RESUMO
ABSTRACT: Many factors that may co-occur with craniosynostosis, such as oral structural anomalies, hearing impairment, visual impairment, cognitive difficulties and psychosocial factors, may predispose this population to communication difficulties. At the Oxford Craniofacial Unit, children's speech, language and communication are regularly monitored in accordance with a systematic developmental screening protocol developed by the Speech and Language Therapists in the 4 United Kingdom (UK) Highly Specialized Craniofacial Centers. In addition to routine assessments, when parents attend routine multidisciplinary clinic appointments, they are asked about their child's communication development, and whether they have any concerns.A retrospective review was undertaken of parental concerns about hearing, speech development, behavior, physical development, concentration, school and friendships as indicated by parents on the Oxford Craniofacial Unit Pre-Clinic Questionnaire. The areas of concern were then correlated with the results of a standardized, guided parent questionnaire about children's language development, (Children's Communication Checklist - 2 (CCC-2)), to determine whether parental concern alone is a reliable way of identifying whether patients require further assessment for Language Disorder associated with Craniosynostosis.Participants were parents of 89 monolingual English-speaking children with craniosynostosis (62 male; 27 female), age range four to 13 years (mean ageâ=â8 years 7 months), receiving active care at the Oxford Craniofacial Unit (June 2017-July 2018). Results of the pre-clinic questionnaire indicated that 6% of parents had concerns about their child's communication development. Results of the CCC-2 indicated that 29/89 (32.6%) of children required further assessment for Language Disorder associated with Craniosynostosis. When language difficulties were identified on the CCC-2, only 14% (nâ=â4/29) parents indicated concern on the pre clinic questionnaire. Results indicated that parental concern about behavior was the most important factor in identifying language disorder (Pâ=â0.023).Results reinforce that the pre-clinic questionnaire is useful for identifying areas of parental concern. Results also indicate that parental concern alone is not sufficient to identify language disorder, and that further, detailed assessment is warranted. The results are consistent with previously reported links between behavior and language in the general population.
Assuntos
Craniossinostoses , Transtornos da Linguagem , Criança , Feminino , Humanos , Lactente , Masculino , Pais , Estudos Retrospectivos , Reino UnidoRESUMO
The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel, H(V)1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes, histamine secretion by basophils, sperm capacitation, and airway pH. The most selective ion channel known, H(V)1 shows no detectable permeability to other ions. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions. Here we identify aspartate 112 as a crucial component of the selectivity filter of H(V)1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H(V)1 requires an acidic group at the selectivity filter.
Assuntos
Ácido Aspártico/metabolismo , Ativação do Canal Iônico/genética , Canais Iônicos/química , Canais Iônicos/metabolismo , Prótons , Ácido Aspártico/genética , Condutividade Elétrica , Histidina/genética , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/genética , Soluções Isotônicas/farmacologia , Lisina/genética , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação/genética , Fases de Leitura Aberta/genética , Concentração Osmolar , Permeabilidade/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos , Sacarose/farmacologiaRESUMO
Insulin resistance, hyperinsulinemia, and hyperproinsulinemia occur early in the pathogenesis of type 2 diabetes (T2D). Elevated levels of proinsulin and proinsulin intermediates are markers of ß-cell dysfunction and are strongly associated with development of T2D in humans. However, the mechanism(s) underlying ß-cell dysfunction leading to hyperproinsulinemia is poorly understood. Here, we show that disruption of insulin receptor (IR) expression in ß cells has a direct impact on the expression of the convertase enzyme carboxypeptidase E (CPE) by inhibition of the eukaryotic translation initiation factor 4 gamma 1 translation initiation complex scaffolding protein that is mediated by the key transcription factors pancreatic and duodenal homeobox 1 and sterol regulatory element-binding protein 1, together leading to poor proinsulin processing. Reexpression of IR or restoring CPE expression each independently reverses the phenotype. Our results reveal the identity of key players that establish a previously unknown link between insulin signaling, translation initiation, and proinsulin processing, and provide previously unidentified mechanistic insight into the development of hyperproinsulinemia in insulin-resistant states.
Assuntos
Carboxipeptidase H/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Células Secretoras de Insulina/fisiologia , Insulina/metabolismo , Animais , Carboxipeptidase H/genética , Células Cultivadas , Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/fisiologia , Fator de Iniciação Eucariótico 4G/genética , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biossíntese de Proteínas/fisiologia , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais/fisiologia , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Transativadores/metabolismoRESUMO
Glucagon-like peptide-1 (GLP-1)-based incretin therapy is becoming central to the treatment of type 2 diabetes. Activation of incretin hormone receptors results in rapid elevation of cAMP followed by enhanced insulin secretion. However, the incretin effect may be significantly impaired in diabetes. The objective of this study is to investigate downregulation of GLP-1 signaling by small ubiquitin-related modifier protein (SUMO). Mouse islets exposed to high glucose showed increased expression of endogenous SUMO transcripts and its conjugating enzyme Ubc-9. Overexpression of SUMO-1 in mouse insulinoma 6 (MIN6) cells and primary mouse ß-cells resulted in reduced static and real-time estimates of intracellular cAMP upon receptor stimulation with exendin-4, a GLP-1 receptor (GLP-1R) agonist. GLP1-R was covalently modified by SUMO. Overexpression of SUMO-1 attenuated cell surface trafficking of GLP-1R, which resulted in significantly reduced insulin secretion when stimulated by exendin-4. Partial knock down of SUMO-conjugating enzyme Ubc-9 resulted in enhanced exendin-4-stimulated insulin secretion in mouse islets exposed to high glucose. Thus, SUMO modification of the GLP-1R could be a contributing factor to reduced incretin responsiveness. Elucidating mechanisms of GLP-1R regulation by sumoylation will help improve our understanding of incretin biology and of GLP-1-based treatment of type 2 diabetes.
Assuntos
GMP Cíclico/biossíntese , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/fisiologia , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Animais , Biotinilação/fisiologia , Células Cultivadas , DNA Complementar/genética , Regulação para Baixo/fisiologia , Ensaio de Imunoadsorção Enzimática , Transferência Ressonante de Energia de Fluorescência , Imunofluorescência , Glucose/farmacologia , Humanos , Imunoprecipitação , Incretinas/farmacologia , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase em Tempo Real , Estimulação Química , Transfecção , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
Voltage-gated proton channels are strongly inhibited by Zn(2+), which binds to His residues. However, in a molecular model, the two externally accessible His are too far apart to coordinate Zn(2+). We hypothesize that high-affinity Zn(2+) binding occurs at the dimer interface between pairs of His residues from both monomers. Consistent with this idea, Zn(2+) effects were weaker in monomeric channels. Mutation of His(193) and His(140) in various combinations and in tandem dimers revealed that channel opening was slowed by Zn(2+) only when at least one His was present in each monomer, suggesting that in wild-type (WT) H(V)1, Zn(2+) binding between His of both monomers inhibits channel opening. In addition, monomeric channels opened exponentially, and dimeric channels opened sigmoidally. Monomeric channel gating had weaker temperature dependence than dimeric channels. Finally, monomeric channels opened 6.6 times faster than dimeric channels. Together, these observations suggest that in the proton channel dimer, the two monomers are closely apposed and interact during a cooperative gating process. Zn(2+) appears to slow opening by preventing movement of the monomers relative to each other that is prerequisite to opening. These data also suggest that the association of the monomers is tenuous and allows substantial freedom of movement. The data support the idea that native proton channels are dimeric. Finally, the idea that monomer-dimer interconversion occurs during activation of phagocytes appears to be ruled out.
Assuntos
Ativação do Canal Iônico/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Zinco/farmacologia , Linhagem Celular , Dimerização , Eletrofisiologia , Proteínas de Fluorescência Verde/metabolismo , Histidina/química , Humanos , Concentração de Íons de Hidrogênio , Ativação do Canal Iônico/genética , Canais Iônicos/química , Canais Iônicos/genética , Cinética , Modelos Moleculares , Mutação/fisiologia , Técnicas de Patch-Clamp , Temperatura , TransfecçãoRESUMO
Permanent neonatal diabetes mellitus is a rare form of insulin-requiring diabetes presenting within the first few weeks or months of life. Mutations in the insulin gene are the second most common cause of this form of diabetes. These mutations are located in critical regions of preproinsulin and are likely to prevent normal processing or folding of the preproinsulin/proinsulin molecule. To characterize these mutations, we transiently expressed proinsulin-GFP fusion proteins in MIN6 mouse insulinoma cells. Our study revealed three groups of mutant proteins: 1) mutations that result in retention of proinsulin in the endoplasmic reticulum (ER) and attenuation of secretion of cotransfected wild-type insulin: C43G, F48C, and C96Y; 2) mutations with partial ER retention, partial recruitment to granules, and attenuation of secretion of wild-type insulin: G32R, G32S, G47V, G90C, and Y108C; and 3) similar to (2) but with no significant attenuation of wild-type insulin secretion: A24D and R89C. The mutant insulin proteins do not prevent targeting of wild-type insulin to secretory granules, but most appear to lead to decreased secretion of wild-type insulin. Each of the mutants triggers the expression of the proapoptotic gene Chop, indicating the presence of ER stress.
Assuntos
Diabetes Mellitus/congênito , Diabetes Mellitus/genética , Resistência à Insulina/genética , Insulina/metabolismo , Mutação , Proinsulina/genética , Animais , Animais Recém-Nascidos , Doença Crônica , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Progressão da Doença , Glucose/metabolismo , Humanos , Recém-Nascido , Insulina/genética , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Erros Inatos do Metabolismo/metabolismo , Camundongos , Modelos Biológicos , Proinsulina/metabolismoRESUMO
I(Ks) voltage-gated K(+) channels contain four pore-forming KCNQ1 subunits and MinK accessory subunits in a number that has been controversial. Here, I(Ks) channels assembled naturally by monomer subunits are compared to those with linked subunits that force defined stoichiometries. Two strategies that exploit charybdotoxin (CTX)-sensitive subunit variants are applied. First, CTX on rate, off rate, and equilibrium affinity are found to be the same for channels of monomers and those with a fixed 2:4 MinK:KCNQ1 valence. Second, 3H-CTX and an antibody are used to directly quantify channels and MinK subunits, respectively, showing 1.97 +/- 0.07 MinK per I(Ks) channel. Additional MinK subunits do not enter channels of monomeric subunits or those with fixed 2:4 valence. We conclude that two MinK subunits are necessary, sufficient, and the norm in I(Ks) channels. This stoichiometry is expected for other K(+) channels that contain MinK or MinK-related peptides (MiRPs).
Assuntos
Charibdotoxina/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio/química , Canais de Potássio/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Relação Dose-Resposta a Droga , Feminino , Humanos , Dados de Sequência Molecular , Mutação , Canais de Potássio/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Ligação Proteica/fisiologia , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Homologia de Sequência de Aminoácidos , XenopusRESUMO
MVP, a Methanococcus jannaschii voltage-gated potassium channel, was cloned and shown to operate in eukaryotic and prokaryotic cells. Like pacemaker channels, MVP opens on hyperpolarization using S4 voltage sensors like those in classical channels activated by depolarization. The MVP S4 span resembles classical sensors in sequence, charge, topology and movement, traveling inward on hyperpolarization and outward on depolarization (via canaliculi in the protein that bring the extracellular and internal solutions into proximity across a short barrier). Thus, MVP opens with sensors inward indicating a reversal of S4 position and pore state compared to classical channels. Homologous channels in mammals and plants are expected to function similarly.
Assuntos
Escherichia coli/metabolismo , Células Eucarióticas/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/isolamento & purificação , Células Procarióticas/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos/genética , Archaea/genética , Archaea/metabolismo , Sequência de Bases/genética , Clonagem Molecular , Cisteína/metabolismo , DNA Complementar/análise , DNA Complementar/genética , Evolução Molecular , Potenciais da Membrana/fisiologia , Mesilatos/farmacologia , Mathanococcus/metabolismo , Dados de Sequência Molecular , Potássio/metabolismo , Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Estrutura Terciária de Proteína/fisiologiaRESUMO
A decade since their discovery, the K2P channels are recognized as pathways dedicated to regulated background leakage of potassium ions that serve to control neuronal excitability. The recent identification of protein partners that directly interact with K2P channels (SUMO, 14-3-3 and Vpu1) has exposed new regulatory pathways. Reversible linkage to SUMO silences K2P1 plasma membrane channels; phosphorylation of K2P3 enables 14-3-3 binding to affect forward trafficking, whereas it decreases open probability of K2P2; and, Vpu1, an HIV encoded partner, mediates assembly-dependent degradation of K2P3. An operational strategy has emerged: tonic inhibition of K2P channels allows baseline neuronal activity until enhanced potassium leak is required to suppress excitability.
Assuntos
Proteínas de Membrana/fisiologia , Canais de Potássio/fisiologia , Animais , Potenciais da Membrana/fisiologia , Neurônios/fisiologiaRESUMO
In glioblastoma, invasion and proliferation are presumed to be mutually exclusive events; however, the molecular mechanisms that mediate this switch at the cellular level remain elusive. Previously, we have shown that phospho-OLIG2, a central-nervous-system-specific transcription factor, is essential for tumor growth and proliferation. Here, we show that the modulation of OLIG2 phosphorylation can trigger a switch between proliferation and invasion. Glioma cells with unphosphorylated OLIG2(S10, S13, S14) are highly migratory and invasive, both in vitro and in vivo. Mechanistically, unphosphorylated OLIG2 induces TGF-ß2 expression and promotes invasive mesenchymal properties in glioma cells. Inhibition of the TGF-ß2 pathway blocks this OLIG2-dependent invasion. Furthermore, ectopic expression of phosphomimetic Olig2 is sufficient to block TGF-ß2-mediated invasion and reduce expression of invasion genes (ZEB1 and CD44). Our results not only provide a mechanistic insight into how cells switch from proliferation to invasion but also offer therapeutic opportunities for inhibiting dissemination of gliomas.
Assuntos
Glioblastoma/genética , Glioblastoma/patologia , Invasividade Neoplásica/genética , Fator de Transcrição 2 de Oligodendrócitos/genética , Processamento de Proteína Pós-Traducional/genética , Fator de Crescimento Transformador beta/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Humanos , Receptores de Hialuronatos/genética , Camundongos , Camundongos Nus , Invasividade Neoplásica/patologia , Fosforilação/genética , Transdução de Sinais/genética , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genéticaRESUMO
OBJECTIVE: Glucagon-like peptide 1 (GLP-1) enhances insulin secretion and protects ß-cell mass. Diabetes therapies targeting the GLP-1 receptor (GLP-1R), expressed in numerous tissues, have diminished dose-response in patients with type 2 diabetes compared with healthy human controls. The aim of this study was to determine the mechanistic causes underlying the reduced efficacy of GLP-1R ligands. METHODS: Using primary mouse islets and the ß-cell line MIN6, outcomes downstream of the GLP-1R were analyzed: Insulin secretion; phosphorylation of the cAMP-response element binding protein (CREB); cAMP responses. Signaling systems were studied by immunoblotting and qRT-PCR, and PKA activity was assayed. Cell surface localization of the GLP-1R was studied by confocal microscopy using a fluorescein-tagged exendin-4 and GFP-tagged GLP-1R. RESULTS: Rodent ß-cells chronically exposed to high glucose had diminished responses to GLP-1R agonists including: diminished insulin secretory response; reduced phosphorylation of (CREB); impaired cAMP response, attributable to chronically increased cAMP levels. GLP-1R signaling systems were affected by hyperglycemia with increased expression of mRNAs encoding the inducible cAMP early repressor (ICER) and adenylyl cyclase 8, reduced PKA activity due to increased expression of the PKA-RIα subunit, reduced GLP-1R mRNA expression and loss of GLP-1R from the cell surface. To specifically examine the loss of GLP-1R from the plasma membrane a GLP-1R-GFP fusion protein was employed to visualize subcellular localization. Under low glucose conditions or when PKA activity was inhibited, GLP-1R-GFP was found at the plasma membrane. Conversely high glucose, expression of a constitutively active PKA subunit, or exposure to exendin-4 or forskolin led to GLP-1R-GFP internalization. Mutation of serine residue 301 of the GLP-1R abolished the glucose-dependent loss of the receptor from the plasma membrane. This was associated with a loss of an interaction between the receptor and the small ubiquitin-related modifier (SUMO), an interaction that was found to be necessary for internalization of the receptor. CONCLUSIONS: These data show that glucose acting, at least in part, via PKA leads to the loss of the GLP-1R from the cell surface and an impairment of GLP-1R signaling, which may underlie the reduced clinical efficacy of GLP-1R based therapies in individuals with poorly controlled hyperglycemia.
RESUMO
Recently, the gene coding for the tandem pore domain K(+)-channel TASK-3 (KCNK9) has been localized to the chromosomal region 8q24. Because mutations in ion channel genes have been recognized as an important factor in the etiology of abnormal neuronal excitability, TASK-3 is an interesting candidate gene for epilepsies linked to 8q24. We therefore performed a mutation analysis of the TASK-3 gene in 65 patients with childhood and juvenile absence epilepsy. Only one silent nucleotide exchange (636C/T) was detected in exon 2 of the TASK-3 coding region. No evidence for an allelic association was found between the exon 2 polymorphism and absence epilepsy. Accordingly, genetic variation of the TASK-3 coding region does not play a major role in the etiology of idiopathic absence epilepsies.
Assuntos
Epilepsia Tipo Ausência/genética , Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Adolescente , Criança , DNA/química , DNA/genética , Análise Mutacional de DNA , Humanos , Polimorfismo de Nucleotídeo Único , Polimorfismo Conformacional de Fita SimplesRESUMO
AIMS/INTRODUCTION: The human insulin gene/preproinsulin protein mutation C43G disrupts disulfide bond formation and causes diabetes in humans. Previous in vitro studies showed that these mutant proteins are retained in the endoplasmic reticulum (ER), are not secreted and are associated with decreased secretion of wild-type insulin. The current study extends this work to an in vivo zebrafish model. We hypothesized that C43G-green fluorescent protein (GFP) would be retained in the ER, disrupt ß-cell function and lead to impaired glucose homeostasis. MATERIALS AND METHODS: Islets from adult transgenic zebrafish expressing GFP-tagged human proinsulin mutant C43G (C43G-GFP) or wild-type human proinsulin (Cpep-GFP) were analyzed histologically across a range of ages. Blood glucose concentration was determined under fasting conditions and in response to glucose injection. Insulin secretion was assessed by measuring circulating GFP and endogenous C-peptide levels after glucose injection. RESULTS: The majority of ß-cells expressing C43G proinsulin showed excessive accumulation of C43G-GFP in the ER. Western blotting showed that C43G-GFP was present only as proinsulin, indicating defective processing. GFP was poorly secreted in C43G mutants compared with controls. Despite these defects, blood glucose homeostasis was normal. Mutant fish maintained ß-cell mass well into maturity and secreted endogenous C-peptide. CONCLUSIONS: In this model, the C43G proinsulin mutation does not impair glucose homeostasis or cause significant loss of ß-cell mass. This model might be useful for identifying potential therapeutic targets for proper trafficking of intracellular insulin or for maintenance of ß-cell mass in early-stage diabetic patients.
RESUMO
Extraordinary selectivity is crucial to all proton-conducting molecules, including the human voltage-gated proton channel (hHV1), because the proton concentration is >10(6) times lower than that of other cations. Here we use "selectivity filter scanning" to elucidate the molecular requirements for proton-specific conduction in hHV1. Asp(112), in the middle of the S1 transmembrane helix, is an essential part of the selectivity filter in wild-type (WT) channels. After neutralizing Asp(112) by mutating it to Ala (D112A), we introduced Asp at each position along S1 from 108 to 118, searching for "second site suppressor" activity. Surprisingly, most mutants lacked even the anion conduction exhibited by D112A. Proton-specific conduction was restored only with Asp or Glu at position 116. The D112V/V116D channel strikingly resembled WT in selectivity, kinetics, and ΔpH-dependent gating. The S4 segment of this mutant has similar accessibility to WT in open channels, because R211H/D112V/V116D was inhibited by internally applied Zn(2+). Asp at position 109 allowed anion permeation in combination with D112A but did not rescue function in the nonconducting D112V mutant, indicating that selectivity is established externally to the constriction at F150. The three positions that permitted conduction all line the pore in our homology model, clearly delineating the conduction pathway. Evidently, a carboxyl group must face the pore directly to enable conduction. Molecular dynamics simulations indicate reorganization of hydrogen bond networks in the external vestibule in D112V/V116D. At both positions where it produces proton selectivity, Asp frequently engages in salt linkage with one or more Arg residues from S4. Surprisingly, mean hydration profiles were similar in proton-selective, anion-permeable, and nonconducting constructs. That the selectivity filter functions in a new location helps to define local environmental features required to produce proton-selective conduction.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/química , Simulação de Dinâmica Molecular , Prótons , Sequência de Aminoácidos , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/genética , Canais Iônicos/metabolismo , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Zinco/farmacologiaRESUMO
The voltage-gated proton channel exists as a dimer, although each protomer has a separate conduction pathway, and when forced to exist as a monomer, most major functions are retained. However, the proton channel protomers appear to interact during gating. Proton channel dimerization is thought to result mainly from coiled-coil interaction of the intracellular C-termini. Several types of evidence are discussed that suggest that the dimer conformation may not be static, but is dynamic and can sample different orientations. Zn(2+) appears to link the protomers in an orientation from which the channel(s) cannot open. A tandem WT-WT dimer exhibits signs of cooperative gating, indicating that despite the abnormal linkage, the correct orientation for opening can occur. We propose that C-terminal interaction functions mainly to tether the protomers together. Comparison of the properties of monomeric and dimeric proton channels speaks against the hypothesis that enhanced gating reflects monomer-dimer interconversion.
Assuntos
Ativação do Canal Iônico , Canais Iônicos/metabolismo , Basófilos/metabolismo , Humanos , Canais Iônicos/química , Canais Iônicos/genética , Cinética , Potenciais da Membrana , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Subunidades Proteicas , Prótons , Relação Estrutura-Atividade , Zinco/metabolismoRESUMO
Reversible, covalent modification with small ubiquitin-related modifier proteins (SUMOs) is known to mediate nuclear import/export and activity of transcription factors. Here, the SUMO pathway is shown to operate at the plasma membrane to control ion channel function. SUMO-conjugating enzyme is seen to be resident in plasma membrane, to assemble with K2P1, and to modify K2P1 lysine 274. K2P1 had not previously shown function despite mRNA expression in heart, brain, and kidney and sequence features like other two-P loop K+ leak (K2P) pores that control activity of excitable cells. Removal of the peptide adduct by SUMO protease reveals K2P1 to be a K+-selective, pH-sensitive, openly rectifying channel regulated by reversible peptide linkage.
Assuntos
Membrana Celular/metabolismo , Ativação do Canal Iônico/fisiologia , Canais de Potássio de Domínios Poros em Tandem/fisiologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Células COS , Membrana Celular/fisiologia , Chlorocebus aethiops , Cisteína Endopeptidases , Eletrofisiologia , Endopeptidases/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Mutação/genética , Oócitos/metabolismo , Oócitos/fisiologia , Peptídeos/metabolismo , Ligação Proteica , Técnicas do Sistema de Duplo-Híbrido , Proteínas de Xenopus/metabolismo , Xenopus laevisRESUMO
Andersen's syndrome, an autosomal dominant disorder related to mutations of the potassium channel Kir2.1, is characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic bone structure. The aim of our study was to find out whether heteromerization of Kir2.1 channels with wild-type Kir2.2 and Kir2.3 channels contributes to the phenotype of Andersen's syndrome. The following results show that Kir2.x channels can form functional heteromers: (i) HEK293 cells transfected with Kir2.x-Kir2.y concatemers expressed inwardly rectifying K(+) channels with a conductance of 28-30 pS. (ii) Expression of Kir2.x-Kir2.y concatemers in Xenopus oocytes produced inwardly rectifying, Ba(2+) sensitive currents. (iii) When Kir2.1 and Kir2.2 channels were coexpressed in Xenopus oocytes the IC(50) for Ba(2+) block of the inward rectifier current differed substantially from the value expected for independent expression of homomeric channels. (iv) Coexpression of nonfunctional Kir2.x constructs, in which the GYG region of the pore region was replaced by AAA, with wild-type Kir2.x channels produced both homomeric and heteromeric dominant-negative effects. (v) Kir2.1 and Kir2.3 channels could be coimmunoprecipitated in membrane extracts from isolated guinea pig cardiomyocytes. (vi) Yeast two-hybrid analysis showed interaction between the N- and C-terminal intracellular domains of different Kir2.x subunits. Coexpression of Kir2.1 mutants related to Andersen's syndrome with wild-type Kir2.x channels showed a dominant negative effect, the extent of which varied between different mutants. Our results suggest that differential tetramerization of the mutant allele of Kir2.1 with wild-type Kir2.1, Kir2.2, and Kir2.3 channels represents the molecular basis of the extraordinary pleiotropy of Andersen's syndrome.
Assuntos
Síndrome do QT Longo/genética , Paralisias Periódicas Familiares/genética , Canais de Potássio Corretores do Fluxo de Internalização/genética , Substituição de Aminoácidos , Animais , Linhagem Celular , Cobaias , Humanos , Modelos Moleculares , Mutagênese Insercional , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/química , Conformação Proteica , Subunidades Proteicas , TransfecçãoRESUMO
The two-pore-domain potassium channels TASK-1, TASK-3 and TASK-5 possess a conserved C-terminal motif of five amino acids. Truncation of the C-terminus of TASK-1 strongly reduced the currents measured after heterologous expression in Xenopus oocytes or HEK293 cells and decreased surface membrane expression of GFP-tagged channel proteins. Two-hybrid analysis showed that the C-terminal domain of TASK-1, TASK-3 and TASK-5, but not TASK-4, interacts with isoforms of the adapter protein 14-3-3. A pentapeptide motif at the extreme C-terminus of TASK-1, RRx(S/T)x, was found to be sufficient for weak but significant interaction with 14-3-3, whereas the last 40 amino acids of TASK-1 were required for strong binding. Deletion of a single amino acid at the C-terminal end of TASK-1 or TASK-3 abolished binding of 14-3-3 and strongly reduced the macroscopic currents observed in Xenopus oocytes. TASK-1 mutants that failed to interact with 14-3-3 isoforms (V411*, S410A, S410D) also produced only very weak macroscopic currents. In contrast, the mutant TASK-1 S409A, which interacts with 14-3-3-like wild-type channels, displayed normal macroscopic currents. Co-injection of 14-3-3zeta cRNA increased TASK-1 current in Xenopus oocytes by about 70 %. After co-transfection in HEK293 cells, TASK-1 and 14-3-3zeta (but not TASK-1DeltaC5 and 14-3-3zeta) could be co-immunoprecipitated. Furthermore, TASK-1 and 14-3-3 could be co-immunoprecipitated in synaptic membrane extracts and postsynaptic density membranes. Our findings suggest that interaction of 14-3-3 with TASK-1 or TASK-3 may promote the trafficking of the channels to the surface membrane.