RESUMO
Cerebral SVDs encompass a group of genetic and sporadic pathological processes leading to brain lesions, cognitive decline, and stroke. There is no specific treatment for SVDs, which progress silently for years before becoming clinically symptomatic. Here, we examine parallels in the functional defects of PAs in CADASIL, a monogenic form of SVD, and in response to SAH, a common type of hemorrhagic stroke that also targets the brain microvasculature. Both animal models exhibit dysregulation of the voltage-gated potassium channel, KV 1, in arteriolar myocytes, an impairment that compromises responses to vasoactive stimuli and impacts CBF autoregulation and local dilatory responses to neuronal activity (NVC). However, the extent to which this channelopathy-like defect ultimately contributes to these pathologies is unknown. Combining experimental data with computational modeling, we describe the role of KV 1 channels in the regulation of myocyte membrane potential at rest and during the modest increase in extracellular potassium associated with NVC. We conclude that PA resting membrane potential and myogenic tone depend strongly on KV 1.2/1.5 channel density, and that reciprocal changes in KV channel density in CADASIL and SAH produce opposite effects on extracellular potassium-mediated dilation during NVC.
Assuntos
Microvasos/patologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Animais , CADASIL/fisiopatologia , Dilatação , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Hemorragia Subaracnóidea/fisiopatologiaRESUMO
Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.
Assuntos
Axônios/enzimologia , Metabolismo dos Lipídeos , Lisossomos/metabolismo , Neuroglia/metabolismo , Doenças do Sistema Nervoso Periférico/fisiopatologia , Peroxissomos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Adrenoleucodistrofia/patologia , Animais , Axônios/ultraestrutura , Modelos Animais de Doenças , Humanos , Camundongos , Microscopia Eletrônica , Receptor 1 de Sinal de Orientação para Peroxissomos/deficiênciaRESUMO
OBJECTIVE: To identify novel combinations of genetic and psychological factors that predicted 12-month postoperative pain and disability outcomes following arthroscopic shoulder surgery. METHODS: A prospective presurgical cohort (n = 150) was recruited to complete validated psychological questionnaires and have their DNA collected from saliva. DNA was genotyped for a priori selected genes involved with pain modulation (ADRB2, OPRM1, AVPR1A, GCH1, and KCNS1) and inflammation (IL1B, TNF/LTA, and IL6). The outcome measures of interest were the Brief Pain Inventory and Disabilities of the Arm, Shoulder, and Hand questionnaire. Followup for the cohort was at 3, 6, and 12 months postoperatively. After controlling for age, sex, race, and preoperative status, genetic and psychological factors were entered as main effects and interaction terms in separate general linear models for predicting postoperative pain and disability outcomes. RESULTS: Seven interactions involving pain-modulatory genes were identified. Three provided strong statistical evidence for different outcomes, including KCNS1 and kinesiophobia for preoperative pain intensity, ADRB2 and depressive symptoms for postoperative course, and GCH1 and anxiety symptoms for 12-month pain-intensity outcome. Ten interactions involving inflammatory genes were identified. Three provided strong statistical evidence for the 12-month postoperative course outcome, including 2 different IL6 single-nucleotide polymorphism and pain catastrophizing, and IL6 and depressive symptoms. CONCLUSION: The current study identified novel genetic and psychological interactions that can be used in future studies to further understand the development of persistent postoperative pain and investigate the effectiveness of tailored treatment.
Assuntos
Artroscopia/efeitos adversos , Dor Pós-Operatória/genética , Dor Pós-Operatória/psicologia , Dor de Ombro/genética , Dor de Ombro/psicologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Ansiedade/genética , Ansiedade/psicologia , Catastrofização/genética , Catastrofização/psicologia , Depressão/genética , Depressão/psicologia , Avaliação da Deficiência , Feminino , Seguimentos , GTP Cicloidrolase/análise , Humanos , Interleucina-6/análise , Masculino , Pessoa de Meia-Idade , Medição da Dor , Dor Pós-Operatória/fisiopatologia , Polimorfismo de Nucleotídeo Único , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Estudos Prospectivos , Receptores Adrenérgicos beta 2/análise , Ombro/cirurgia , Dor de Ombro/fisiopatologia , Inquéritos e Questionários , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
Voltage-gated K+ channel (VKC) plays important roles in biology procession, especially in nervous system. Different subfamilies of VKCs have different biological functions. Thus, knowing VKCs' subfamilies has become a meaningful job because it can guide the direction for the disease diagnosis and drug design. However, the traditional wet-experimental methods were costly and time-consuming. It is highly desirable to develop an effective and powerful computational tool for identifying different subfamilies of VKCs. In this study, a predictor, called iVKC-OTC, has been developed by incorporating the optimized tripeptide composition (OTC) generated by feature selection technique into the general form of pseudo-amino acid composition to identify six subfamilies of VKCs. One of the remarkable advantages of introducing the optimized tripeptide composition is being able to avoid the notorious dimension disaster or over fitting problems in statistical predictions. It was observed on a benchmark dataset, by using a jackknife test, that the overall accuracy achieved by iVKC-OTC reaches to 96.77% in identifying the six subfamilies of VKCs, indicating that the new predictor is promising or at least may become a complementary tool to the existing methods in this area. It has not escaped our notice that the optimized tripeptide composition can also be used to investigate other protein classification problems.
Assuntos
Algoritmos , Biologia Computacional , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Bases de Dados de Proteínas , Internet , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Máquina de Vetores de Suporte , Interface Usuário-ComputadorRESUMO
Gastritis cystica profunda (GCP) is a rare condition caused by ectopic entrapment of gastric glands, probably secondary to the disruption of muscularis mucosae. GCP is often associated with gastric adenocarcinoma, and loss of the KCNE2 subunit from potassium channel complexes is considered a common primary target molecule leads to both GCP and malignancy. In this study, we, for the first time, analyzed the expression of KCNE2 in surgically excised tissue from human gastric cancer associated with GCP and confirmed that reduced KCNE2 expression correlates with disease formation.
Assuntos
Adenocarcinoma/química , Biomarcadores Tumorais/análise , Gastrite/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Neoplasias Gástricas/química , Adenocarcinoma/patologia , Adenocarcinoma/cirurgia , Biópsia , Regulação para Baixo , Gastrectomia , Mucosa Gástrica/química , Mucosa Gástrica/patologia , Gastrite/patologia , Gastrite/cirurgia , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgiaRESUMO
KCNE genes encode for a small family of Kv channel ancillary subunits that form heteromeric complexes with Kv channel alpha subunits to modify their functional properties. Mutations in KCNE genes have been found in patients with cardiac arrhythmias such as the long QT syndrome and/or atrial fibrillation. However, the precise molecular pathophysiology that leads to these diseases remains elusive. In previous studies the electrophysiological properties of the disease causing mutations in these genes have mostly been studied in heterologous expression systems and we cannot be sure if the reported effects can directly be translated into native cardiomyocytes. In our laboratory we therefore use a different approach. We directly study the effects of KCNE gene deletion in isolated cardiomyocytes from knockout mice by cellular electrophysiology - a unique technique that we describe in this issue of the Journal of Visualized Experiments. The hearts from genetically engineered KCNE mice are rapidly excised and mounted onto a Langendorff apparatus by aortic cannulation. Free Ca(2+) in the myocardium is bound by EGTA, and dissociation of cardiac myocytes is then achieved by retrograde perfusion of the coronary arteries with a specialized low Ca(2+) buffer containing collagenase. Atria, free right ventricular wall and the left ventricle can then be separated by microsurgical techniques. Calcium is then slowly added back to isolated cardiomyocytes in a multiple step comprising washing procedure. Atrial and ventricular cardiomyocytes of healthy appearance with no spontaneous contractions are then immediately subjected to electrophysiological analyses by patch clamp technique or other biochemical analyses within the first 6 hours following isolation.
Assuntos
Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Cálcio/metabolismo , Átrios do Coração/citologia , Átrios do Coração/metabolismo , Ventrículos do Coração/citologia , Ventrículos do Coração/metabolismo , Camundongos , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análiseRESUMO
Human placental expression of K(V)9.3, a voltage-gated K channel linked to tissue oxygenation responses, has been suggested at the messenger RNA level but tissue localisation has not been described. We aimed to: (1) produce an antibody to human K(V)9.3 and (2) assess channel expression and distribution in human placental tissue. We determined human placental protein expression and localisation using an antibody to K(V)9.3. Antibody specificity was confirmed by Western blotting. Staining was observed in syncytiotrophoblast microvillous membrane, endothelial cells (in intermediate, stem villi and chorionic plate blood vessels) and vascular smooth muscle cells (large diameter vessels only) by immunohistochemistry. Expression was unchanged in tissue from women with small-for-gestational age babies. It was concluded that K(V)9.3 is localised to human placental vascular tissues and syncytiotrophoblast.
Assuntos
Placenta/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Antígenos/química , Antígenos/imunologia , Western Blotting , Células Endoteliais/química , Feminino , Expressão Gênica , Idade Gestacional , Humanos , Imuno-Histoquímica , Microvilosidades/química , Dados de Sequência Molecular , Placenta/irrigação sanguínea , Canais de Potássio de Abertura Dependente da Tensão da Membrana/imunologia , Gravidez , RNA Mensageiro/análise , Coelhos , Trofoblastos/química , Trofoblastos/ultraestruturaRESUMO
KCNE2 functions as an auxiliary subunit in voltage-gated K and HCN channels in the heart. Genetic variations in KCNE2 have been linked to long QT syndrome. The underlying mechanisms are not entirely clear. One of the issues is whether KCNE2 protein is expressed in ventricles. We use adenovirus-mediated genetic manipulations of adult cardiac myocytes to validate two antibodies (termed Ab1 and Ab2) for their ability to detect native KCNE2 in the heart. Ab1 faithfully detects native KCNE2 proteins in spontaneously hypertensive rat and guinea pig hearts. In both cases, KCNE2 protein is more abundant in ventricles than in atria. In both ventricular and atrial myocytes, KCNE2 protein is preferentially distributed on the cell surface. Ab1 can detect a prominent KCNE2 band in human ventricular muscle from nonfailing hearts. The band intensity is much fainter in atria and in failing ventricles. Ab2 specifically detects S98 phosphorylated KCNE2. Through exploring the functional significance of S98 phosphorylation, we uncover a novel mechanism by which KCNE2 modulates the human ether-a-go-go related gene (hERG) current amplitude: by accelerating hERG protein degradation and thus reducing the hERG protein level on the cell surface. S98 phosphorylation appears to be required for this modulation, so that S98 dephosphorylation leads to an increase in hERG/rapid delayed rectifier current amplitude. Our data confirm that KCNE2 protein is expressed in the ventricles of human and animal models. Furthermore, KCNE2 can modulate its partner channel function not only by altering channel conductance and/or gating kinetics, but also by affecting protein stability.
Assuntos
Átrios do Coração/metabolismo , Ventrículos do Coração/metabolismo , Miócitos Cardíacos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteólise , Transativadores/metabolismo , Sequência de Aminoácidos , Animais , Células Cultivadas , Cães , Feminino , Cobaias , Átrios do Coração/citologia , Ventrículos do Coração/citologia , Humanos , Masculino , Modelos Animais , Dados de Sequência Molecular , Miócitos Cardíacos/citologia , Técnicas de Patch-Clamp , Fosforilação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Ratos , Ratos Endogâmicos SHR , Canais de Potássio Shal , Regulador Transcricional ERGRESUMO
Kv1 channels are concentrated at specific sites in the axonal membrane, where they regulate neuronal excitability. Establishing these distributions requires regulated dissociation of Kv1 channels from the neuronal trafficking machinery and their subsequent insertion into the axonal membrane. We find that the auxiliary Kvß2 subunit of Kv1 channels purified from brain is phosphorylated on serine residues 9 and 31, and that cyclin-dependent kinase (Cdk)-mediated phosphorylation at these sites negatively regulates the interaction of Kvß2 with the microtubule plus end-tracking protein EB1. Endogenous Cdks, EB1, and Kvß2 phosphorylated at serine 31 are colocalized in the axons of cultured hippocampal neurons, with enrichment at the axon initial segment (AIS). Acute inhibition of Cdk activity leads to intracellular accumulation of EB1, Kvß2, and Kv1 channel subunits within the AIS. These studies reveal a new regulatory mechanism for the targeting of Kv1 complexes to the axonal membrane through the reversible Cdk phosphorylation-dependent binding of Kvß2 to EB1.
Assuntos
Axônios/metabolismo , Quinases Ciclina-Dependentes/fisiologia , Neurônios/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Superfamília Shaker de Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Axônios/química , Cromatografia Líquida , Hipocampo/química , Hipocampo/metabolismo , Humanos , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Dados de Sequência Molecular , Neurônios/química , Fosforilação , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Subunidades Proteicas/análise , Subunidades Proteicas/química , Subunidades Proteicas/fisiologia , Transporte Proteico/fisiologia , Ratos , Nervo Isquiático/química , Nervo Isquiático/metabolismo , Serina/química , Espectrometria de Massas em TandemRESUMO
Delayed rectifier potassium currents such as I (Kr) and I (Ks) play an important role in the repolarization phase of the action potential in cardiac myocytes. Electrophysiological studies have shown that the pig is a useful animal not only for clinical use as a good candidate for humans, but also for basic research in heart function or arrhythmia. However, no studies concerning the potassium channels on a molecular level have been done. To elucidate the expression level and distribution of delayed rectifier potassium channels in pigs, we quantitatively investigated the I (Kr) and I (Ks) channel subunits using the real-time polymerase chain reaction (PCR) method. The hearts from Clawn miniature pigs were separated into the apical and basal regions, and subsequently excised into transmural trisections within each of the left ventricular walls, epicardium, midcardium, and endocardium. After RNA extraction from these sites, real-time PCR was executed with reverse transcriptional products for quantitative analysis. The expression level of KCNE1 was significantly higher than those of KCNQ1, KCNH2, and KCNE2, which were comparable in all sites. Transmural heterogeneity of these potassium channel subunits was not detected on the mRNA level. These results indicate that KCNE1 is a dominant subunit on the post-transcriptional level in the miniature pig.
Assuntos
Ventrículos do Coração/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Análise de Variância , Animais , Canais de Potássio Éter-A-Go-Go/análise , Regulação da Expressão Gênica , Canal de Potássio KCNQ1/análise , Masculino , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Suínos , Porco Miniatura , Transcrição GênicaRESUMO
Transient cerebral ischemia is known to induce endogenous mechanisms that can prevent or delay neuronal injury, such as the activation of mitochondrial potassium channels. However, the molecular mechanism of this effect remains unclear. In this study, the single-channel activity was measured using the patch-clamp technique of the mitoplasts isolated from gerbil hippocampus. In 70% of all patches, a potassium-selective current with the properties of a voltage-gated Kv-type potassium channel was recorded with mean conductance 109+/-6pS in a symmetrical solution. The channel was blocked at negative voltages and irreversibly by margatoxin, a specific Kv1.3 channel inhibitor. The ATP/Mg(2+) complex and Ca(2+) ions had no effect on channel activity. Additionally, agitoxin-2, a potent inhibitor of voltage-gated potassium channels, had no effect on mitochondrial channel activity. This observation suggests that in contrast to surface membrane channels, the mitochondrial voltage-gated potassium channel could have a different molecular structure with no affinity to agitoxin-2. Western blots of gerbil hippocampal mitochondria and immunohistochemistry on gerbil brain sections confirmed the expression of the Kv1.3 protein in mitochondria. Our findings indicate that gerbil brain mitochondria contain a voltage-gated potassium channel that can influence the function of mitochondria in physiological and pathological conditions and that has properties similar to the surface membrane Kv1.3 channel.
Assuntos
Hipocampo/metabolismo , Canal de Potássio Kv1.3/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Gerbillinae , Hipocampo/química , Canal de Potássio Kv1.3/análise , Mitocôndrias/química , Membranas Mitocondriais/química , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análiseRESUMO
Pannexin 1 (Panx1), a member of the second gap junction protein family identified in vertebrates, appears to preferentially form non-junctional membrane channels. A candidate regulatory protein of Panx1 is the potassium channel subunit Kvbeta3, previously identified by bacterial two-hybrid strategies. Here, we report on the physical association of Panx1 with Kvbeta3 by immunoprecipitation when co-expressed in a neuroblastoma cell line (Neuro2A). Furthermore, in vivo co-expression of Panx1 and Kvbeta3 was shown to occur in murine hippocampus and cerebellum. Kvbeta3 is known to accelerate inactivation of otherwise slowly inactivating potassium channels under reducing conditions. We subsequently found that Panx1 channel currents exhibit a significant reduction when exposed to reducing agents, and that this effect is attenuated in the presence of Kvbeta3. Apparently, Kvbeta3 is involved in regulating the susceptibility of Panx1 channels to redox potential. Furthermore, the Panx1 channel blockers carbenoxolone and Probenecid were less effective in inhibiting Panx1 currents when Kvbeta3 was co-expressed. The influence of Kvbeta3 on Panx1 is the first example of modulation of Panx1 channel function(s) by interacting proteins, and suggests the physiological importance of sensing changes in redox potentials.
Assuntos
Conexinas/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Animais , Química Encefálica , Linhagem Celular , Conexina 43/fisiologia , Conexinas/análise , Conexinas/química , Camundongos , Proteínas do Tecido Nervoso/análise , Proteínas do Tecido Nervoso/química , Oxirredução , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Ratos , XenopusRESUMO
Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structure-function analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cycle-coupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Regulação Alostérica , Ativação do Canal Iônico , Ligação Proteica , Subunidades Proteicas/análise , Subunidades Proteicas/metabolismo , TermodinâmicaRESUMO
Voltage-gated potassium (K(V)) channels can form heteromultimeric complexes with a variety of accessory subunits, including KCNE proteins. Heterologous expression studies have demonstrated diverse functional effects of KCNE subunits on several K(V) channels, including KCNQ1 (K(V)7.1) that, together with KCNE1, generates the slow-delayed rectifier current (I(Ks)) important for cardiac repolarization. In particular, KCNE4 exerts a strong inhibitory effect on KCNQ1 and other K(V) channels, raising the possibility that this accessory subunit is an important potassium current modulator. A polyclonal KCNE4 antibody was developed to determine the human tissue expression pattern and to investigate the biochemical associations of this protein with KCNQ1. We found that KCNE4 is widely and variably expressed in several human tissues, with greatest abundance in brain, liver and testis. In heterologous expression experiments, immunoprecipitation followed by immunoblotting was used to establish that KCNE4 directly associates with KCNQ1, and can co-associate together with KCNE1 in the same KCNQ1 complex to form a 'triple subunit' complex (KCNE1-KCNQ1-KCNE4). We also used cell surface biotinylation to demonstrate that KCNE4 does not impair plasma membrane expression of either KCNQ1 or the triple subunit complex, indicating that biophysical mechanisms probably underlie the inhibitory effects of KCNE4. The observation that multiple KCNE proteins can co-associate with and modulate KCNQ1 channels to produce biochemically diverse channel complexes has important implications for understanding K(V) channel regulation in human physiology.
Assuntos
Canal de Potássio KCNQ1/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Animais , Anticorpos/imunologia , Membrana Celular/metabolismo , Humanos , Imunoprecipitação , Canal de Potássio KCNQ1/análise , Canal de Potássio KCNQ1/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Coelhos , Distribuição TecidualRESUMO
Ion channels are multisubunit proteins responsible for the generation and propagation of action potentials in nerve, skeletal muscle, and heart as well as maintaining salt and water homeostasis in epithelium. The subunit composition and stoichiometry of these membrane protein complexes underlies their physiological function, as different cells pair ion-conducting alpha-subunits with specific regulatory beta-subunits to produce complexes with diverse ion-conducting and gating properties. However, determining the number of alpha- and beta-subunits in functioning ion channel complexes is challenging and often fraught with contradictory results. Here we describe the synthesis of a chemically releasable, irreversible K(+) channel inhibitor and its iterative application to tally the number of beta-subunits in a KCNQ1/KCNE1 K(+) channel complex. Using this inhibitor in electrical recordings, we definitively show that there are two KCNE subunits in a functioning tetrameric K(+) channel, breaking the apparent fourfold arrangement of the ion-conducting subunits. This digital determination rules out any measurable contribution from supra, sub, and multiple stoichiometries, providing a uniform structural picture to interpret KCNE beta-subunit modulation of voltage-gated K(+) channels and the inherited mutations that cause dysfunction. Moreover, the architectural asymmetry of the K(+) channel complex affords a unique opportunity to therapeutically target ion channels that coassemble with KCNE beta-subunits.
Assuntos
Membrana Celular/química , Charibdotoxina/análogos & derivados , Dissulfetos/farmacologia , Canal de Potássio KCNQ1/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Subunidades Proteicas/análise , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Charibdotoxina/síntese química , Charibdotoxina/química , Charibdotoxina/farmacologia , Dissulfetos/síntese química , Dissulfetos/química , Humanos , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Potássio KCNQ1/metabolismo , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , XenopusRESUMO
KCNE1, KCNA5 and KCNK5 have been identified, by using specific blockers, as K(+)-channels involved in sperm volume regulation under physiological conditions. All three channels were localised on the cytoplasmic droplets and tail of human ejaculated spermatozoa by fluorescence microscopy. Using flow cytometric quantification, KCNE1 was found to be present in 80% or more spermatozoa and KCNK5 in only about 20%, with KCNA5 expressed by 20-90% of cells. Whereas the extents of such protein expression did not differ statistically between semen donors and subfertile patients, the former group exhibited higher capacities for sperm volume regulation which were correlated with other sperm qualities including normal morphology and motile sperm number in the ejaculate. Channel identification was further confirmed at the protein level using Western blotting. RT-PCR analysis of testicular and sperm RNA of proven quality indicated the presence of Kcne1, Kcna5 and Kcnk5 transcripts. Subsequent sequencing of PCR products demonstrated that the nucleotide sequences of the entire encoding regions of Kcne1 and Kcnk5 were identical to those published in the database, whereas that of Kcna5 mRNA showed a single nucleotide synonymous deviation that agrees with the published genomic sequence. Quantitative real-time PCR analysis of sperm RNA revealed the amounts of Kcne1 > Kcna5 > Kcnk5, in the same order as for protein expression. Thus, KCNE1 is probably the major K(+)-channel involved in regulatory volume decrease in human spermatozoa, and channel activity is regulated beyond the extent of protein expression.
Assuntos
Canal de Potássio Kv1.5/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , RNA Mensageiro/genética , Espermatozoides/fisiologia , Sequência de Bases , Tamanho Celular , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Canal de Potássio Kv1.5/análise , Canal de Potássio Kv1.5/fisiologia , Masculino , Dados de Sequência Molecular , Canais de Potássio de Domínios Poros em Tandem/análise , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , RNA Mensageiro/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/química , Espermatozoides/citologia , Testículo/química , Testículo/fisiologiaRESUMO
BACKGROUND AND OBJECTIVES: The aim of this study was to evaluate the expression and prognostic roles of human ether à go-go related gene (hERG1) potassium channels in resected esophageal squamous cell carcinoma. METHODS: Expression of hERG1 protein and mRNA was detected by immunohistochemistry and reverse transcription PCR in resected esophageal squamous cell carcinomas (ESCCs) and non-cancerous matched tissues, and the correlation was examined between expression of hERG1 protein and clinicopathological factors and prognosis of ESCC patients. RESULTS: Frequency of positive expression of hERG1 protein was 77.9% (53/68), mRNA was 81.8% (9/11). hERG1 protein and mRNA were negatively expressed in all non-cancerous matched tissues. There was no significant correlation between hERG1 protein expression and lymph node metastases, depth of penetration, stage, and other clinicopathological factors. Completeness was 92.5% for hERG1-positive group and 92.0% for hERG1-negative group. ESCC patients with positive expression of hERG1 protein had a significantly shorter postoperative survival time than those with negative expression (median, 30 vs. 56 months). Survival rates at each time-point for hERG1-positive group were lower than that for hERG1-negative group, and hERG1 was identified as an independent prognostic factor of long-term survival by multivariate analysis. CONCLUSION: In ESCC, hERG1 was aberrantly expressed and correlated with poor prognosis after surgery.
Assuntos
Carcinoma de Células Escamosas/química , Neoplasias Esofágicas/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Adulto , Idoso , Biomarcadores Tumorais , Carcinoma de Células Escamosas/mortalidade , Carcinoma de Células Escamosas/patologia , Canal de Potássio ERG1 , Neoplasias Esofágicas/mortalidade , Neoplasias Esofágicas/patologia , Canais de Potássio Éter-A-Go-Go , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Análise Multivariada , PrognósticoRESUMO
INTRODUCTION: Long QT syndrome (LQTS), an inherited cardiac arrhythmia, is a disorder of ventricular repolarisation characterised by electrocardiographic abnormalities and the onset of torsades de pointes leading to syncope and sudden death. Genetic polymorphisms in 5 well-characterised cardiac ion channel genes have been identified to be responsible for the disorder. The aim of this study is to identify disease-causing mutations in these candidate genes in a LQTS family. MATERIALS AND METHODS: The present study systematically screens the coding region of the LQTS-associated genes (KCNQ1, HERG, KCNE1, KCNE2 and SCN5A) for mutations using DNA sequencing analysis. RESULTS: The mutational analysis revealed 7 synonymous and 2 non-synonymous polymorphisms in the 5 ion channel genes screened. CONCLUSION: We did not identify any clear identifiable genetic marker causative of LQTS, suggesting the existence of LQTS-associated genes awaiting discovery.
Assuntos
Canais de Potássio Éter-A-Go-Go/genética , Canal de Potássio KCNQ1/genética , Síndrome do QT Longo/genética , Proteínas Musculares/genética , Polimorfismo Genético/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Sódio/genética , Adolescente , Adulto , Criança , Análise Mutacional de DNA , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/análise , Feminino , Mutação da Fase de Leitura , Humanos , Canal de Potássio KCNQ1/análise , Masculino , Pessoa de Meia-Idade , Proteínas Musculares/análise , Canal de Sódio Disparado por Voltagem NAV1.5 , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Sódio/análise , TransativadoresRESUMO
BACKGROUND: Atrial fibrillation is a common arrhythmia with multi-factorial pathogenesis. Recently, a single nucleotide polymorphism (G/T) at position 1057 in the KCNE4 gene, resulting in a glutamic acid (Glu, E)/aspartic acid (Asp, D) substitution at position 145 of the KCNE4 peptide, was found in our laboratory to be associated with idiopathic atrial fibrillation (atrial fibrillation more frequent with KCNE4 145D). However, the functional effect of the KCNE4 145E/D polymorphism is still unknown. METHODS: We constructed KCNE4 (145E/D) expression plasmids and transiently co-transfected them with the KCNQ1 gene into Chinese hamster ovary-K1 cells and performed whole-cell patch-clamping recording to identify the possible functional consequences of the single nucleotide polymorphism. Quantitative data were analyzed by Student;s t test. Probability values less than 0.05 were considered statistically significant. RESULTS: A slowly activating, non-inactivating voltage-dependent current ((24.0 +/- 2.9) pA/pF, at +60 mV)) could be recorded in the cells transfected with KCNQ1 alone. Co-expression of wild type KCNE4 inhibited the KCNQ1 current ((7.3 +/- 1.1) pA/pF)). By contrast, co-expression of KCNE4 (145D) augment the KCNQ1 current ((42.9 +/- 7) pA/pF)). The V(1/2) of activation for the KCNQ1/KCNE4 (145D) current was shifted significantly towards the depolarizing potential compared to that for the KCNQ1 current ((-2.3 +/- 0.2) mv vs (-13.0 +/- 1.5) mv, P < 0.01)) without changing the slope factorkappa. Furthermore, KCNE4 (145D) also affected the activation and deactivation kinetics of KCNQ1 channels. CONCLUSION: We provide experimental evidence that the KCNE4 (145E/D) polymorphism exerts the effect of "gain of function" on the KCNQ1 channel. It may underlie the genetic mechanism of atrial fibrillation. Further studies on the functional association between I(Ks) and KCNE4 (145D) polymorphism in cardiac myocytes are suggested.
Assuntos
Canal de Potássio KCNQ1/fisiologia , Polimorfismo de Nucleotídeo Único , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/análise , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologiaRESUMO
Potassium channels play a critical role in defining the electrophysiological properties accounting for the unique response patterns of auditory neurons. Serial analysis of gene expression (SAGE), microarrays, RT-PCR, and real-time RT-PCR were used to generate a broad profile of potassium channel expression in the rat cochlear nucleus. This study identified mRNAs for 51 different potassium channel subunits or channel interacting proteins. The relative expression levels of 27 of these transcripts among the AVCN, PVCN, and DCN were determined by real-time RT-PCR. Four potassium channel transcripts showed substantial levels of differential expression. Kcnc2 was expressed more than 15-fold higher in the DCN as compared to AVCN and PVCN. In contrast, Kcnj13 had an approximate 10-fold higher expression in AVCN and PVCN than in DCN. Two subunits that modify the activity of other channels were inversely expressed between ventral and dorsal divisions. Kcns1 was over 15-fold higher in DCN than AVCN or PVCN, while Kcns3 was about 25-fold higher in AVCN than in DCN. The expression patterns of potassium channels in the subdivisions of the cochlear nucleus provide a basis for understanding the electrophysiological mechanisms which sub-serve central auditory processing and provide targets for further investigations into neural plastic changes that occur with hearing loss.