RESUMO
Spalt-like proteins are Zinc finger transcription factors from Caenorhabditis elegans to vertebrates, with critical roles in development. In vertebrates, four paralogues have been identified (SALL1-4), and SALL2 is the family's most dissimilar member. SALL2 is required during brain and eye development. It is downregulated in cancer and acts as a tumor suppressor, promoting cell cycle arrest and cell death. Despite its critical functions, information about SALL2 regulation is scarce. Public data indicate that SALL2 is ubiquitinated and phosphorylated in several residues along the protein, but the mechanisms, biological consequences, and enzymes responsible for these modifications remain unknown. Bioinformatic analyses identified several putative phosphorylation sites for Casein Kinase II (CK2) located within a highly conserved C-terminal PEST degradation motif of SALL2. CK2 is a serine/threonine kinase that promotes cell proliferation and survival and is often hyperactivated in cancer. We demonstrated that CK2 phosphorylates SALL2 residues S763, T778, S802, and S806 and promotes SALL2 degradation by the proteasome. Accordingly, pharmacological inhibition of CK2 with Silmitasertib (CX-4945) restored endogenous SALL2 protein levels in SALL2-deficient breast MDA-MB-231, lung H1299, and colon SW480 cancer cells. Silmitasertib induced a methuosis-like phenotype and cell death in SW480 cells. However, the phenotype was significantly attenuated in CRISPr/Cas9-mediated SALL2 knockout SW480 cells. Similarly, Sall2-deficient tumor organoids were more resistant to Silmitasertib-induced cell death, confirming that SALL2 sensitizes cancer cells to CK2 inhibition. We identified a novel CK2-dependent mechanism for SALL2 regulation and provided new insights into the interplay between these two proteins and their role in cell survival and proliferation.
Assuntos
Caseína Quinase II , Neoplasias do Colo , Animais , Humanos , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Neoplasias do Colo/genética , Linhagem Celular TumoralRESUMO
We investigated the role of PDZ proteins (GRIP, ABP, and PICK1) interacting with the C-terminal GluR2 by infusing a ct-GluR2 peptide ("pep2-SVKI") into CA1 pyramidal neurons in hippocampal slices using whole-cell recordings. Pep2-SVKI, but not a control or PICK1 selective peptide, caused AMPAR-mediated EPSC amplitude to increase in approximately one-third of control neurons and in most neurons following the prior induction of LTD. Pep2-SVKI also blocked LTD; however, this occurred in all neurons. A PKC inhibitor prevented these effects of pep2-SVKI on synaptic transmission and LTD. We propose a model in which the maintenance of LTD involves the binding of AMPARs to PDZ proteins to prevent their reinsertion. We also present evidence that PKC regulates AMPAR reinsertion during dedepression.
Assuntos
Hipocampo/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteína Quinase C/metabolismo , Receptores de AMPA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Peptídeos beta-Amiloides/metabolismo , Animais , Proteínas de Transporte/metabolismo , Células Cultivadas , Proteínas do Citoesqueleto , Inibidores Enzimáticos/farmacologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Hipocampo/citologia , Técnicas In Vitro , Peptídeos e Proteínas de Sinalização Intracelular , Modelos Neurológicos , Proteínas do Tecido Nervoso/metabolismo , Inibição Neural/efeitos dos fármacos , Inibição Neural/fisiologia , Plasticidade Neuronal/fisiologia , Proteínas Nucleares/metabolismo , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/genética , Proteína Quinase C/antagonistas & inibidores , Estrutura Terciária de Proteína/genética , Células Piramidais/citologia , Células Piramidais/efeitos dos fármacos , Células Piramidais/metabolismo , Ratos , Receptores de AMPA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Transmissão Sináptica/efeitos dos fármacosRESUMO
We investigated whether the interaction between the N-ethyl-maleimide-sensitive fusion protein (NSF) and the AMPA receptor (AMPAR) subunit GluR2 is involved in synaptic plasticity in the CA1 region of the hippocampus. Blockade of the NSF-GluR2 interaction by a specific peptide (pep2m) introduced into neurons prevented homosynaptic, de novo long-term depression (LTD). Moreover, saturation of LTD prevented the pep2m-induced reduction in AMPAR-mediated excitatory postsynaptic currents (EPSCs). Minimal stimulation experiments indicated that both pep2m action and LTD were due to changes in quantal size and quantal content but were not associated with changes in AMPAR single-channel conductance or EPSC kinetics. These results suggest that there is a pool of AMPARs dependent on the NSF-GluR2 interaction and that LTD expression involves the removal of these receptors from synapses.
Assuntos
Proteínas de Transporte/metabolismo , Hipocampo/fisiologia , Potenciação de Longa Duração/fisiologia , Receptores de AMPA/metabolismo , Proteínas de Transporte Vesicular , Animais , Eletrofisiologia , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Proteínas Sensíveis a N-Etilmaleimida , Peptídeos/farmacologia , RatosRESUMO
The two P domain K+ channel mTWIK-1 has been cloned from mouse brain. In Xenopus oocytes, mTWIK-1 currents are K+-selective, instantaneous, and weakly inward rectifying. These currents are blocked by Ba2+ and quinine, decreased by protein kinase C and increased by internal acidification. The apparent molecular weight of mTWIK-1 in brain is 81 kDa. A 40 kDa form is revealed after treatment with a reducing agent, strongly suggesting that native mTWIK-1 subunits dimerize via a disulfide bridge. TWIK-1 mRNA is expressed abundantly in brain and at lower levels in lung, kidney, and skeletal muscle. In situ hybridization shows that mTWIK-1 expression is restricted to a few brain regions, with the highest levels in cerebellar granule cells, brainstem, hippocampus and cerebral cortex.
Assuntos
Encéfalo/metabolismo , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/química , Sequência de Aminoácidos , Animais , Bário/farmacologia , Sequência de Bases , Western Blotting , DNA Complementar/genética , Dimerização , Hibridização In Situ , Potenciais da Membrana , Camundongos , Dados de Sequência Molecular , Peso Molecular , Oócitos , Canais de Potássio/genética , Canais de Potássio/metabolismo , Quinina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , XenopusRESUMO
MbIRK3, mbGIRK2 and mbGIRK3 K+ channels cDNAs have been cloned from adult mouse brain. These cDNAs encode polypeptides of 445, 414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane-spanning domains M1 and M2 and a pore-forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMK1 and GIRK1. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRK1 (60%) than to ROMK1 and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K(+)-selective currents very similar to IRK1 and GIRK1, respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G-protein coupled inward rectifier K+ channels which has been observed through their functional coupling with co-expressed delta-opioid receptors. These results provide the first evidence that the GIRK family, as the IRK family, is composed of multiple genes with members specifically expressed in the nervous system.
Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Camundongos , Dados de Sequência Molecular , Canais de Potássio/metabolismo , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , XenopusRESUMO
Chimeras have been constructed using three different fragments (N-terminal, central and C-terminal) of IRK3, a constitutive inward rectifier K+ channel subunit, and GIRK2, a G-protein activated inward rectifier K+ channel subunit and have been coinjected into Xenopus oocytes together with IRK3 or IRK1 (another constitutive inward rectifier) cRNA. Both IRK1 and IRK3 expression was inhibited by coinjection with chimeras containing a N-terminal fragment of IRK3 suggesting that subunits of K+ channels in the IRK family form a functional multimeric assembly where the N-terminal end has an important role. In situ hybridization shows that IRK1 and IRK3 are coexpressed in the same areas of the brain and probably in the same cells. Taken together both the localization and the oocyte expression results suggest that not only homomultimeric IRK1 or homomultimeric IRK3 assemblies take place but that heteromultimeric IRK1/IRK3 assemblies are also formed.
Assuntos
Fragmentos de Peptídeos/química , Canais de Potássio/química , Proteínas Recombinantes de Fusão , Animais , Química Encefálica , Feminino , Proteínas de Ligação ao GTP/fisiologia , Expressão Gênica , Hibridização In Situ , Substâncias Macromoleculares , Oócitos/metabolismo , Fragmentos de Peptídeos/genética , Canais de Potássio/genética , RNA Mensageiro/análise , Distribuição Tecidual , XenopusRESUMO
Postoperative subconjonctival 5-Fluorouracil (5 FU) may enhance the surgical outcome of trabeculectomies in eyes at high risk of operative failure. 23 patients who received postoperative low doses of subconjonctival 5 Fluorouracil (5 FU) have been studied with a mean follow-up of 10 months. Our indications were: risk factors for a trabeculectomy; postoperative pressure remaining high or absence of filtering bleb. The results were: 42% of complete success (IOP less than 21 mmHg without addition therapy); 29% of qualified success (IOP less than 25 mmHg with additional therapy, or 21 mmHg less than IOP less than 25 mmHg without therapy); 29% of failure. The most frequent complications are corneal, but healed within a short period. The efficacity of the drug remains even with low doses, and with less complications.
Assuntos
Fluoruracila/administração & dosagem , Complicações Pós-Operatórias/prevenção & controle , Trabeculectomia , Feminino , Fluoruracila/efeitos adversos , Fluoruracila/uso terapêutico , Humanos , Injeções , Masculino , Pessoa de Meia-Idade , Trabeculectomia/efeitos adversosRESUMO
The authors report a case of Clostridium perfringens endophthalmitis occurring after a corneo-limb al injury without intraocular foreign body. The diagnosis was made on CT scan prior to bacteriological identification of the bacteria. Rapid and adapted treatment allowed preservation of the eyeball. The clinical signs, often stereotyped, as well as the combined medical--with systemic antibiotics--and surgical--vitrectomy--treatments are described.
Assuntos
Infecções por Clostridium , Clostridium perfringens , Endoftalmite/microbiologia , Antibacterianos/uso terapêutico , Endoftalmite/diagnóstico , Endoftalmite/terapia , Humanos , Masculino , Pessoa de Meia-Idade , VitrectomiaRESUMO
We report a case of haemorrhagic macular choroidopathy in young adult occurring in a 24-year-old woman. The aetiology of the affection remains unknown in Europe, but many authors suggest arterial dissemination of an infectious agent may be involved, as in the American form. In the case described here, it could be Chlamydia Psittaci.
Assuntos
Chlamydophila psittaci , Hemorragia da Coroide/etiologia , Macula Lutea , Adulto , Fatores Etários , Hemorragia da Coroide/diagnóstico , Hemorragia da Coroide/microbiologia , Feminino , Humanos , Neovascularização Retiniana/diagnóstico , Neovascularização Retiniana/etiologia , Neovascularização Retiniana/microbiologiaRESUMO
We report a case of birdshot retinochoroidopathy following a cataract surgery in a 78-year-old man. Low-grade forms of the affection may be discovered lately on a recurrence of the inflammation following any event as an ocular surgical procedure. Medical management of cataract surgery in uveitis is then discussed.
Assuntos
Coriorretinite/diagnóstico , Lentes Intraoculares , Complicações Pós-Operatórias/diagnóstico , Idoso , Angiofluoresceinografia , Humanos , Masculino , Acuidade VisualRESUMO
The place of Chlamydia trachomatis in acute or chronic conjunctivitis is certainly underestimated due to the difficulties involved in the diagnosis. Three diagnostic techniques for Chlamydia trachomatis conjunctivitis were compared: conjunctival sac swab for ELISA technique, scraping of the tarsal conjunctiva for direct immunofluorescence and for culture. Two groups of patients were studied: 73 patients with acute or chronic conjunctivitis and 44 asymptomatic patients. 19.2% of the patients in the first group had a positive result with the two techniques and 6.8% of the patients in the second group also had positive results with the same criteria. Our results show that indirect immunofluorescence is the technique most frequently positive, followed by ELISA, while culture remains the most specific but the least sensitive technique. The existence of healthy carriers Chlamydia trachomatis is demonstrated.
Assuntos
Infecções por Chlamydia/diagnóstico , Chlamydia trachomatis , Conjuntivite Bacteriana/diagnóstico , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Técnicas Bacteriológicas , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
This study firstly shows with in situ hybridization on human pancreas that TALK-1 and TALK-2, two members of the 2P domain potassium channel (K(2P)) family, are highly and specifically expressed in the exocrine pancreas and absent in Langherans islets. On the contrary, expression of TASK-2 in mouse pancreas is found both in the exocrine pancreas and in the Langherans islets. This study also shows that TALK-1 and TALK-2 channels, expressed in Xenopus oocytes, are strongly and specifically activated by nitric oxide (obtained with a mixture of sodium nitroprussate (SNP) and dithiothreitol (DTT)), superoxide anion (obtained with xanthine and xanthine oxidase) and singlet oxygen (obtained upon photoactivation of rose bengal, and with chloramine T). Other nitric oxide and reactive oxygen species (NOS and ROS) donors, as well as reducing conditions were found to be ineffective on TALK-1, TALK-2 and TASK-2 (sin-1, angeli's salt, SNP alone, tBHP, H(2)O(2), and DTT). These results suggest that, in the exocrine pancreas, specific members of the NOS and ROS families could act as endogenous modulators of TALK channels with a role in normal secretion as well as in disease states such as acute pancreatitis and apoptosis.
Assuntos
Óxido Nítrico/farmacologia , Pâncreas/fisiologia , Canais de Potássio/efeitos dos fármacos , Espécies Reativas de Oxigênio/farmacologia , Animais , Eletrofisiologia , Humanos , Radical Hidroxila/farmacologia , Hibridização In Situ , Camundongos , Oócitos/metabolismo , Oxidantes/farmacologia , Canais de Potássio de Domínios Poros em Tandem/efeitos dos fármacos , Xenopus laevisRESUMO
The authors report a study of the modifications observed with sutures of PTFE (Goretex) and polyglactin (Vicryl) at the level of the sclera and the conjunctival. Inflammatory cells are fewer with PTFE both in the rabbit eyes (14 cases) from 3 to 110 days and in the human eye in a clinical study of 28 cases.
Assuntos
Túnica Conjuntiva/citologia , Poliglactina 910/normas , Polímeros/normas , Politetrafluoretileno/normas , Esclera/citologia , Suturas/normas , Animais , Túnica Conjuntiva/imunologia , Eosinófilos/análise , Estudos de Avaliação como Assunto , Histiócitos/análise , Humanos , Poliglactina 910/efeitos adversos , Politetrafluoretileno/efeitos adversos , Coelhos , Esclera/imunologiaRESUMO
Riluzole (RP 54274) is a potent neuroprotective agent with anticonvulsant, sedative, and anti-ischemic properties. It is currently used in the treatment of amyotrophic lateral sclerosis. This article reports that riluzole is an activator of TREK-1 and TRAAK, two important members of a new structural family of mammalian background K(+) channels with four transmembrane domains and two pore regions. Whereas riluzole activation of TRAAK is sustained, activation of TREK-1 is transient and is followed by an inhibition. The inhibitory process is attributable to an increase of the intracellular cAMP concentration by riluzole that produces a protein kinase A-dependent inhibition of TREK-1. Mutants of TREK-1 lacking the Ser residue where the kinase A phosphorylation takes place are activated in a sustained manner by riluzole. TRAAK is permanently activated by riluzole because, unlike TREK-1, it lacks the negative regulation by cAMP.
Assuntos
Fármacos Neuroprotetores/farmacologia , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/metabolismo , Riluzol/farmacologia , Animais , Células COS , AMP Cíclico/metabolismo , Canais de Potássio/química , Canais de Potássio/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , TransfecçãoRESUMO
A new human weakly inward rectifying K+ channel, TWIK-1, has been isolated. This channel is 336 amino acids long and has four transmembrane domains. Unlike other mammalian K+ channels, it contains two pore-forming regions called P domains. Genes encoding structural homologues are present in the genome of Caenorhabditis elegans. TWIK-1 currents expressed in Xenopus oocytes are time-independent and present a nearly linear I-V relationship that saturated for depolarizations positive to O mV in the presence of internal Mg2+. This inward rectification is abolished in the absence of internal Mg2+. TWIK-1 has a unitary conductance of 34 pS and a kinetic behaviour that is dependent on the membrane potential. In the presence of internal Mg2+, the mean open times are 0.3 and 1.9 ms at -80 and +80 mV, respectively. The channel activity is up-regulated by activation of protein kinase C and down-regulated by internal acidification. Both types of regulation are indirect. TWIK-1 channel activity is blocked by Ba2+(IC50=100 microM), quinine (IC50=50 microM) and quinidine (IC50=95 microM). This channel is of particular interest because its mRNA is widely distributed in human tissues, and is particularly abundant in brain and heart. TWIK-1 channels are probably involved in the control of background K+ membrane conductances.
Assuntos
Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Clonagem Molecular , DNA Complementar/genética , Genes de Helmintos , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Estrutura Molecular , Oócitos/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Xenopus laevisRESUMO
Cloned K+ channel beta subunits are hydrophilic proteins which associate to pore-forming alpha subunits of the Shaker subfamily. The resulting alphabeta heteromultimers K+ channels have inactivation kinetics significantly more rapid than those of the corresponding alpha homomultimers. This paper reports the cloning and the brain localization of mKvbeta4 (m for mouse), a new beta subunit. This new beta subunit is highly expressed in the nervous system but is also present in other tissues such as kidney. In contrast with other beta subunits, coexpression of the mKvbeta4 subunit with alpha subunits of Shaker-type K+ channel does not modify the kinetic properties or voltage-dependence of these channels in Xenopus oocytes. Instead, mKvbeta4 associates to Kv2.2 (CDRK), a Shab K+ channel, to specifically enhance (a factor of up to 6) its expression level without changing its elementary conductance or kinetics. It is without effect on another closely related Shab K+ channel Kv2.1 (DRK1). Chimeras between Kv2.1 and Kv2. 2 indicate that the COOH-terminal end of the Kv2.2 protein is essential for its mKvbeta4 sensitivity. The functional results associated with the observation of the co-localization of mKvbeta4 and Kv2.2 transcripts in most brain areas strongly suggest that both subunits interact in vivo to form a slowly-inactivating K+ channel. A chaperone-like effect of mKvbeta4 seems to permit the integration of a larger number of Kv2.2 channels at the plasma membrane.
Assuntos
Encéfalo/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/biossíntese , Sequência de Aminoácidos , Animais , Autorradiografia , Sequência de Bases , Northern Blotting , Clonagem Molecular , DNA Complementar , Canais de Potássio de Retificação Tardia , Feminino , Canal de Potássio Kv1.3 , Canal de Potássio Kv1.5 , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta , Camundongos , Dados de Sequência Molecular , Oócitos , Canais de Potássio/química , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio Shab , Superfamília Shaker de Canais de Potássio , Canais de Potássio Shaw , Distribuição Tecidual , Transcrição Gênica , XenopusRESUMO
YORK is a newly cloned K+ channel from yeast. Unlike all other cloned K+ channels, it has two pore domains instead of one. It displays eight transmembrane segments arranged like a covalent assembly of a Shaker-type voltage-dependent K+ channel (without S4 transmembrane segments) with an inward rectifier K+ channel. When expressed in Xenopus oocytes, YORK does not pass inward currents; it conducts only K+-selective outward currents. However, the mechanism responsible for this strict outward rectification is unusual. Like inward rectifiers, its activation potential threshold closely follows the K+ equilibrium potential. Unlike inward rectifiers, the rectification is not due to a voltage-dependent Mg2+ block. The blocking element is probably intrinsic to the YORK protein itself. YORK activity is decreased at acidic internal pH, with a pKa of 6.5. Pharmacological and regulation properties were analyzed. Ba2+ ions and quinine block YORK currents through high and low affinity sites, while tetraethylammonium displays only one affinity for blocking. Activation of protein kinase C indirectly produces an increase of the current, while protein kinase A activation has no effect.
Assuntos
Ativação do Canal Iônico , Canais de Potássio/química , Canais de Potássio/fisiologia , Estrutura Secundária de Proteína , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/fisiologia , Sequência de Aminoácidos , Animais , Membrana Celular/fisiologia , Clonagem Molecular , Feminino , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Cinética , Potenciais da Membrana , Dados de Sequência Molecular , Oócitos/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , XenopusRESUMO
Prophylaxis of rhegmatogenous retinal detachment consists of measures both non surgical and/or surgical that are effective in preventing some risk-factors that are part of the disease. The procedure must avoid any types of danger. This paper describes a new reliable surgical procedure of circular buckling used for prophylaxis on predisposed fellow-eye of giant retinal tear.
Assuntos
Descolamento Retiniano/prevenção & controle , Recurvamento da Esclera/métodos , HumanosRESUMO
Human TWIK-1, which has been cloned recently, is a new structural type of weak inward rectifier K+ channel. Here we report the structural and functional properties of TREK-1, a mammalian TWIK-1-related K+ channel. Despite a low amino acid identity between TWIK-1 and TREK-1 (approximately 28%), both channel proteins share the same overall structural arrangement consisting of two pore-forming domains and four transmembrane segments (TMS). This structural similarity does not give rise to a functional analogy. K+ currents generated by TWIK-1 are inwardly rectifying while K+ currents generated by TREK-1 are outwardly rectifying. These channels have a conductance of 14 pS. TREK-1 currents are insensitive to pharmacological agents that block TWIK-1 activity such as quinine and quinidine. Extensive inhibitions of TREK-1 activity are observed after activation of protein kinases A and C. TREK-1 currents are sensitive to extracellular K+ and Na+. TREK-1 mRNA is expressed in most tissues and is particularly abundant in the lung and in the brain. Its localization in this latter tissue has been studied by in situ hybridization. TREK-1 expression is high in the olfactory bulb, hippocampus and cerebellum. These results provide the first evidence for the existence of a K+ channel family with four TMS and two pore domains in the nervous system of mammals. They also show that different members in this structural family can have totally different functional properties.
Assuntos
Encéfalo/metabolismo , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Clonagem Molecular , DNA Complementar , Humanos , Dados de Sequência Molecular , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , XenopusRESUMO
TWIK-1 is a new type of K+ channel with two P domains and is abundantly expressed in human heart and brain. Here we show that TWIK-1 subunits can self-associate to give dimers containing an interchain disulfide bridge. This assembly involves a 34 amino acid domain that is localized to the extracellular M1P1 linker loop. Cysteine 69 which is part of this interacting domain is implicated in the formation of the disulfide bond. Replacing this cysteine with a serine residue results in the loss of functional K+ channel expression. This is the first example of a covalent association of functional subunits in voltage-sensitive channels via a disulfide bridge.