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1.
Genet Med ; 2018 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-30449888

RESUMO

PURPOSE: Precision health initiatives and reduced sequencing costs are driving large-scale human genome analyses. Genetic variant curation is a bottleneck in clinical applications. The burden of variant curation can be high for newly discovered variants because they are less likely to have undergone previous clinical annotation; the rate of discovery of genetic variants in large clinical populations has not been empirically determined. METHODS: We determined the rate of accrual of unique sequence variants in 90,000 exome sequences. Separate analyses were done for 17,267 autosomal genes and a subset of 74 actionable genes; the effect of relatedness in the cohort was also determined. RESULTS: Variant discovery showed a nonlinear growth pattern. The rate of unique variant accrual decreased as the database size increased; by 90,000 exomes 97% of all projected coding and splicing variants had been observed. Variants in 74 actionable genes showed a similar pattern. Family relatedness slightly reduced the rate of discovery of unique variants. CONCLUSION: The heaviest burden of interpretation for genetic variants occurs early and diminishes as the database size increases. Our data provide a framework for scaling pathogenic genetic variant discovery and curation, a critical element of patient care in the era of precision health.

2.
Nat Commun ; 9(1): 2252, 2018 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-29899519

RESUMO

Angiopoietin-like 4 (ANGPTL4) is an endogenous inhibitor of lipoprotein lipase that modulates lipid levels, coronary atherosclerosis risk, and nutrient partitioning. We hypothesize that loss of ANGPTL4 function might improve glucose homeostasis and decrease risk of type 2 diabetes (T2D). We investigate protein-altering variants in ANGPTL4 among 58,124 participants in the DiscovEHR human genetics study, with follow-up studies in 82,766 T2D cases and 498,761 controls. Carriers of p.E40K, a variant that abolishes ANGPTL4 ability to inhibit lipoprotein lipase, have lower odds of T2D (odds ratio 0.89, 95% confidence interval 0.85-0.92, p = 6.3 × 10-10), lower fasting glucose, and greater insulin sensitivity. Predicted loss-of-function variants are associated with lower odds of T2D among 32,015 cases and 84,006 controls (odds ratio 0.71, 95% confidence interval 0.49-0.99, p = 0.041). Functional studies in Angptl4-deficient mice confirm improved insulin sensitivity and glucose homeostasis. In conclusion, genetic inactivation of ANGPTL4 is associated with improved glucose homeostasis and reduced risk of T2D.

3.
Circ Arrhythm Electrophysiol ; 11(5): e005859, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29752375

RESUMO

BACKGROUND: Heterologous functional validation studies of putative long-QT syndrome subtype 2-associated variants clarify their pathological potential and identify disease mechanism(s) for most variants studied. The purpose of this study is to clarify the pathological potential for rare nonsynonymous KCNH2 variants seemingly associated with sudden infant death syndrome. METHODS: Genetic testing of 292 sudden infant death syndrome cases identified 9 KCNH2 variants: E90K, R181Q, A190T, G294V, R791W, P967L, R1005W, R1047L, and Q1068R. Previous studies show R181Q-, P967L-, and R1047L-Kv11.1 channels function similar to wild-type Kv11.1 channels, whereas Q1068R-Kv11.1 channels accelerate inactivation gating. We studied the biochemical and biophysical properties for E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels expressed in human embryonic kidney 293 cells; examined the electronic health records of patients who were genotype positive for the sudden infant death syndrome-linked KCNH2 variants; and simulated their functional impact using computational models of the human ventricular action potential. RESULTS: Western blot and voltage-clamping analyses of cells expressing E90K-, G294V-, R791W-, and R1005W-Kv11.1 channels demonstrated these variants express and generate peak Kv11.1 current levels similar to cells expressing wild-type-Kv11.1 channels, but R791W- and R1005W-Kv11.1 channels accelerated deactivation and activation gating, respectively. Electronic health records of patients with the sudden infant death syndrome-linked KCNH2 variants showed that the patients had median heart rate-corrected QT intervals <480 ms and none had been diagnosed with long-QT syndrome or experienced cardiac arrest. Simulating the impact of dysfunctional gating variants predicted that they have little impact on ventricular action potential duration. CONCLUSIONS: We conclude that these rare Kv11.1 missense variants are not long-QT syndrome subtype 2-causative variants and therefore do not represent the pathogenic substrate for sudden infant death syndrome in the variant-positive infants.

4.
N Engl J Med ; 378(12): 1096-1106, 2018 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-29562163

RESUMO

BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10-12) and AST (P=6.2×10-10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).


Assuntos
17-Hidroxiesteroide Desidrogenases/genética , Fígado Gorduroso/genética , Predisposição Genética para Doença , Hepatopatias/genética , Mutação com Perda de Função , 17-Hidroxiesteroide Desidrogenases/metabolismo , Alanina Transaminase/sangue , Aspartato Aminotransferases/sangue , Biomarcadores/sangue , Doença Crônica , Progressão da Doença , Feminino , Variação Genética , Genótipo , Humanos , Modelos Lineares , Fígado/patologia , Hepatopatias/patologia , Masculino , Análise de Sequência de RNA , Sequenciamento Completo do Exoma
5.
BMC Genomics ; 17 Suppl 2: 445, 2016 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-27358062

RESUMO

BACKGROUND: Alzheimer's disease (AD) represents the most common form of dementia in elder populations with approximately 30 million cases worldwide. Genome wide genotyping and sequencing studies have identified many genetic variants associated with late-onset Alzheimer's disease (LOAD). While most of these variants are associated with increased risk of developing LOAD, only limited number of reports focused on variants that are protective against the disease. METHODS: Here we applied a novel approach to uncover protective alleles against AD by analyzing genetic and phenotypic data in Mount Sinai Biobank and Electronic Medical Record (EMR) databases. RESULTS: We discovered a likely loss-of-function small deletion variant in the caspase 7 (CASP7) gene associated with significantly reduced incidence of LOAD in carriers of the high-risk APOE ε4 allele. Further investigation of four independent cohorts of European ancestry revealed the protective effect of the CASP7 variant against AD is most significant in homozygous APOE ε4 allele carriers. Meta analysis of multiple datasets shows overall odds ratio = 0.45 (p = 0.004). Analysis of RNA sequencing derived gene expression data indicated the variant correlates with reduced caspase 7 expression in multiple brain tissues we examined. CONCLUSIONS: Taken together, these results are consistent with the notion that caspase 7 plays a key role in microglial activation driving neuro-degeneration during AD pathogenesis, and may explain the underlying genetic mechanisms that anti-inflammatory interventions in AD show greater benefit in APOE ε4 carriers than non-carriers. Our findings inform potential novel therapeutic opportunities for AD and warrant further investigations.


Assuntos
Doença de Alzheimer/genética , Apolipoproteínas E/genética , Caspase 7/genética , Perfilação da Expressão Gênica/métodos , Análise de Sequência de RNA/métodos , Idade de Início , Alelos , Regulação para Baixo , Registros Eletrônicos de Saúde , Predisposição Genética para Doença , Homozigoto , Humanos , Razão de Chances , Deleção de Sequência
7.
PLoS One ; 9(4): e93629, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24705671

RESUMO

BACKGROUND: The melanocortin 4 receptor (MC4R) critically regulates feeding and satiety. Rare variants in MC4R are predominantly found in obese individuals. Though some rare variants in MC4R discovered in patients have defects in localization, ligand binding and signaling to cAMP, many have no recognized defects. SUBJECTS/METHODS: In our cohort of 1433 obese subjects that underwent Roux-en-Y Gastric Bypass (RYGB) surgery, we found fifteen variants of MC4R. We matched rare variant carriers to patients with the MC4R reference alleles for gender, age, starting BMI and T2D to determine the variant effect on weight-loss post-RYGB. In vitro, we determined expression of mutant receptors by ELISA and western blot, and cAMP production by microscopy. RESULTS: While carrying a rare MC4R allele is associated with obesity, carriers of rare variants exhibited comparable weight-loss after RYGB to non-carriers. However, subjects carrying three of these variants, V95I, I137T or L250Q, lost less weight after surgery. In vitro, the R305Q mutation caused a defect in cell surface expression while only the I137T and C326R mutations showed impaired cAMP signaling. Despite these apparent differences, there was no correlation between in vitro signaling and pre- or post-surgery clinical phenotype. CONCLUSIONS: These data suggest that subtle differences in receptor signaling conferred by rare MC4R variants combined with additional factors predispose carriers to obesity. In the absence of complete MC4R deficiency, these differences can be overcome by the powerful weight-reducing effects of bariatric surgery. In a complex disorder such as obesity, genetic variants that cause subtle defects that have cumulative effects can be overcome after appropriate clinical intervention.


Assuntos
Derivação Gástrica , Obesidade/genética , Obesidade/cirurgia , Receptor Tipo 4 de Melanocortina/genética , Perda de Peso/genética , Adolescente , Adulto , Idoso , Alelos , Peso Corporal/genética , Estudos de Casos e Controles , Feminino , Derivação Gástrica/estatística & dados numéricos , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/epidemiologia , Polimorfismo de Nucleotídeo Único , Período Pós-Operatório , Fatores de Tempo , Resultado do Tratamento , Adulto Jovem
8.
J Biol Chem ; 288(26): 18842-52, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23671286

RESUMO

Ionotropic glutamate receptor (iGluR) channels control synaptic activity. The crystallographic structure of GluA2, the prototypical iGluR, reveals a clamshell-like ligand-binding domain (LBD) that closes in the presence of glutamate to open a gate on the pore lining α-helix. How LBD closure leads to gate opening remains unclear. Here, we show that bending the pore helix at a highly conserved alanine residue (Ala-621) below the gate is responsible for channel opening. Substituting Ala-621 with the smaller more flexible glycine resulted in a basally active, nondesensitizing channel with ∼39-fold increase in glutamate potency without affecting surface expression or binding. On GluA2(A621G), the partial agonist kainate showed efficacy similar to a full agonist, and competitive antagonists CNQX and DNQX acted as a partial agonists. Met-629 in GluA2 sits above the gate and is critical in transmitting LBD closure to the gate. Substituting Met-629 with the flexible glycine resulted in reduced channel activity and glutamate potency. The pore regions in potassium channels are structurally similar to iGluRs. Whereas potassium channels typically use glycines as a hinge for gating, iGluRs use the less flexible alanine as a hinge at a similar position to maintain low basal activity allowing for ligand-mediated gating.


Assuntos
Alanina/química , Ativação do Canal Iônico , Receptores de AMPA/química , Animais , Membrana Celular/metabolismo , Glicina/química , Células HEK293 , Humanos , Ácido Caínico/química , Ligantes , Neurônios/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/química , Ligação Proteica , Estrutura Terciária de Proteína , Receptores de Glutamato/metabolismo , Proteínas Recombinantes/química , Xenopus laevis
9.
Gastroenterology ; 144(3): 580-590.e7, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23159449

RESUMO

BACKGROUND & AIMS: Roux-en-Y gastric bypass (RYGB) improves glucose homeostasis independently of changes in body weight by unknown mechanisms. Melanocortin-4 receptors (MC4R) have weight-independent effects on glucose homeostasis, via autonomic neurons, and also might contribute to weight loss after RYGB. We investigated whether MC4Rs mediate effects of RYGB, such as its weight-independent effects on glucose homeostasis, in mice and humans. METHODS: We studied C57BL/6 mice with diet-induced obesity, MC4R-deficient mice, and mice that re-express MC4R specifically in autonomic neurons after RYGB or sham surgeries. We also sequenced the MC4R locus in patients undergoing RYGB to investigate diabetes resolution in carriers of rare MC4R variants. RESULTS: MC4Rs in autonomic brainstem neurons (including the parasympathetic dorsal motor vagus) mediated improved glucose homeostasis independent of changes in body weight. In contrast, MC4Rs in cholinergic preganglionic motor neurons (sympathetic and parasympathetic) mediated RYGB-induced increased energy expenditure and weight loss. Increased energy expenditure after RYGB is the predominant mechanism of weight loss and confers resistance to weight gain from a high-fat diet, the effects of which are MC4R-dependent. MC4R-dependent effects of RYGB still occurred in mice with Mc4r haplosufficiency, and early stage diabetes resolved at a similar rate in patients with rare variants of MC4R and noncarriers. However, carriers of MC4R (I251L), a rare variant associated with increased weight loss after RYGB and increased basal activity in vitro, were more likely to have early and weight-independent resolution of diabetes than noncarriers, indicating a role for MC4Rs in the effects of RYGB. CONCLUSIONS: MC4Rs in autonomic neurons mediate beneficial effects of RYGB, including weight-independent improved glucose homeostasis, in mice and humans.


Assuntos
Glicemia/metabolismo , Derivação Gástrica , Homeostase , Neurônios Motores/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismo , Nervo Vago/metabolismo , Perda de Peso , Animais , Neurônios Colinérgicos/metabolismo , Dieta Hiperlipídica/efeitos adversos , Metabolismo Energético , Heterozigoto , Humanos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Receptor Tipo 4 de Melanocortina/genética
10.
J Biol Chem ; 287(10): 7121-33, 2012 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-22207761

RESUMO

The functions of different G-protein αßγ subunit combinations are traditionally ascribed to their various α components. However, the discovery of similarly diverse γ subtypes raises the possibility that they may also contribute to specificity. To test this possibility, we used a gene targeting approach to determine whether the closely related γ(3) and γ(7) subunits can perform functionally interchangeable roles in mice. In contrast to single knock-out mice that show normal survival, Gng3(-/-)Gng7(-/-) double knock-out mice display a progressive seizure disorder that dramatically reduces their median life span to only 75 days. Biochemical analyses reveal that the severe phenotype is not due to redundant roles for the two γ subunits in the same signaling pathway but rather is attributed to their unique actions in different signaling pathways. The results suggest that the γ(3) subunit is a component of a G(i/o) protein that is required for γ-aminobutyric acid, type B, receptor-regulated neuronal excitability, whereas the γ(7) subunit is a component of a G(olf) protein that is responsible for A(2A) adenosine or D(1) dopamine receptor-induced neuro-protective response. The development of this mouse model offers a novel experimental framework for exploring how signaling pathways integrate to produce normal brain function and how their combined dysfunction leads to spontaneous seizures and premature death. The results underscore the critical role of the γ subunit in this process.


Assuntos
Encéfalo/enzimologia , Epilepsia/enzimologia , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Transdução de Sinais , Animais , Encéfalo/patologia , Epilepsia/genética , Epilepsia/patologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/genética , Predisposição Genética para Doença , Camundongos , Camundongos Knockout , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptores Dopaminérgicos/genética , Receptores Dopaminérgicos/metabolismo , Receptores de GABA-B/genética , Receptores de GABA-B/metabolismo
11.
J Clin Endocrinol Metab ; 96(12): E2088-96, 2011 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21976721

RESUMO

CONTEXT: Factors that influence long-term weight loss after Roux-en Y gastric bypass (RYGB) surgeries are poorly defined. The melanocortin system plays an important role in regulating energy homeostasis, satiety, and glucose metabolism. Variations of the MC4R comprise the most prevalent monogenetic obesity disorder. OBJECTIVE: The objective of the study was to examine the role of MC4R variants and diabetic status in long-term weight loss after RYGB. PARTICIPANTS AND METHODS: In 1433 extremely obese patients who underwent RYGB, we sequenced for genetic variants of MC4R. We examined the MC4R genotype and its relationship with weight loss profile, and clinical phenotypes accumulated during a 48-month period before and after surgery. RESULTS: We found 80 subjects with rare and common variants of MC4R in the RYGB cohort. Among these, 26 and 36 patients carry the I251L and V103I variants, respectively. These common alleles are negatively associated with obesity. Remarkably, after the 12-month presurgery caloric restriction and RYGB, I251L allele carriers lost 9% more weight (∼9 kg) compared with the noncarriers, continued rapid weight loss longer, regained less weight, and had lower presurgery homeostatic model assessment for insulin resistance values. Normoglycemic, I251L allele carriers lost more weight compared with their diabetic and prediabetic counterparts and maintained their weight loss. Among noncarriers, normoglycemic individuals initially lost more weight compared with dysglycemics, but this difference was not maintained in the long term. CONCLUSIONS: Individuals carrying the I251L common allele are predisposed to better clinical outcome, reduced risk of type 2 diabetes, and better weight loss during diet and surgical interventions. Diabetic status has only a small, short-term effect on weight loss after RYGB.


Assuntos
Derivação Gástrica/métodos , Obesidade/genética , Receptor Tipo 4 de Melanocortina/genética , Perda de Peso/genética , Adulto , Alelos , Feminino , Seguimentos , Frequência do Gene , Genótipo , Homeostase/genética , Humanos , Masculino , Pessoa de Meia-Idade , Obesidade/metabolismo , Obesidade/cirurgia , Fenótipo , Estudos Prospectivos , Receptor Tipo 4 de Melanocortina/metabolismo
12.
Obesity (Silver Spring) ; 19(8): 1676-83, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21311511

RESUMO

Genome-wide association and linkage studies have identified multiple susceptibility loci for obesity. We hypothesized that such loci may affect weight loss outcomes following dietary or surgical weight loss interventions. A total of 1,001 white individuals with extreme obesity (BMI >35 kg/m(2)) who underwent a preoperative diet/behavioral weight loss intervention and Roux-en-Y gastric bypass surgery were genotyped for single-nucleotide polymorphisms (SNPs) in or near the fat mass and obesity-associated (FTO), insulin induced gene 2 (INSIG2), melanocortin 4 receptor (MC4R), and proprotein convertase subtilisin/kexin type 1 (PCSK1) obesity genes. Association analysis was performed using recessive and additive models with pre- and postoperative weight loss data. An increasing number of obesity SNP alleles or homozygous SNP genotypes was associated with increased BMI (P < 0.0006) and excess body weight (P < 0.0004). No association between the amounts of weight lost from a short-term dietary intervention and any individual obesity SNP or cumulative number of obesity SNP alleles or homozygous SNP genotypes was observed. Linear mixed regression analysis revealed significant differences in postoperative weight loss trajectories across groups with low, intermediate, and high numbers of obesity SNP alleles or numbers of homozygous SNP genotypes (P < 0.0001). Initial BMI interacted with genotype to influence weight loss with initial BMI <50 kg/m(2), with evidence of a dosage effect, which was not present in individuals with initial BMI ≥50 kg/m(2). Differences in metabolic rate, binge eating behavior, and other clinical parameters were not associated with genotype. These data suggest that response to a surgical weight loss intervention is influenced by genetic susceptibility and BMI.


Assuntos
Tecido Adiposo , Alelos , Derivação Gástrica , Genótipo , Obesidade/genética , Polimorfismo de Nucleotídeo Único , Perda de Peso/genética , Adulto , Idoso , Dioxigenase FTO Dependente de alfa-Cetoglutarato , Índice de Massa Corporal , Feminino , Homozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Proteínas de Membrana/genética , Pessoa de Meia-Idade , Neuropeptídeos/genética , Obesidade/cirurgia , Proteínas/genética , Receptor Tipo 4 de Melanocortina/genética , Análise de Regressão
13.
J Biol Chem ; 285(53): 41290-9, 2010 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-21044958

RESUMO

G protein-activated inwardly rectifying potassium (GIRK or Kir3) channels are directly gated by the ßγ subunits of G proteins and contribute to inhibitory neurotransmitter signaling pathways. Paradoxically, volatile anesthetics such as halothane inhibit these channels. We find that neuronal Kir3 currents are highly sensitive to inhibition by halothane. Given that Kir3 currents result from increased Gßγ available to the channels, we asked whether reducing available Gßγ to the channel would adversely affect halothane inhibition. Remarkably, scavenging Gßγ using the C-terminal domain of ß-adrenergic receptor kinase (cßARK) resulted in channel activation by halothane. Consistent with this effect, channel mutants that impair Gßγ activation were also activated by halothane. A single residue, phenylalanine 192, occupies the putative Gßγ gate of neuronal Kir3.2 channels. Mutation of Phe-192 at the gate to other residues rendered the channel non-responsive, either activated or inhibited by halothane. These data indicated that halothane predominantly interferes with Gßγ-mediated Kir3 currents, such as those functioning during inhibitory synaptic activity. Our report identifies the molecular correlate for anesthetic inhibition of Kir3 channels and highlights the significance of these effects in modulating neurotransmitter-mediated inhibitory signaling.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Anestésicos , Animais , Sítios de Ligação , Linhagem Celular , Halotano/química , Hipocampo/metabolismo , Humanos , Neurotransmissores/química , Oócitos/metabolismo , Técnicas de Patch-Clamp , Estrutura Terciária de Proteína , Xenopus , Quinases de Receptores Adrenérgicos beta/metabolismo
14.
Mol Pharmacol ; 73(4): 1185-94, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18202303

RESUMO

Kir2.0 channels play a significant role in setting the resting membrane potential, modulating action potential wave form, and buffering extracellular potassium. One member of this family, Kir2.3, is highly expressed in the heart and brain and is modulated by a variety of factors, including arachidonic acid (AA). Using two-electrode voltage clamp and inside-out patch clamp recordings from Xenopus laevis oocytes expressing Kir2.3 channels, we found that AA selectively activated Kir2.3 channels with an EC(50) of 0.59 muM and that this activation required phosphatidyl inositol 4,5-bisphosphate (PIP(2)). We found that AA activated Kir2.3 by enhancing channel-PIP(2) interactions as demonstrated by a shift in PIP(2) activation curve. EC(50) for channel activation by PIP(2) were 36 and 12 muM in the absence and presence of AA, respectively. A single point mutation on the channel C terminus that enhanced basal channel-PIP(2) interactions reduced the sensitivity of the channel to AA. Effects of AA are mediated through cytoplasmic sites on the channel by increasing the open probability, mainly due to more frequent bursts of opening in the presence of PIP(2). Therefore, enhanced interaction with PIP(2) is the molecular mechanism for Kir2.3 channel activation by AA.


Assuntos
Ácido Araquidônico/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo , Animais , Bovinos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Diglicerídeos/farmacologia , Humanos , Cinética , Camundongos , Oócitos/citologia , Oócitos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização/antagonistas & inibidores , Receptor Muscarínico M1/metabolismo , Soroalbumina Bovina , Fatores de Tempo , Xenopus laevis
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