RESUMO
Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by amyloid plaques and cognitive decline, the latter of which is thought to be driven by soluble oligomeric amyloid-ß (oAß). The dysregulation of G protein-gated inwardly rectifying K+ (GIRK; also known as Kir3) channels has been implicated in rodent models of AD. Here, seeking mechanistic insights, we uncovered a sex-dependent facet of GIRK-dependent signaling in AD-related amyloid pathophysiology. Synthetic oAß1-42 suppressed GIRK-dependent signaling in hippocampal neurons from male mice, but not from female mice. This effect required cellular prion protein, the receptor mGluR5, and production of arachidonic acid by the phospholipase PLA2. Although oAß suppressed GIRK channel activity only in male hippocampal neurons, intrahippocampal infusion of oAß or genetic suppression of GIRK channel activity in hippocampal pyramidal neurons impaired performance on a memory test in both male and female mice. Moreover, genetic enhancement of GIRK channel activity in hippocampal pyramidal neurons blocked oAß-induced cognitive impairment in both male and female mice. In APP/PS1 AD model mice, GIRK-dependent signaling was diminished in hippocampal CA1 pyramidal neurons from only male mice before cognitive deficit was detected. However, enhancing GIRK channel activity rescued cognitive deficits in older APP/PS1 mice of both sexes. Thus, whereas diminished GIRK channel activity contributes to cognitive deficits in male mice with increased oAß burden, enhancing its activity may have therapeutic potential for both sexes.
Assuntos
Doença de Alzheimer , Peptídeos beta-Amiloides , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Hipocampo , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Peptídeos beta-Amiloides/metabolismo , Feminino , Masculino , Hipocampo/metabolismo , Camundongos , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Neurônios/metabolismo , Camundongos Transgênicos , Camundongos Endogâmicos C57BL , Receptor de Glutamato Metabotrópico 5/metabolismo , Receptor de Glutamato Metabotrópico 5/genética , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/genética , Caracteres Sexuais , Modelos Animais de Doenças , Fosfolipases A2/metabolismo , HumanosRESUMO
Aldosterone-producing adenoma is a subtype of primary aldosteronism. Recent advancements in multi-omics research have led to significant progress in understanding primary aldosteronism at the genetic level. Among the various genes associated with the development of aldosterone-producing adenomas, the KCNJ5 (potassium inwardly rectifying channel, subfamily J, member 5) gene has received considerable attention due to its prevalence as the most common somatic mutation gene in primary aldosteronism. This paper aims to integrate the existing evidence on the involvement of KCNJ5 gene in the pathogenesis of aldosterone-producing adenomas, to enhance the understanding of the underlying mechanisms of aldosterone-producing adenomas from the perspective of genetics, and to provide novel insights for the clinical diagnosis and treatment of aldosterone-producing adenomas.
Assuntos
Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Aldosterona , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Hiperaldosteronismo , Humanos , 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 , Aldosterona/metabolismo , Aldosterona/biossíntese , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Adenoma/genética , Adenoma/metabolismo , MutaçãoRESUMO
CONTEXT: The search for somatic mutations in adrenals resected from patients with primary aldosteronism (PA) is performed by Sanger sequencing, often implemented with immunohistochemistry (IHC)-guidance focused on aldosterone-producing (CYP11B2-positive) areas. OBJECTIVE: To investigate the impact of double IHC for CYP11B1 and CYP11B2 on Sanger and next-generation sequencing (NGS). METHODS: We investigated 127 consecutive adrenal aldosterone-producing adenomas from consenting surgically cured PA patients using double IHC for CYP11B1 and CYP11B2, by Sanger sequencing and NGS. RESULTS: Double IHC for CYP11B2 and CYP11B1 revealed 3 distinct patterns: CYP11B2-positive adenoma (pattern 1), mixed CYP11B1/CYP11B2-positive adenoma (pattern 2), and adrenals with multiple small CYP11B2-positive nodules (pattern 3). Sanger sequencing allowed detection of KCNJ5 mutations in 44% of the adrenals; NGS revealed such mutations in 10% of those negative at Sanger and additional mutations in 61% of the cases. Importantly the rate of KCNJ5 mutations differed across patterns: 17.8% in pattern 1, 71.4% in pattern 2, and 10.7% in pattern 3 (χ2 = 22.492, P < .001). CONCLUSION: NGS allowed detection of mutations in many adrenals that tested negative at Sanger sequencing. Moreover, the different distribution of KCNJ5 mutations across IHC patterns indicates that IHC-guided sequencing protocols selecting CYP11B2-positive areas could furnish results that might not be representative of the entire mutational status of the excised adrenal, which is important at a time when KCNJ5 mutations are suggested to drive management of patients with aldosterone-producing adenomas.
Assuntos
Citocromo P-450 CYP11B2 , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Hiperaldosteronismo , Imuno-Histoquímica , Mutação , Esteroide 11-beta-Hidroxilase , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/metabolismo , 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 , Esteroide 11-beta-Hidroxilase/genética , Esteroide 11-beta-Hidroxilase/metabolismo , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , Feminino , Masculino , Pessoa de Meia-Idade , Adulto , Sequenciamento de Nucleotídeos em Larga Escala , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Adenoma Adrenocortical/patologia , Adenoma Adrenocortical/diagnóstico , Idoso , Neoplasias do Córtex Suprarrenal/genética , Neoplasias do Córtex Suprarrenal/metabolismo , Neoplasias do Córtex Suprarrenal/diagnóstico , Neoplasias do Córtex Suprarrenal/patologiaRESUMO
This manuscript explores the intricate role of acetylcholine-activated inward rectifier potassium (KACh) channels in the pathogenesis of atrial fibrillation (AF), a common cardiac arrhythmia. It delves into the molecular and cellular mechanisms that underpin AF, emphasizing the vital function of KACh channels in modulating the atrial action potential and facilitating arrhythmogenic conditions. This study underscores the dual nature of KACh activation and its genetic regulation, revealing that specific variations in potassium channel genes, such as Kir3.4 and K2P3.1, significantly influence the electrophysiological remodeling associated with AF. Furthermore, this manuscript identifies the crucial role of the KACh-mediated current, IKACh, in sustaining arrhythmia through facilitating shorter re-entry circuits and stabilizing the re-entrant circuits, particularly in response to vagal nerve stimulation. Experimental findings from animal models, which could not induce AF in the absence of muscarinic activation, highlight the dependency of AF induction on KACh channel activity. This is complemented by discussions on therapeutic interventions, where KACh channel blockers have shown promise in AF management. Additionally, this study discusses the broader implications of KACh channel behavior, including its ubiquitous presence across different cardiac regions and species, contributing to a comprehensive understanding of AF dynamics. The implications of these findings are profound, suggesting that targeting KACh channels might offer new therapeutic avenues for AF treatment, particularly in cases resistant to conventional approaches. By integrating genetic, cellular, and pharmacological perspectives, this manuscript offers a holistic view of the potential mechanisms and therapeutic targets in AF, making a significant contribution to the field of cardiac arrhythmia research.
Assuntos
Fibrilação Atrial , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Fibrilação Atrial/genética , Humanos , Animais , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Potenciais de Ação , Acetilcolina/metabolismoRESUMO
Context: The prevalence of unilateral primary aldosteronism (UPA) with cortisol co-secretion varies geographically. Objective: To investigate the prevalence and clinical characteristics of UPA with cortisol co-secretion in a Chinese population. Design: Retrospective cohort study. Methods: We recruited 580 patients with UPA who underwent cosyntropin stimulation test (CST) after the 1-mg dexamethasone suppression test (DST) and retrospectively analyzed the clinical characteristics and postoperative outcomes of UPA with and without cortisol co-secretion. Results: UPA with cortisol co-secretion (1 mg DST>1.8 ug/dL) was identified in 65 of 580 (11.2%) patients. These patients were characterized by older age, longer duration of hypertension, higher concentration of plasma aldosterone and midnight cortisol, lower adrenocorticotropic hormone (ACTH) and dehydroepiandrosterone sulfate (DHEAS), larger tumor diameter, and more history of diabetes mellitus. Cortisol and aldosterone levels were higher and DHEAS level was lower in UPA with cortisol co-secretion at 0-120 min after CST. Among 342 UPA patients with KCNJ5 gene sequencing and follow-up results, the complete clinical success rate was lower in UPA with cortisol co-secretion (33.3% vs. 56.4%, P<0.05); the complete biochemical success rate and KCNJ5 mutation did not differ between the two groups. Age, tumor size, and ACTH were independent predictors of UPA with cortisol co-secretion. Sex, BMI, duration of hypertension, KCNJ5 mutation, and cortisol co-secretion were independent predictors for complete clinical success in UPA after surgery. Conclusions: UPA with cortisol co-secretion is not uncommon in China, but the clinical features were distinctly different from those without co-secretion. Cortisol co-secretion is an independent risk factor for incomplete clinical success after surgery in UPA.
Assuntos
Hidrocortisona , Hiperaldosteronismo , Humanos , Hiperaldosteronismo/cirurgia , Hiperaldosteronismo/metabolismo , Hiperaldosteronismo/sangue , Masculino , Feminino , Pessoa de Meia-Idade , Hidrocortisona/sangue , Estudos Retrospectivos , Adulto , Aldosterona/sangue , Adrenalectomia , China/epidemiologia , Resultado do Tratamento , Hormônio Adrenocorticotrópico/sangue , 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 , Seguimentos , PrognósticoAssuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Hiperaldosteronismo , Mutação , Humanos , Hiperaldosteronismo/genética , Hiperaldosteronismo/diagnóstico , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Masculino , Feminino , Pessoa de Meia-Idade , AdultoRESUMO
BACKGROUND: While clinical features of KCNJ5-mutated aldosterone-producing adenoma (APA) have been reported, evidence of its clinical outcomes is lacking. We aimed to synthesize available literature about the associations between KCNJ5 mutation with cardiovascular and metabolic outcomes among patients with APA. METHODS: In this systematic review of observational studies, MEDLINE and Embase were searched through August 2022. Two independent authors screened the search results and extracted data from eligible observational studies investigating cardiovascular or metabolic outcomes between KCNJ5-mutated APAs and KCNJ5-non-mutated APAs. Risk of Bias In Non-randomized Studies of Interventions was used to assess the quality of the included studies. RESULTS: A total of 573 titles/abstracts were screened and after the expert opinion of the literature, full text was read in 20 titles/abstracts, of which 12 studies were included. Across 3 studies comparing the baseline or change in the cardiac function between KCNJ5-mutated APAs and KCNJ5-non-mutated APAs, all studies reported the association between impaired cardiac functions and KCNJ5 mutation status. Among 6 studies evaluating the cure of hypertension after surgery, all studies showed that KCNJ5 mutation was significantly associated with the cure of hypertension. In quality assessment, 7 studies were at serious risk of bias, while the remaining studies were at moderate risk of bias. CONCLUSIONS: This systematic review provided evidence of the significant association between KCNJ5 mutation and unfavorable cardiovascular outcomes in patients with primary aldosteronism. Further research is needed to improve the quality of evidence on this topic and elucidate the underlying mechanisms of the potential burden of KCNJ5 mutation.
Assuntos
Aldosterona , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Mutação , Humanos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Aldosterona/metabolismo , Aldosterona/biossíntese , Doenças Cardiovasculares/genética , Neoplasias do Córtex Suprarrenal/genética , Hiperaldosteronismo/genética , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/metabolismo , Adenoma/genética , Adenoma/metabolismoRESUMO
G-protein-gated inward rectifier K+ (GIRK) channels play a critical role in the regulation of the excitability of cardiomyocytes and neurons and include GIRK1, GIRK2, GIRK3 and GIRK4 subfamily members. BD1047 dihydrobromide (BD1047) is one of the representative antagonists of the multifunctional Sigma-1 receptor (S1R). In the analysis of the effect of BD1047 on the regulation of Gi-coupled receptors by S1R using GIRK channel as an effector, we observed that BD1047, as well as BD1063, directly inhibited GIRK currents even in the absence of S1R and in a voltage-independent manner. Thus, we aimed to clarify the effect of BD1047 on GIRK channels and identify the structural determinants. By electrophysiological recordings in Xenopus oocytes, we observed that BD1047 directly inhibited GIRK channel currents, producing a much stronger inhibition of GIRK4 compared to GIRK2. It also inhibited ACh-induced native GIRK current in isolated rat atrial myocytes. Chimeric and mutagenesis studies of GIRK2 and GIRK4 combined with molecular docking analysis demonstrated the importance of Leu77 and Leu84 within the cytoplasmic, proximal N-terminal region and Glu147 within the pore-forming region of GIRK4 for inhibition by BD1047. The activator of GIRK channels, ivermectin, competed with BD1047 at Leu77 on GIRK4. This study provides us with a novel inhibitor of GIRK channels and information for developing pharmacological treatments for GIRK4-associated diseases.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Receptores sigma , Receptor Sigma-1 , Animais , Ratos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , 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/química , Simulação de Acoplamento Molecular , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Oócitos/metabolismo , Receptores sigma/metabolismo , Receptores sigma/antagonistas & inibidores , Receptores sigma/genética , Receptores sigma/química , Xenopus laevis , Ratos WistarRESUMO
The inwardly rectifying potassium channel Kir3.2, a member of the inward rectifier potassium (Kir) channel family, exerts important biological functions through transporting potassium ions outside of the cell, during which a large-scale synergistic movement occurs among its different domains. Currently, it is not fully understood how the binding of the ligand to the Kir3.2 channel leads to the structural changes and which key residues are responsible for the channel gating and allosteric dynamics. Here, we construct the Gaussian network model (GNM) of the Kir3.2 channel with the secondary structure and covalent interaction information considered (sscGNM), which shows a better performance in reproducing the channel's flexibility compared with the traditional GNM. In addition, the sscANM-based perturbation method is used to simulate the channel's conformational transition caused by the activator PIP2's binding. By applying certain forces to the PIP2 binding pocket, the coarse-grained calculations generate the similar conformational changes to the experimental observation, suggesting that the topology structure as well as PIP2 binding are crucial to the allosteric activation of the Kir3.2 channel. We also utilize the sscGNM-based thermodynamic cycle method developed by us to identify the key residues whose mutations significantly alter the channel's binding free energy with PIP2. We identify not only the residues important for the specific binding but also the ones critical for the allosteric transition coupled with PIP2 binding. This study is helpful for understanding the working mechanism of Kir3.2 channels and can provide important information for related drug design.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Potássio , 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 , Mutação , Estrutura Secundária de Proteína , Fenômenos Biofísicos , Potássio/metabolismoRESUMO
BACKGROUND: KCNJ3 encodes a subunit of G-protein-coupled inwardly rectifying potassium channels, which are important for cellular excitability and inhibitory neurotransmission. However, the genetic basis of KCNJ3 in epilepsy has not been determined. This study aimed to identify the pathogenic KCNJ3 variants in patients with epilepsy. METHODS: Trio exome sequencing was performed to determine potential variants of epilepsy. Individuals with KCNJ3 variants were recruited for this study. Detailed clinical information and genetic data were obtained and systematically reviewed. Whole-cell patch-clamp recordings were performed to evaluate the functional consequences of the identified variants. RESULTS: Two de novo missense variants (c.998T>C (p.Leu333Ser) and c.938G>A (p. Arg313Gln)) in KCNJ3 were identified in two unrelated families with epilepsy. The variants were absent from the gnomAD database and were assumed to be damaging or probably damaging using multiple bioinformatics tools. They were both located in the C-terminal domain. The amino acid residues were highly conserved among various species. Clinically, the seizures occurred at a young age and were under control after combined treatment. Electrophysiological analysis revealed that the KCNJ3 Leu333Ser and Arg313Gln variants significantly compromised the current activities and exhibited loss-of-function (LOF) effects. CONCLUSION: Our findings suggest that de novo LOF variants in KCNJ3 are associated with early-onset epilepsy. Genetic testing of KCNJ3 in patients with epilepsy may serve as a strategy for precision medicine.
Assuntos
Epilepsia , Mutação de Sentido Incorreto , Humanos , Mutação de Sentido Incorreto/genética , Epilepsia/genética , Fenômenos Eletrofisiológicos , Canais de Potássio/genética , Testes Genéticos , 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/metabolismoRESUMO
BACKGROUND: Recent advances in omics techniques have allowed detailed genetic characterization of aldosterone-producing adenoma (APA). The pathogenesis of APA is characterized by tumorigenesis-associated aldosterone synthesis. The pathophysiological intricacies of APAs have not yet been elucidated at the level of individual cells. Therefore, a single-cell level analysis is speculated to be valuable in studying the differentiation process of APA. METHODS: We conducted single-nucleus RNA sequencing of APAs with KCNJ5 mutation and nonfunctional adenomas obtained from 3 and 2 patients, respectively. RESULTS: The single-nucleus RNA sequencing revealed the intratumoral heterogeneity of APA and identified cell populations consisting of a shared cluster of nonfunctional adenoma and APA. In addition, we extracted 2 cell fates in APA and obtained a cell population specialized in aldosterone synthesis. Genes related to ribosomes and neurodegenerative diseases were upregulated in 1 of these fates, whereas those related to the regulation of glycolysis were upregulated in the other fate. Furthermore, the total RNA reads in the nucleus were higher in hormonally activated clusters, indicating a marked activation of transcription per cell. CONCLUSIONS: The single-nucleus RNA sequencing revealed intratumoral heterogeneity of APA with KCNJ5 mutation. The observation of 2 cell fates in KCNJ5-mutated APAs provides the postulation that a heterogeneous process of cellular differentiation was implicated in the pathophysiological mechanisms underlying APA tumors.
Assuntos
Adenoma , Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Hiperaldosteronismo , Humanos , Aldosterona , Adenoma Adrenocortical/genética , Adenoma Adrenocortical/patologia , Adenoma/genética , Adenoma/patologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Mutação , Neoplasias do Córtex Suprarrenal/genética , Hiperaldosteronismo/genéticaRESUMO
Primary aldosteronism (PA) causes 5-10% of hypertension cases, but only a minority of patients are currently diagnosed and treated because of a complex, stepwise, and partly invasive workup. We tested the performance of urine steroid metabolomics, the computational analysis of 24-hour urine steroid metabolome data by machine learning, for the identification and subtyping of PA. Mass spectrometry-based multi-steroid profiling was used to quantify the excretion of 34 steroid metabolites in 24-hour urine samples from 158 adults with PA (88 with unilateral PA [UPA] due to aldosterone-producing adenomas [APAs]; 70 with bilateral PA [BPA]) and 65 sex- and age-matched healthy controls. All APAs were resected and underwent targeted gene sequencing to detect somatic mutations associated with UPA. Patients with PA had increased urinary metabolite excretion of mineralocorticoids, glucocorticoids, and glucocorticoid precursors. Urine steroid metabolomics identified patients with PA with high accuracy, both when applied to all 34 or only the three most discriminative steroid metabolites (average areas under the receiver-operating characteristics curve [AUCs-ROC] 0.95-0.97). Whilst machine learning was suboptimal in differentiating UPA from BPA (average AUCs-ROC 0.65-0.73), it readily identified APA cases harbouring somatic KCNJ5 mutations (average AUCs-ROC 0.79-85). These patients showed a distinctly increased urine excretion of the hybrid steroid 18-hydroxycortisol and its metabolite 18-oxo-tetrahydrocortisol, the latter identified by machine learning as by far the most discriminative steroid. In conclusion, urine steroid metabolomics is a non-invasive candidate test for the accurate identification of PA cases and KCNJ5-mutated APAs.
Assuntos
Adenoma , Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Hiperaldosteronismo , Adulto , Humanos , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Hiperaldosteronismo/metabolismo , Adenoma Adrenocortical/genética , Adenoma/diagnóstico , Esteroides , Espectrometria de Massas , Aldosterona/metabolismo , Mutação , 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 , Neoplasias do Córtex Suprarrenal/genéticaRESUMO
Genetic abnormalities have been associated with primary aldosteronism, a major cause of secondary hypertension. This includes mutations in the KCNJ5 gene, which encodes G protein-gated inwardly rectifying K+ channel 4 (GIRK4). For example, the substitution of glycine with glutamic acid gives rise to the pathogenic GIRK4G151E mutation, which alters channel selectivity, making it more permeable to Na+ and Ca2+. While tertiapin and tertiapin-Q are well-known peptide inhibitors of the GIRK4WT channel, clinically, there is a need for the development of selective modulators of mutated channels, including GIRK4G151E. Using in silico methods, including homology modeling, protein-peptide docking, ligand-binding site prediction, and molecular docking, we aimed to explore potential modulators of GIRK4WT and GIRK4G151E. Firstly, protein-peptide docking was performed to characterize the binding site of tertiapin and its derivative to the GIRK4 channels. In accordance with previous studies, the peptide inhibitors preferentially bind to the GIRK4WT channel selectivity filter compared to GIRK4G151E. A ligand-binding site analysis was subsequently performed, resulting in the identification of two potential regions of interest: the central cavity and G-loop gate. Utilizing curated chemical libraries, we screened over 700 small molecules against the central cavity of the GIRK4 channels. Flavonoids, including luteolin-7-O-rutinoside and rutin, and the macrolides rapamycin and troleandomycin bound strongly to the GIRK4 channels. Similarly, xanthophylls, particularly luteoxanthin, bound to the central cavity with a strong preference towards the mutated GIRK4G151E channel compared to GIRK4WT. Overall, our findings suggest potential lead compounds for further investigation, particularly luteoxanthin, that may selectively modulate GIRK4 channels.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Hipertensão , Humanos , 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 , Ligantes , Simulação de Acoplamento Molecular , Proteínas de Ligação ao GTP/metabolismo , Peptídeos/metabolismo , Descoberta de DrogasRESUMO
Mutations in the KCNJ5 gene, encoding one of the major subunits of cardiac G-protein-gated inwardly rectifying K+ (GIRK) channels, have been recently linked to inherited forms of sinus node dysfunction. Here, the pathogenic mechanism of the W101C KCNJ5 mutation underlying sinus bradycardia in a patient-derived cellular disease model of sinus node dysfunction (SND) was investigated. A human-induced pluripotent stem cell (hiPSCs) line of a mutation carrier was generated, and CRISPR/Cas9-based gene targeting was used to correct the familial mutation as a control line. Both cell lines were further differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed GIRK channels which underly the acetylcholine-regulated K+ current (IK,ACh). hiPSC-CMs with the W101C KCNJ5 mutation (hiPSCW101C-CM) had a constitutively active IK,ACh under baseline conditions; the application of carbachol was able to increase IK,ACh, further indicating that not all available cardiac GIRK channels were open at baseline. Additionally, hiPSCW101C-CM had a more negative maximal diastolic potential (MDP) and a slower pacing frequency confirming the bradycardic phenotype. Of note, the blockade of the constitutively active GIRK channel with XAF-1407 rescued the phenotype. These results provide further mechanistic insights and may pave the way for the treatment of SND patients with GIRK channel dysfunction.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Humanos , Miócitos Cardíacos/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do Nó Sinusal/genética , Mutação , Arritmias Cardíacas/metabolismo , Acetilcolina/metabolismo , 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/metabolismoAssuntos
Adenoma , Adenoma Adrenocortical , Hiperaldosteronismo , Humanos , Aldosterona , Adrenalectomia , Adenoma Adrenocortical/genética , Mutação , Hiperaldosteronismo/complicações , Hiperaldosteronismo/genética , Hiperaldosteronismo/fisiopatologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genéticaRESUMO
Primary aldosteronism (PA) is the most common form of endocrine hypertension and is characterized by inappropriately elevated aldosterone production via a renin-independent mechanism. Driver somatic mutations for aldosterone excess have been found in approximately 90% of aldosterone-producing adenomas (APAs). Other causes of lateralized adrenal PA include aldosterone-producing nodules (APNs). Using next-generation sequencing, we identified recurrent in-frame deletions in SLC30A1 in four APAs and one APN (p.L51_A57del, n = 3; p.L49_L55del, n = 2). SLC30A1 encodes the ubiquitous zinc efflux transporter ZnT1 (zinc transporter 1). The identified SLC30A1 variants are situated close to the zinc-binding site (His43 and Asp47) in transmembrane domain II and probably cause abnormal ion transport. Cases of PA with SLC30A1 mutations showed male dominance and demonstrated increased aldosterone and 18-oxocortisol concentrations. Functional studies of the SLC30A151_57del variant in a doxycycline-inducible adrenal cell system revealed pathological Na+ influx. An aberrant Na+ current led to depolarization of the resting membrane potential and, thus, to the opening of voltage-gated calcium (Ca2+) channels. This resulted in an increase in cytosolic Ca2+ activity, which stimulated CYP11B2 mRNA expression and aldosterone production. Collectively, these data implicate zinc transporter alterations as a dominant driver of aldosterone excess in PA.
Assuntos
Adenoma , Neoplasias do Córtex Suprarrenal , Adenoma Adrenocortical , Proteínas de Transporte de Cátions , Hiperaldosteronismo , Masculino , Humanos , Aldosterona/genética , Adenoma Adrenocortical/genética , Hiperaldosteronismo/genética , Adenoma/genética , Adenoma/complicações , Mutação , Zinco/metabolismo , Neoplasias do Córtex Suprarrenal/genética , 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 , Proteínas de Transporte de Cátions/genéticaRESUMO
It is well known that the neuropeptide Y (NPY)/agouti-related peptide (AgRP) neurons increase appetite and decrease thermogenesis. Previous studies demonstrated that optogenetic and/or chemogenetic manipulations of NPY/AgRP neuronal activity alter food intake and/or energy expenditure (EE). However, little is known about intrinsic molecules regulating NPY/AgRP neuronal excitability to affect long-term metabolic function. Here, we found that the G protein-gated inwardly rectifying K+ (GIRK) channels are key to stabilize NPY/AgRP neurons and that NPY/AgRP neuron-selective deletion of the GIRK2 subunit results in a persistently increased excitability of the NPY/AgRP neurons. Interestingly, increased body weight and adiposity observed in the NPY/AgRP neuron-selective GIRK2 knockout mice were due to decreased sympathetic activity and EE, while food intake remained unchanged. The conditional knockout mice also showed compromised adaptation to coldness. In summary, our study identified GIRK2 as a key determinant of NPY/AgRP neuronal excitability and driver of EE in physiological and stress conditions.
Assuntos
Adiposidade , Proteína Relacionada com Agouti , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Obesidade , Animais , Camundongos , Proteína Relacionada com Agouti/genética , Peso Corporal , Camundongos Knockout , Neurônios , Peptídeos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genéticaRESUMO
The relationship of KCNJ5 mutation with vascular function and vascular structure in aldosterone-producing adenoma (APA) patients before and after adrenalectomy remains unclear. The purpose of this study was to evaluate the influence of KCNJ5 mutation on vascular function and vascular structure in APA and the effects of adrenalectomy on vascular function and vascular structure in APA patients with and those without KCNJ5 mutation. Flow-mediated vasodilation (FMD), nitroglycerine-induced vasodilation (NID), brachial artery intima-media thickness (IMT), and brachial-ankle pulse wave velocity (baPWV) were measured to assess vascular function and vascular structure in 46 APA patients with KCNJ5 mutation and 23 APA patients without KCNJ5 mutation and in 69 matched pairs of patients with essential hypertension (EHT). FMD, NID, brachial IMT and baPVW were evacuated before adrenalectomy and at 12 weeks after adrenalectomy in APA patients with KCNJ5 mutation and APA patients without KCNJ5 mutation. FMD and NID were significantly lower in APA patients than in patients with EHT. There was no significant difference in FMD or NID between patients with and those without KCNJ5 mutation. In APA patients with KCNJ5 mutation, FMD and NID after adrenalectomy were significantly higher than those before adrenalectomy. In APA patients without KCNJ5 mutation, only NID after adrenalectomy was significantly higher than that before adrenalectomy. Endothelial function in APA patients with KCNJ5 mutation was improved by adrenalectomy in the early postoperative period. KCNJ5 mutation is a predictor for early resolution of endothelial function by adrenalectomy. This study was approved by principal authorities and ethical issues in Japan (URL for Clinical Trial: http://www.umin.ac.jp/ctr/index.htm Registration Number for Clinical Trial: UMIN000003409).
Assuntos
Adenoma , Hiperaldosteronismo , Humanos , Aldosterona , Índice Tornozelo-Braço , Adrenalectomia , Hiperaldosteronismo/genética , Hiperaldosteronismo/cirurgia , Análise de Onda de Pulso , Hipertensão Essencial , Mutação , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genéticaRESUMO
Abnormalities in G-protein-gated inwardly rectifying potassium (GIRK) channels have been implicated in diseased states of the cardiovascular system; however, the role of GIRK4 (Kir3.4) in cardiac physiology and pathophysiology has yet to be completely understood. Within the heart, the KACh channel, consisting of two GIRK1 and two GIRK4 subunits, plays a major role in modulating the parasympathetic nervous system's influence on cardiac physiology. Being that GIRK4 is necessary for the functional KACh channel, KCNJ5, which encodes GIRK4, it presents as a therapeutic target for cardiovascular pathology. Human variants in KCNJ5 have been identified in familial hyperaldosteronism type III, long QT syndrome, atrial fibrillation, and sinus node dysfunction. Here, we explore the relevance of KCNJ5 in each of these diseases. Further, we address the limitations and complexities of discerning the role of KCNJ5 in cardiovascular pathophysiology, as identical human variants of KCNJ5 have been identified in several diseases with overlapping pathophysiology.
Assuntos
Hiperaldosteronismo , Síndrome do QT Longo , Humanos , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Coração , Síndrome do Nó SinusalRESUMO
Sepsis has emerged as a global health burden associated with multiple organ dysfunction and 20% mortality rate in patients. Numerous clinical studies over the past two decades have correlated the disease severity and mortality in septic patients with impaired heart rate variability (HRV), as a consequence of impaired chronotropic response of sinoatrial node (SAN) pacemaker activity to vagal/parasympathetic stimulation. However, the molecular mechanism(s) downstream to parasympathetic inputs have not been investigated yet in sepsis, particularly in the SAN. Based on electrocardiography, fluorescence Ca2+ imaging, electrophysiology, and protein assays from organ to subcellular level, we report that impaired muscarinic receptor subtype 2-G protein-activated inwardly-rectifying potassium channel (M2R-GIRK) signaling in a lipopolysaccharide-induced proxy septic mouse model plays a critical role in SAN pacemaking and HRV. The parasympathetic responses to a muscarinic agonist, namely IKACh activation in SAN cells, reduction in Ca2+ mobilization of SAN tissues, lowering of heart rate and increase in HRV, were profoundly attenuated upon lipopolysaccharide-induced sepsis. These functional alterations manifested as a direct consequence of reduced expression of key ion-channel components (GIRK1, GIRK4, and M2R) in the mouse SAN tissues and cells, which was further evident in the human right atrial appendages of septic patients and likely not mediated by the common proinflammatory cytokines elevated in sepsis.