologia , Hipocampo/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos , Muscarina/farmacologia , Agonistas Muscarínicos/farmacologia , Antagonistas Muscarínicos/farmacologia , Técnicas de Patch-Clamp , Canais de Potássio/efeitos dos fármacosRESUMO
Cardiac G protein-activated Kir (GIRK) channels may assemble as heterotetrameric polypeptides from two subunits, Kir3.1 and Kir3.4. For a functional comparison with native channels in the CNS we investigated all possible combinations of heteromeric channel formation from brain Kir3.1, Kir3.2, Kir3.3, and Kir3.4 subunits in mRNA-injected Xenopus oocytes. Analysis of macroscopic current amplitudes and channel gating kinetics indicated that individual subunits or combinations of Kir3.2, Kir3.3, and Kir3.4 formed functional channels ineffectively. Each of these subunits gave rise to prominent currents with distinct characteristics only in the presence of Kir3.1 subunits. Functional expression of concatemeric constructs between Kir3.1 and Kir3.2/3.4 subunits as well as coimmunoprecipitations with subunit-specific antibodies confirmed heteromeric channel formation. Mutational swapping between subunits of a single pore loop residue (Kir3.1F137S; Kir3.3S114F; a phenylalanine confers slow channel gating in Kir3.1 subunits) revealed that Kir3.1 subunits are an important constituent for native heteromeric channels and dominate their functional properties. However, homomeric channels from Kir3.1 subunits in vivo may not exist due to the spatial conflict of bulky phenylalanines in the pore structure.
Assuntos
Encéfalo/fisiologia , Neurônios/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/fisiologia , Sequência de Aminoácidos , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Humanos , Dados de Sequência Molecular , Oócitos/metabolismo , Ratos , XenopusRESUMO
Strongly rectifying IRK-type inwardly rectifying K+ channels are involved in the control of neuronal excitability in the mammalian brain. Whole-cell patch-clamp experiments show that cloned rat IRK1 (Kir 2.1) channels, when heterologously expressed in mammalian COS-7 cells, are inhibited following the activation of coexpressed serotonin (5-hydroxytryptamine) type 1A receptors by receptor agonists. Inhibition is mimicked by internal perfusion with GTP[gamma-S] and elevation of internal cAMP concentrations. Addition of the catalytic subunits of protein kinase A (PKA) to the internal recording solution causes complete inhibition of wild-type IRK1 channels, but not of mutant IRK1(S425N) channels in which a C-terminal PKA phosphorylation site has been removed. Our data suggest that in the nervous system serotonin may negatively control IRK1 channel activity by direct PKA-mediated phosphorylation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Bloqueadores dos Canais de Potássio , Canais de Potássio Corretores do Fluxo de Internalização , Animais , Linhagem Celular , Mutação , Fosforilação , Canais de Potássio/genética , Canais de Potássio/metabolismo , Ratos , Receptores de Serotonina/genética , Agonistas do Receptor de Serotonina/farmacologia , Células Tumorais CultivadasRESUMO
We have cloned by homology screening from a rat brain cDNA library a GIRK3-type (Kir 3.3) inwardly rectifying K+ channel subunit with high structural similarity to other subfamily members whose activity is thought to be controlled by receptor-stimulated G proteins. When heterologously expressed both in Xenopus oocytes and in mammalian COS-7 cells, rbGIRK3 subunits individually fail to form functional channels. In contrast, when coexpressed with other GIRK subunits, rbGIRK3 gives rise to prominent currents which are enhanced by the stimulation of coexpressed 5-HT1A receptors. In situ hybridizations show that of all GIRK subunits rbGIRK3 is most widely distributed and strongly expressed throughout the rat brain and thus may play an important role in central signal processing.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/biossíntese , RNA Mensageiro/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Chlorocebus aethiops , DNA Complementar , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Biblioteca Gênica , Hibridização In Situ , Dados de Sequência Molecular , Oócitos/fisiologia , Fases de Leitura Aberta , Canais de Potássio/fisiologia , RNA Mensageiro/análise , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , XenopusRESUMO
Two splice variants of the ligand-gated 5-hydroxytryptamine or serotonin 5-HT3 receptor that differ in a six-amino-acid deletion were cloned by polymerase chain reaction from the hippocampus x neuroblastoma cell line HN9.10e. When expressed in Xenopus oocytes, both variants individually formed 5-HT3 receptors that revealed no significant differences in current responses to the agonists 5-HT and 1-phenylbiguanide and block by the specific antagonist LY-278, 584-maleate. For both receptors, the monovalent cations Na+, K+, Rb+ and Li+ showed the same relative permeability; NH4(+)permeated approximately 2.7 times better than Na+, and Tris+ was only poorly permeable. In contrast to other reports, the receptors were completely and reversibly blocked by extracellular Cs+ in both oocytes and native HN9.10 cells. Moreover, Ca2+ was not permeant and exhibited a concentration-dependent decrease (0.9-18 mM) of the 5-HT-induced currents without affecting the inward rectification of the current/voltage relation. The two receptors were reversibly inhibited by nanomolar concentrations of the specific inhibitor of protein kinase C (PKC) bisindolylmaleimide, but not by the equipotent and less specific inhibitor staurosporine. A regulatory effect on both 5-HT3 receptor subunits by PKC-mediated protein phosphorylation might be possible, however, a functional role of the two splice variants present in one cell remains to be determined.
Assuntos
DNA Recombinante , Variação Genética , Hipocampo/metabolismo , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Animais , Sequência de Bases , Cálcio/farmacologia , Clonagem Molecular , Ativação Enzimática , Hipocampo/citologia , Injeções , Ativação do Canal Iônico , Canais Iônicos/efeitos dos fármacos , Camundongos , Dados de Sequência Molecular , Sondas de Oligonucleotídeos/genética , Oócitos/metabolismo , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , RNA Complementar , Proteínas Recombinantes , Serotonina/farmacologia , Células Tumorais CultivadasRESUMO
Transcripts of a gene, GIRK4, that encodes for a 419-amino-acid protein and shows high structural similarity to other subfamily members of G-protein-activated inwardly rectifying K+ channels (GIRK) have been identified in the human hippocampus. When expressed in Xenopus oocytes, GIRK4 yielded functional GIRK channels with activity that was enhanced by the stimulation of coexpressed serotonin 1A receptors. GIRK4 potentiated basal and agonist-induced currents mediated by other GIRK channels, possibly because of channel heteromerization. Despite the structural similarity to a putative rat KATP channel, no ATP sensitivity or KATP-typical pharmacology was observed for GIRK4 alone or GIRK4 transfected in conjunction with other GIRK channels in COS-7 cells. In rat brain, GIRK4 is expressed together with three other subfamily members, GIRK1-3, most likely in identical hippocampal neurons. Thus, heteromerization or an unknown molecular interaction may cause the physiological diversity observed within this class of K+ channels.