Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 426
Filtrar
1.
Nat Commun ; 10(1): 2008, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043612

RESUMO

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/ultraestrutura , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/ultraestrutura , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Ativação do Canal Iônico/fisiologia , Técnicas de Transferência de Energia por Ressonância de Bioluminescência , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/isolamento & purificação , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/isolamento & purificação , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestrutura
2.
Hypertension ; 73(6): 1283-1290, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31006333

RESUMO

Peripheral 18-oxocortisol (18oxoF) level could contribute to the detection of aldosterone-producing adenoma (APA) in patients with primary aldosteronism. However, peripheral 18oxoF varies among such patients, which is a big drawback concerning its clinical application. We studied 48 cases of APA, 35 harboring KCNJ5 mutation, to clarify the significance of clinical and pathological parameters about peripheral 18oxoF. Peripheral 18oxoF concentration ranged widely from 0.50 to 183.13 ng/dL and correlated positively with intratumoral areas stained positively for steroidogenic enzymes ( P<0.0001). The peripheral 18oxoF level also correlated significantly with that of circulating aldosterone ( P<0.0001) but not with that of cortisol, a precursor of 18oxoF. However, a significant correlation was detected between peripheral 18oxoF and intratumoral glucocorticoids ( P<0.05). In addition, peripheral 18oxoF correlated positively with the number of hybrid cells double positive for 11ß-hydroxylase and aldosterone synthase ( P<0.0001). Comparing between the cases with and those without KCNJ5 mutation, the KCNJ5-mutated group demonstrated a significantly higher concentration of peripheral 18oxoF (28.4±5.6 versus 3.0±0.9 ng/dL, P<0.0001) and a larger intratumoral environment including the hybrid cells ( P<0.001), possibly representing a deviation from normal aldosterone biosynthesis. After multivariate analysis, KCNJ5 mutation status turned out to be the most associated factor involved in 18oxoF synthesis in APA ( P<0.0001). Results of our present study first revealed that enhanced 18oxoF synthesis in APA could come from a functional deviation of aldosterone biosynthesis from the normal zona glomerulosa and the utility of peripheral 18oxoF measurement could be influenced by the prevalence of KCNJ5 mutation in an APA.


Assuntos
Neoplasias do Córtex Suprarrenal/genética , Adenoma Adrenocortical/genética , Aldosterona/metabolismo , DNA de Neoplasias/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Hidrocortisona/análogos & derivados , Mutação/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma Adrenocortical/metabolismo , Análise Mutacional de DNA , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Humanos , Hidrocortisona/biossíntese , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
3.
PLoS One ; 14(4): e0215213, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30978208

RESUMO

Type 1 diabetic Akita mice develop severe cardiac parasympathetic dysfunction that we have previously demonstrated is due at least in part to an abnormality in the response of the end organ to parasympathetic stimulation. Specifically, we had shown that hypoinsulinemia in the diabetic heart results in attenuation of the G-protein coupled inward rectifying K channel (GIRK) which mediates the negative chronotropic response to parasympathetic stimulation due at least in part to decreased expression of the GIRK1 and GIRK4 subunits of the channel. We further demonstrated that the expression of GIRK1 and GIRK4 is under the control of the Sterol Regulatory element Binding Protein (SREBP-1), which is also decreased in response to hypoinsulinemia. Finally, given that hyperactivity of Glycogen Synthase Kinase (GSK)3ß, had been demonstrated in the diabetic heart, we demonstrated that treatment of Akita mice with Li+, an inhibitor of GSK3ß, increased parasympathetic responsiveness and SREBP-1 levels consistent with the conclusion that GSK3ß might regulate IKACh via an effect on SREBP-1. However, inhibitor studies were complicated by lack of specificity for GSK3ß. Here we generated an Akita mouse with cardiac specific inducible knockout of GSK3ß. Using this mouse, we demonstrate that attenuation of GSK3ß expression is associated with an increase in parasympathetic responsiveness measured as an increase in the heart rate response to atropine from 17.3 ± 3.5% (n = 8) prior to 41.2 ± 5.4% (n = 8, P = 0.017), an increase in the duration of carbamylcholine mediated bradycardia from 8.43 ± 1.60 min (n = 7) to 12.71 ± 2.26 min (n = 7, P = 0.028) and an increase in HRV as measured by an increase in the high frequency fraction from 40.78 ± 3.86% to 65.04 ± 5.64 (n = 10, P = 0.005). Furthermore, patch clamp measurements demonstrated a 3-fold increase in acetylcholine stimulated peak IKACh in atrial myocytes from GSK3ß deficiency mice compared with control. Finally, western blot analysis of atrial extracts from knockout mice demonstrated increased levels of SREBP-1, GIRK1 and GIRK4 compared with control. Taken together with our prior observations, these data establish a role of increased GSK3ß activity in the pathogenesis of parasympathetic dysfunction in type 1 diabetes via the regulation of IKACh and GIRK1/4 expression.


Assuntos
Diabetes Mellitus Tipo 1/fisiopatologia , Glicogênio Sintase Quinase 3 beta/deficiência , Miócitos Cardíacos/enzimologia , Sistema Nervoso Parassimpático/fisiopatologia , Animais , Diabetes Mellitus Tipo 1/enzimologia , Diabetes Mellitus Tipo 1/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Átrios do Coração/inervação , Átrios do Coração/fisiopatologia , Frequência Cardíaca/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/fisiologia , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo
4.
Nature ; 567(7746): 127-131, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814734

RESUMO

The GABAB (γ-aminobutyric acid type B) receptor is one of the principal inhibitory neurotransmitter receptors in the brain, and it signals through heterotrimeric G proteins to activate a variety of effectors, including G-protein-coupled inwardly rectifying potassium channels (GIRKs)1,2. GABAB-receptor signalling is tightly regulated by auxiliary subunits called KCTDs, which control the kinetics of GIRK activation and desensitization3-5. However, the mechanistic basis for KCTD modulation of GABAB signalling remains incompletely understood. Here, using a combination of X-ray crystallography, electron microscopy, and functional and biochemical experiments, we reveal the molecular details of KCTD binding to both GABAB receptors and G-protein ßγ subunits. KCTDs associate with the receptor by forming an asymmetric pentameric ring around a region of the receptor carboxy-terminal tail, while a second KCTD domain, H1, engages in a symmetric interaction with five copies of Gßγ in which the G-protein subunits also interact directly with one another. We further show that KCTD binding to Gßγ is highly cooperative, defining a model in which KCTD proteins cooperatively strip G proteins from GIRK channels to induce rapid desensitization following receptor activation. These results provide a framework for understanding the molecular basis for the precise temporal control of GABAB signalling by KCTD proteins.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas do Tecido Nervoso/química , Proteínas/química , Receptores de GABA-B/química , Receptores de GABA-B/metabolismo , Transdução de Sinais , Cristalografia por Raios X , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/química , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/química , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Subunidades beta da Proteína de Ligação ao GTP/ultraestrutura , Subunidades gama da Proteína de Ligação ao GTP/química , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Subunidades gama da Proteína de Ligação ao GTP/ultraestrutura , Humanos , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Proteínas do Tecido Nervoso/ultraestrutura , Ligação Proteica , Domínios Proteicos , Proteínas/metabolismo , Proteínas/ultraestrutura , Receptores de GABA-B/ultraestrutura
5.
Nat Commun ; 10(1): 1352, 2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30902995

RESUMO

G-protein-gated inward rectifying potassium channels (GIRKs) require Gßγ subunits and phosphorylated phosphatidylinositides (PIPs) for gating. Although studies have provided insight into these interactions, the mechanism of how these events are modulated by Gßγ and the binding affinity between PIPs and GIRKs remains poorly understood. Here, native ion mobility mass spectrometry is employed to directly monitor small molecule binding events to mouse GIRK2. GIRK2 binds the toxin tertiapin Q and PIPs selectively and with significantly higher affinity than other phospholipids. A mutation in GIRK2 that causes a rotation in the cytoplasmic domain, similarly to Gßγ-binding to the wild-type channel, revealed differences in the selectivity towards PIPs. More specifically, PIP isoforms known to weakly activate GIRKs have decreased binding affinity. Taken together, our results reveal selective small molecule binding and uncover a mechanism by which rotation of the cytoplasmic domain can modulate GIRK•PIP interactions.


Assuntos
Venenos de Abelha/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Mamíferos/metabolismo , Fosfatidilinositóis/metabolismo , Animais , Espectrometria de Massas , Camundongos , Mutação/genética , Fosforilação , Ligação Proteica
6.
Int J Mol Sci ; 20(5)2019 Mar 07.
Artigo em Inglês | MEDLINE | ID: mdl-30866445

RESUMO

Imbalances of excitatory/inhibitory synaptic transmission occur early in the pathogenesis of Alzheimer's disease (AD), leading to hippocampal hyperexcitability and causing synaptic, network, and cognitive dysfunctions. G-protein-gated potassium (GirK) channels play a key role in the control of neuronal excitability, contributing to inhibitory signaling. Here, we evaluate the relationship between GirK channel activity and inhibitory hippocampal functionality in vivo. In a non-transgenic mouse model of AD, field postsynaptic potentials (fPSPs) from the CA3⁻CA1 synapse in the dorsal hippocampus were recorded in freely moving mice. Intracerebroventricular (ICV) injections of amyloid-ß (Aß) or GirK channel modulators impaired ionotropic (GABAA-mediated fPSPs) and metabotropic (GirK-mediated fPSPs) inhibitory signaling and disrupted the potentiation of synaptic inhibition. However, the activation of GirK channels prevented Aß-induced changes in GABAA components. Our data shows, for the first time, the presence of long-term potentiation (LTP) for both the GABAA and GirK-mediated inhibitory postsynaptic responses in vivo. In addition, our results support the importance of an accurate level of GirK-dependent signaling for dorsal hippocampal performance in early amyloid pathology models by controlling the excess of excitation that disrupts synaptic plasticity processes.


Assuntos
Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/administração & dosagem , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Sinapses/fisiologia , Doença de Alzheimer/metabolismo , Animais , Modelos Animais de Doenças , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Injeções , Potenciação de Longa Duração , Masculino , Camundongos , Transdução de Sinais/efeitos dos fármacos , Sinapses/metabolismo
7.
Circulation ; 139(18): 2157-2169, 2019 04 30.
Artigo em Inglês | MEDLINE | ID: mdl-30764634

RESUMO

BACKGROUND: Bradyarrhythmia is a common clinical manifestation. Although the majority of cases are acquired, genetic analysis of families with bradyarrhythmia has identified a growing number of causative gene mutations. Because the only ultimate treatment for symptomatic bradyarrhythmia has been invasive surgical implantation of a pacemaker, the discovery of novel therapeutic molecular targets is necessary to improve prognosis and quality of life. METHODS: We investigated a family containing 7 individuals with autosomal dominant bradyarrhythmias of sinus node dysfunction, atrial fibrillation with slow ventricular response, and atrioventricular block. To identify the causative mutation, we conducted the family-based whole exome sequencing and genome-wide linkage analysis. We characterized the mutation-related mechanisms based on the pathophysiology in vitro. After generating a transgenic animal model to confirm the human phenotypes of bradyarrhythmia, we also evaluated the efficacy of a newly identified molecular-targeted compound to upregulate heart rate in bradyarrhythmias by using the animal model. RESULTS: We identified one heterozygous mutation, KCNJ3 c.247A>C, p.N83H, as a novel cause of hereditary bradyarrhythmias in this family. KCNJ3 encodes the inwardly rectifying potassium channel Kir3.1, which combines with Kir3.4 (encoded by KCNJ5) to form the acetylcholine-activated potassium channel ( IKACh channel) with specific expression in the atrium. An additional study using a genome cohort of 2185 patients with sporadic atrial fibrillation revealed another 5 rare mutations in KCNJ3 and KCNJ5, suggesting the relevance of both genes to these arrhythmias. Cellular electrophysiological studies revealed that the KCNJ3 p.N83H mutation caused a gain of IKACh channel function by increasing the basal current, even in the absence of m2 muscarinic receptor stimulation. We generated transgenic zebrafish expressing mutant human KCNJ3 in the atrium specifically. It is interesting to note that the selective IKACh channel blocker NIP-151 repressed the increased current and improved bradyarrhythmia phenotypes in the mutant zebrafish. CONCLUSIONS: The IKACh channel is associated with the pathophysiology of bradyarrhythmia and atrial fibrillation, and the mutant IKACh channel ( KCNJ3 p.N83H) can be effectively inhibited by NIP-151, a selective IKACh channel blocker. Thus, the IKACh channel might be considered to be a suitable pharmacological target for patients who have bradyarrhythmia with a gain-of-function mutation in the IKACh channel.


Assuntos
Fibrilação Atrial , Bloqueio Atrioventricular , Bradicardia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Doenças Genéticas Inatas , Mutação de Sentido Incorreto , Substituição de Aminoácidos , Animais , Animais Geneticamente Modificados , Fibrilação Atrial/genética , Fibrilação Atrial/metabolismo , Fibrilação Atrial/patologia , Fibrilação Atrial/fisiopatologia , Bloqueio Atrioventricular/genética , Bloqueio Atrioventricular/metabolismo , Bloqueio Atrioventricular/patologia , Bloqueio Atrioventricular/fisiopatologia , Benzopiranos/farmacologia , Bradicardia/genética , Bradicardia/metabolismo , Bradicardia/patologia , Bradicardia/fisiopatologia , Técnicas Eletrofisiológicas Cardíacas , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/metabolismo , Doenças Genéticas Inatas/patologia , Doenças Genéticas Inatas/fisiopatologia , Humanos , Masculino , Xenopus laevis , Peixe-Zebra
8.
Mol Cell Endocrinol ; 479: 123-132, 2019 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-30261212

RESUMO

Although there is growing evidence that cortistatin regulates several functions in different tissues, its role in the endocrine pancreas is not totally known. Here, we aim to study the effect of cortistatin on pancreatic beta-cells and glucose-stimulated insulin secretion (GSIS). Exposure of isolated mouse islets to cortistatin inhibited GSIS. This effect was prevented using a somatostatin receptor antagonist. Additionally, cortistatin hyperpolarized the membrane potential and reduced glucose-induced action potentials in isolated pancreatic beta-cells. Cortistatin did not modify ATP-dependent K+ (KATP) channel activity. In contrast, cortistatin increased the activity of a small conductance channel with characteristics of G protein-coupled inwardly rectifying K+ (GIRK) channels. The cortistatin effects on membrane potential and GSIS were largely reduced in the presence of a GIRK channel antagonist and by down-regulation of GIRK2 with small interfering RNA. Thus, cortistatin acts as an inhibitory signal for glucose-induced electrical activity and insulin secretion in the mouse pancreatic beta-cell.


Assuntos
Fenômenos Eletrofisiológicos/efeitos dos fármacos , Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Neuropeptídeos/farmacologia , Animais , Venenos de Abelha/farmacologia , Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Exocitose/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Canais KATP/metabolismo , Masculino , Camundongos Endogâmicos C57BL
9.
Br J Pharmacol ; 176(3): 451-465, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30447001

RESUMO

BACKGROUND AND PURPOSE: 5-HT1B receptors are widely expressed GPCRs and a target of triptans, the most commonly prescribed anti-migraine drugs. There is very limited information about the acute, agonist-induced regulation of 5-HT1B receptor signalling and so we sought to characterize this in a neuron-like system. EXPERIMENTAL APPROACH: Epitope-tagged human 5-HT1B receptors were expressed in mouse AtT20 cells. 5-HT1B receptor signalling was assessed using whole-cell patch-clamp recordings of endogenous G protein-gated inwardly rectified potassium (GIRK) channels, and receptor localization measured using immunofluorescence. KEY RESULTS: 5-HT (EC50 65 nM) and sumatriptan (EC50 165 nM) activated GIRK channels in AtT20 cells expressing 5-HT1B receptors. Continuous application of both 5-HT (EC50 120 nM) and sumatriptan (EC50 280 nM) produced profound desensitization of 5-HT1B receptor signalling within a few minutes. Complete recovery from desensitization was observed after 10 min. Both 5-HT and sumatriptan induced significant heterologous desensitization of SRIF (somatostatin)-activated GIRK currents, with the 5-HT-induced heterologous desensitization being blocked by the protein kinase inhibitor staurosporine. Both agonists induced modest 5-HT1B receptor internalization, with a time course much slower than receptor desensitization. CONCLUSIONS AND IMPLICATIONS: In AtT-20 cells, 5-HT1B receptors undergo rapid and reversible desensitization at concentrations of agonist similar to those required to activate the receptor. Desensitization is incomplete, and the continued signalling of the receptor in the presence of the agonist may lead to cellular adaptations. Finally, 5-HT1B receptor activation causes significant heterologous desensitization, which may lead to a reduced effectiveness of unrelated drugs in vivo.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptor 5-HT1B de Serotonina/biossíntese , Animais , Células HEK293 , Humanos , Camundongos , Células Tumorais Cultivadas
10.
Sleep ; 42(3)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30535004

RESUMO

STUDY OBJECTIVES: A major challenge in treating insomnia is to find effective medicines with fewer side effects. Activation of G-protein-gated inward rectifying K+ channels (GIRKs) by GABAB agonists baclofen or γ-hydroxybutyric acid (GHB) promotes nonrapid eye movement (NREM) sleep and consolidates sleep. However, baclofen has poor brain penetration, GHB possesses abuse liability, and in rodents both drugs cause spike-wave discharges (SWDs), an absence seizure activity. We tested the hypothesis that direct GIRK activation promotes sleep without inducing SWD using ML297, a potent and selective GIRK activator. METHODS: Whole-cell patch-clamp recordings from hypocretin/orexin or hippocampal neurons in mouse brain slices were made to study neuronal excitability and synaptic activity; spontaneous activity, locomotion, contextual and tone-conditioned memory, and novel object recognition were assessed. Electroencephalogram/electromyogram (EEG/EMG) recordings were used to study GIRK modulation of sleep. RESULTS: ML297, like baclofen, caused membrane hyperpolarization, decreased input resistance, and blockade of spontaneous action potentials. Unlike baclofen, ML297 (5-10 µM) did not cause significant depression of postsynaptic excitatory and inhibitory currents (EPSCs-IPSCs), indicating preferential postsynaptic inhibition. ML297 (30 mg/kg, i.p.) inhibited wake activity and locomotion, and preferentially increased NREM sleep without altering EEG delta power, REM sleep, inducing SWDs, or impairing conditioned memory and novel object recognition. CONCLUSIONS: This study finds that direct activation of neuronal GIRK channels modulates postsynaptic membrane excitability and prolongs NREM sleep without changing sleep intensity, inducing SWDs, or impairing memory in rodents. These results suggest that direct GIRK activation with a selective compound may present an innovative approach for the treatment of chronic insomnia.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Compostos de Fenilureia/farmacologia , Pirazóis/farmacologia , Fases do Sono/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Animais , Eletromiografia/efeitos dos fármacos , Eletromiografia/métodos , Feminino , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Técnicas de Patch-Clamp/métodos , Fases do Sono/efeitos dos fármacos
11.
J Lipid Res ; 60(1): 19-29, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30420402

RESUMO

Cholesterol, a critical component of the cellular plasma membrane, is essential for normal neuronal function. Cholesterol content is highest in the brain, where most cholesterol is synthesized de novo; HMG-CoA reductase controls the synthesis rate. Despite strict control, elevated blood cholesterol levels are common and are associated with various neurological disorders. G protein-gated inwardly rectifying potassium (GIRK) channels mediate the actions of inhibitory brain neurotransmitters. Loss of GIRK function enhances neuron excitability; gain of function reduces neuronal activity. However, the effect of dietary cholesterol or HMG-CoA reductase inhibition (i.e., statin therapy) on GIRK function remains unknown. Using a rat model, we compared the effects of a high-cholesterol versus normal diet both with and without atorvastatin, a widely prescribed HMG-CoA reductase inhibitor, on neuronal GIRK currents. The high-cholesterol diet increased hippocampal CA1 region cholesterol levels and correspondingly increased neuronal GIRK currents. Both phenomena were reversed by cholesterol depletion in vitro. Atorvastatin countered the high-cholesterol diet effects on neuronal cholesterol content and GIRK currents; these effects were reversed by cholesterol enrichment in vitro. Our findings suggest that high-cholesterol diet and atorvastatin therapy affect ion channel function in the brain by modulating neuronal cholesterol levels.


Assuntos
Atorvastatina/farmacologia , Colesterol na Dieta/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Animais , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/efeitos dos fármacos , Região CA1 Hipocampal/fisiologia , Suplementos Nutricionais , Relação Dose-Resposta a Droga , Interações de Medicamentos , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-Dawley
12.
ACS Chem Neurosci ; 10(1): 358-370, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30136838

RESUMO

G protein-gated, inwardly rectifying, potassium (GIRK) channels are important regulators of cellular excitability throughout the body. GIRK channels are heterotetrameric and homotetrameric combinations of the Kir3.1-4 (GIRK1-4) subunits. Different subunit combinations are expressed throughout the central nervous system (CNS) and the periphery, and most of these combinations contain a GIRK1 subunit. For example, the predominance of GIRK channels in the CNS are composed of GIRK1 and GIRK2 subunits, while the GIRK channels in cardiac atrial myocytes are made up mostly of GIRK1 and GIRK4 subunits. Although the vast majority of GIRK channels contain a GIRK1 subunit, discrete populations of cells that express non-GIRK1-containing GIRK (non-GIRK1/X) channels do exist. For instance, dopaminergic neurons in the ventral tegmental area of the brain, associated with addiction and reward, do not express the GIRK1 subunit. Targeting these non-GIRK1/X channels with subunit-selective pharmacological probes could lead to important insights into how GIRK channels are involved in reward and addiction. Such insights may, in turn, reveal therapeutic opportunities for the treatment or prevention of addiction. Previously, our laboratory discovered small molecules that can specifically modulate the activity of GIRK1-containing GIRK channels. However, efforts to generate compounds active on non-GIRK1/X channels from these scaffolds have been unsuccessful. Recently, ivermectin was shown to modulate non-GIRK1/X channels, and historically, ivermectin is known to modulate a wide variety of neuronal channels and receptors. Further, ivermectin is a complex natural product, which makes it a challenging starting point for development of more selective, effective, and potent compounds. Thus, while ivermectin provides proof-of-concept as a non-GIRK1/X channel activator, it is of limited utility. Therefore, we sought to discover a synthetic small molecule that would serve as a starting point for the development of non-GIRK1/X channel modulators. To accomplish this, we used a high-throughput thallium flux assay to screen a 100 000-compound library in search of activators of homomeric GIRK2 channels. Using this approach, we discovered VU0529331, the first synthetic small molecule reported to activate non-GIRK1/X channels, to our knowledge. This discovery represents the first step toward developing potent and selective non-GIRK1/X channel probes. Such molecules will help elucidate the role of GIRK channels in addiction, potentially establishing a foundation for future development of therapies utilizing targeted GIRK channel modulation.


Assuntos
Descoberta de Drogas/métodos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/agonistas , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Ativação do Canal Iônico/efeitos dos fármacos , Pirazinas/química , Pirazinas/farmacologia , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
13.
Elife ; 72018 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-30526853

RESUMO

Stimulated muscarinic acetylcholine receptors (M2Rs) release Gßγ subunits, which slow heart rate by activating a G protein-gated K+ channel (GIRK). Stimulated ß2 adrenergic receptors (ß2ARs) also release Gßγ subunits, but GIRK is not activated. This study addresses the mechanism underlying this specificity of GIRK activation by M2Rs. K+ currents and bioluminescence resonance energy transfer between labelled G proteins and GIRK show that M2Rs catalyze Gßγ subunit release at higher rates than ß2ARs, generating higher Gßγ concentrations that activate GIRK and regulate other targets of Gßγ. The higher rate of Gßγ release is attributable to a faster G protein coupled receptor - G protein trimer association rate in M2R compared to ß2AR. Thus, a rate difference in a single kinetic step accounts for specificity.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais , Animais , Células CHO , Simulação por Computador , Cricetinae , Cricetulus , Feminino , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Cinética , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Ligação Proteica
14.
Elife ; 72018 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-30556810

RESUMO

Dopamine (D2) receptors provide autoinhibitory feedback onto dopamine neurons through well-known interactions with voltage-gated calcium channels and G protein-coupled inwardly-rectifying potassium (GIRK) channels. Here, we reveal a third major effector involved in D2R modulation of dopaminergic neurons - the sodium leak channel, NALCN. We found that activation of D2 receptors robustly inhibits isolated sodium leak currents in wild-type mice but not in NALCN conditional knockout mice. Intracellular GDP-ßS abolished the inhibition, indicating a G protein-dependent signaling mechanism. The application of dopamine reliably slowed pacemaking even when GIRK channels were pharmacologically blocked. Furthermore, while spontaneous activity was observed in nearly all dopaminergic neurons in wild-type mice, neurons from NALCN knockouts were mainly silent. Both observations demonstrate the critical importance of NALCN for pacemaking in dopaminergic neurons. Finally, we show that GABA-B receptor activation also produces inhibition of NALCN-mediated currents. Therefore, we identify NALCN as a core effector of inhibitory G protein-coupled receptors.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Canais Iônicos/genética , Proteínas do Tecido Nervoso/genética , Receptores de Dopamina D2/metabolismo , Receptores de GABA-B/metabolismo , 6-Ciano-7-nitroquinoxalina-2,3-diona/farmacologia , Animais , Canais de Cálcio Tipo N/genética , Dopamina/farmacologia , Neurônios Dopaminérgicos/citologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/antagonistas & inibidores , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Expressão Gênica , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacologia , Canais Iônicos/deficiência , Transporte de Íons/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtomia , Proteínas do Tecido Nervoso/deficiência , Técnicas de Patch-Clamp , Picrotoxina/farmacologia , Receptores de Dopamina D2/genética , Receptores de GABA-B/genética , Tionucleotídeos/farmacologia , Técnicas de Cultura de Tecidos , Valina/análogos & derivados , Valina/farmacologia , Área Tegmentar Ventral/citologia , Área Tegmentar Ventral/metabolismo
15.
PLoS One ; 13(9): e0204447, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30240440

RESUMO

G protein-gated inwardly rectifying K+ (GIRK) channel regulates cellular excitability upon activation of Gi/o-coupled receptors. In Gi/o-coupled muscarinic M2R, the intracellular third loop (i3) is known as a key domain for Gi/o coupling, because replacement of i3 of Gq-coupled muscarinic M1R with that of M2R enables the chimeric receptor (MC9) to activate the GIRK channel. In the present study, we showed that MC9, but not M1R, co-localizes with the GIRK channel and Gαi1 by Förster resonance energy transfer (FRET) analysis. When M1R was forced to stay adjacent to the channel through ligation with short linkers, M1R activated the GIRK channel. FRET analysis further suggested that the efficacy of channel activation is correlated with the linker length between M1R and the GIRK channel. The results show that co-localization is an important factor for activating the GIRK channel. In contrast, for MC9 and M2R, the GIRK channel was activated even when they were connected by long linkers, suggesting the formation of a molecular complex even in the absence of a linker. We also observed that replacement of 13 amino acid residues at the N-terminal end of i3 of MC9 with those of M1R impaired the co-localization with the GIRK channel as well as channel activation. These results show that localization of the receptor near the GIRK channel is a key factor in efficiently activating the channel and that the N-terminal end of i3 of M2R plays an important role in co-localization.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Receptores Muscarínicos/metabolismo , Sequência de Aminoácidos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Células HEK293 , Humanos , Mutação , Transporte Proteico , Receptores Muscarínicos/química
16.
Proc Natl Acad Sci U S A ; 115(40): E9479-E9488, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30228121

RESUMO

GABABR-dependent activation of G protein-gated inwardly rectifying potassium channels (GIRK or KIR3) provides a well-known source of inhibition in the brain, but the details on how this important inhibitory pathway affects neural circuits are lacking. We used sorting nexin 27 (SNX27), an endosomal adaptor protein that associates with GIRK2c and GIRK3 subunits, to probe the role of GIRK channels in reward circuits. A conditional knockout of SNX27 in both substantia nigra pars compacta and ventral tegmental area (VTA) dopamine neurons leads to markedly smaller GABABR- and dopamine D2R-activated GIRK currents, as well as to suprasensitivity to cocaine-induced locomotor sensitization. Expression of the SNX27-insensitive GIRK2a subunit in SNX27-deficient VTA dopamine neurons restored GIRK currents and GABABR-dependent inhibition of spike firing, while also resetting the mouse's sensitivity to cocaine-dependent sensitization. These results establish a link between slow inhibition mediated by GIRK channels in VTA dopamine neurons and cocaine addiction, revealing a therapeutic target for treating addiction.


Assuntos
Transtornos Relacionados ao Uso de Cocaína/metabolismo , Cocaína/toxicidade , Neurônios Dopaminérgicos/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Locomoção/efeitos dos fármacos , Animais , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/patologia , Neurônios Dopaminérgicos/patologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Camundongos , Camundongos Knockout , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo
17.
J Neurosci ; 38(41): 8737-8744, 2018 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-30150362

RESUMO

Regulators of G-protein signaling (RGS) proteins negatively modulate presynaptic µ-opioid receptor inhibition of GABA release in the ventrolateral periaqueductal gray (vlPAG). Paradoxically, we find that G-protein-coupled receptor (GPCR) activation of G-protein-gated inwardly rectifying K+ channels (GIRKs) in the vlPAG is reduced in an agonist- and receptor-dependent manner in transgenic knock-in mice of either sex expressing mutant RGS-insensitive Gαo proteins. µ-Opioid receptor agonist activation of GIRK currents was reduced for DAMGO and fentanyl but not for [Met5]-enkephalin acetate salt hydrate (ME) in the RGS-insensitive heterozygous (Het) mice compared with wild-type mice. The GABAB agonist baclofen-induced GIRK currents were also reduced in the Het mice. We confirmed the role of Gαo proteins in µ-opioid receptor and GABAB receptor signaling pathways in wild-type mice using myristoylated peptide inhibitors of Gαo1 and Gαi1-3 The results using these inhibitors indicate that receptor activation of GIRK channels is dependent on the preference of the agonist-stimulated receptor for Gαo versus that for Gαi. DAMGO and fentanyl-mediated GIRK currents were reduced in the presence of the Gαo1 inhibitor, but not the Gαi1-3 inhibitors. In contrast, the Gαo1 peptide inhibitor did not affect ME activation of GIRK currents, which is consistent with results in the Het mice, but the Gαi1-3 inhibitors significantly reduced ME-mediated GIRK currents. Finally, the reduction in GIRK activation in the Het mice plays a role in opioid- and baclofen-mediated spinal antinociception, but not supraspinal antinociception. Thus, our studies indicate that RGS proteins have multiple mechanisms of modulating GPCR signaling that produce negative and positive regulation of signaling depending on the effector.SIGNIFICANCE STATEMENT Regulators of G-protein signaling (RGS) proteins positively modulate GPCR coupling to GIRKs, and this coupling is critical for opioid- and baclofen-mediated spinal antinociception, whereas µ-opioid receptor-mediated supraspinal antinociception depends on presynaptic inhibition that is negatively regulated by RGS proteins. The identification of these opposite roles for RGS proteins has implications for signaling via other GPCRs.


Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Neurônios/metabolismo , Substância Cinzenta Periaquedutal/metabolismo , Proteínas RGS/metabolismo , Analgésicos/administração & dosagem , Animais , Baclofeno/administração & dosagem , Feminino , Agonistas dos Receptores de GABA-B/administração & dosagem , Locomoção/efeitos dos fármacos , Masculino , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Substância Cinzenta Periaquedutal/efeitos dos fármacos , Receptores de GABA-B/metabolismo , Receptores Opioides mu/agonistas
18.
Pflugers Arch ; 470(12): 1765-1776, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30155776

RESUMO

The acetylcholine (ACh)-gated inwardly rectifying K+ current (IKACh) plays a vital role in cardiac excitability by regulating heart rate variability and vulnerability to atrial arrhythmias. These crucial physiological contributions are determined principally by the inwardly rectifying nature of IKACh. Here, we investigated the relative contribution of two distinct mechanisms of IKACh inward rectification measured in atrial myocytes: a rapid component due to KACh channel block by intracellular Mg2+ and polyamines; and a time- and concentration-dependent mechanism. The time- and ACh concentration-dependent inward rectification component was eliminated when IKACh was activated by GTPγS, a compound that bypasses the muscarinic-2 receptor (M2R) and directly stimulates trimeric G proteins to open KACh channels. Moreover, the time-dependent component of IKACh inward rectification was also eliminated at ACh concentrations that saturate the receptor. These observations indicate that the time- and concentration-dependent rectification mechanism is an intrinsic property of the receptor, M2R; consistent with our previous work demonstrating that voltage-dependent conformational changes in the M2R alter the receptor affinity for ACh. Our analysis of the initial and time-dependent components of IKACh indicate that rapid Mg2+-polyamine block accounts for 60-70% of inward rectification, with M2R voltage sensitivity contributing 30-40% at sub-saturating ACh concentrations. Thus, while both inward rectification mechanisms are extrinsic to the KACh channel, to our knowledge, this is the first description of extrinsic inward rectification of ionic current attributable to an intrinsic voltage-sensitive property of a G protein-coupled receptor.


Assuntos
Potenciais de Ação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Miócitos Cardíacos/metabolismo , Receptor Muscarínico M2/metabolismo , Acetilcolina/metabolismo , Animais , Gatos , Células Cultivadas , Feminino , Átrios do Coração/citologia , Magnésio/metabolismo , Masculino , Miócitos Cardíacos/fisiologia , Poliaminas/metabolismo
19.
Cell Physiol Biochem ; 49(1): 65-77, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30134221

RESUMO

BACKGROUND/AIMS: Cardiac arrhythmias are triggered by environmental stimuli that may modulate expression of cardiac ion channels. Underlying epigenetic regulation of cardiac electrophysiology remains incompletely understood. Histone deacetylases (HDACs) control gene expression and cardiac integrity. We hypothesized that class I/II HDACs transcriptionally regulate ion channel expression and determine action potential duration (APD) in cardiac myocytes. METHODS: Global class I/II HDAC inhibition was achieved by administration of trichostatin A (TSA). HDAC-mediated effects on K+ channel expression and electrophysiological function were evaluated in murine atrial cardiomyocytes (HL-1 cells) using real-time PCR, Western blot, and patch clamp analyses. Electrical tachypacing was employed to recapitulate arrhythmia-related effects on ion channel remodeling in the absence and presence of HDAC inhibition. RESULTS: Global HDAC inhibition increased histone acetylation and prolonged APD90 in atrial cardiomyocytes compared to untreated control cells. Transcript levels of voltage-gated or inwardly rectifying K+ channels Kcnq1, Kcnj3 and Kcnj5 were significantly reduced, whereas Kcnk2, Kcnj2 and Kcnd3 mRNAs were upregulated. Ion channel remodeling was similarly observed at protein level. Short-term tachypacing did not induce significant transcriptional K+ channel remodeling. CONCLUSION: The present findings link class I/II HDAC activity to regulation of ion channel expression and action potential duration in atrial cardiomyocytes. Clinical implications for HDAC-based antiarrhythmic therapy and cardiac safety of HDAC inhibitors require further investigation.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio/metabolismo , Animais , Linhagem Celular , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Inibidores de Histona Desacetilases/química , Histona Desacetilases/química , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Transcrição Genética/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
20.
Neuropharmacology ; 139: 217-225, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30025920

RESUMO

Endogenous cannabinoids are diffusible lipid ligands of the main cannabinoid receptors type 1 and 2 (CB1R and CB2R). In the central nervous system endocannabinoids are produced in an activity-dependent manner and have been identified as retrograde modulators of synaptic transmission. Additionally, some neurons display a cell-autonomous slow self-inhibition (SSI) mediated by endocannabinoids. In these neurons, repetitive action potential firing triggers the production of endocannabinoids, which induce a long-lasting hyperpolarization of the membrane potential, rendering the cells less excitable. Different endocannabinoid receptors and effector mechanisms have been described underlying SSI in different cell types and brain areas. Here, we investigate SSI in neurons of layer 2/3 in the somatosensory cortex. High-frequency bursts of action potentials induced SSI in pyramidal cells (PC) and regular spiking non-pyramidal cells (RSNPC), but not in fast-spiking interneurons (FS). In RSNPCs the hyperpolarization was accompanied by a change in input resistance due to the activation of G protein-coupled inward-rectifying K+ (GIRK) channels. A CB2R-specific agonist induced the long-lasting hyperpolarization, whereas preincubation with a CB2R-specific inverse agonist suppressed SSI. Additionally, using cannabinoid receptor knockout mice, we found that SSI was still intact in CB1R-deficient but abolished in CB2R-deficient mice. Taken together, we describe an additional SSI mechanism in which the activity-induced release of endocannabinoids activates GIRK channels via CB2Rs. These findings expand our knowledge about cell type-specific differential neuronal cannabinoid receptor signaling and suggest CB2R-selective compounds as potential therapeutic approaches.


Assuntos
Inibição Neural/fisiologia , Neurônios/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Córtex Somatossensorial/metabolismo , Animais , Moduladores de Receptores de Canabinoides/farmacologia , Endocanabinoides/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Inibição Neural/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptor CB1 de Canabinoide/deficiência , Receptor CB1 de Canabinoide/genética , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/agonistas , Receptor CB2 de Canabinoide/deficiência , Receptor CB2 de Canabinoide/genética , Córtex Somatossensorial/efeitos dos fármacos , Técnicas de Cultura de Tecidos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA