RESUMO
Voltage-sensitive potassium channels play an important role in controlling membrane potential and ionic homeostasis in the gut and have been implicated in gastrointestinal (GI) cancers. Through large-scale analysis of 897 patients with gastro-oesophageal adenocarcinomas (GOAs) coupled with in vitro models, we find KCNQ family genes are mutated in â¼30% of patients, and play therapeutically targetable roles in GOA cancer growth. KCNQ1 and KCNQ3 mediate the WNT pathway and MYC to increase proliferation through resultant effects on cadherin junctions. This also highlights novel roles of KCNQ3 in non-excitable tissues. We also discover that activity of KCNQ3 sensitises cancer cells to existing potassium channel inhibitors and that inhibition of KCNQ activity reduces proliferation of GOA cancer cells. These findings reveal a novel and exploitable role of potassium channels in the advancement of human cancer, and highlight that supplemental treatments for GOAs may exist through KCNQ inhibitors.
Assuntos
Adenocarcinoma , Canais de Potássio KCNQ , Humanos , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Canal de Potássio KCNQ2/fisiologia , Adenocarcinoma/genéticaRESUMO
BACKGROUND/AIMS: Tea, produced from the evergreen Camellia sinensis, has reported therapeutic properties against multiple pathologies, including hypertension. Although some studies validate the health benefits of tea, few have investigated the molecular mechanisms of action. The KCNQ5 voltage-gated potassium channel contributes to vascular smooth muscle tone and neuronal M-current regulation. METHODS: We applied electrophysiology, myography, mass spectrometry and in silico docking to determine effects and their underlying molecular mechanisms of tea and its components on KCNQ channels and arterial tone. RESULTS: A 1% green tea extract (GTE) hyperpolarized cells by augmenting KCNQ5 activity >20-fold at resting potential; similar effects of black tea were inhibited by milk. In contrast, GTE had lesser effects on KCNQ2/Q3 and inhibited KCNQ1/E1. Tea polyphenols epicatechin gallate (ECG) and epigallocatechin-3-gallate (EGCG), but not epicatechin or epigallocatechin, isoform-selectively hyperpolarized KCNQ5 activation voltage dependence. In silico docking and mutagenesis revealed that activation by ECG requires KCNQ5-R212, at the voltage sensor foot. Strikingly, ECG and EGCG but not epicatechin KCNQ-dependently relaxed rat mesenteric arteries. CONCLUSION: KCNQ5 activation contributes to vasodilation by tea; ECG and EGCG are candidates for future anti-hypertensive drug development.
Assuntos
Catequina/análogos & derivados , Canais de Potássio KCNQ/química , Canal de Potássio KCNQ1/química , Artérias Mesentéricas/efeitos dos fármacos , Extratos Vegetais/farmacologia , Chá/química , Animais , Sítios de Ligação , Catequina/química , Catequina/farmacologia , Canais de Potássio KCNQ/agonistas , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Canal de Potássio KCNQ1/antagonistas & inibidores , Canal de Potássio KCNQ1/genética , Canal de Potássio KCNQ1/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/fisiologia , Leite/química , Simulação de Acoplamento Molecular , Miografia , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Extratos Vegetais/química , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Técnicas de Cultura de Tecidos , Vasodilatação/efeitos dos fármacos , Vasodilatação/fisiologia , Xenopus laevisRESUMO
BACKGROUND: Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. METHODS: Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)-specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. RESULTS: Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. CONCLUSION: Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.
Assuntos
Biomarcadores Tumorais/sangue , DNA Tumoral Circulante/sangue , Citosina/química , Metilação de DNA , Idoso , Biomarcadores Tumorais/química , DNA Tumoral Circulante/química , Neoplasias Colorretais/sangue , DNA Antissenso/sangue , DNA Antissenso/química , Feminino , Humanos , Canais de Potássio KCNQ/genética , Masculino , Proteínas de Membrana/genética , Reação em Cadeia da Polimerase/métodos , Sulfitos/químicaRESUMO
The ß-secretase BACE1 is widely known for its pivotal role in the amyloidogenic pathway leading to Alzheimer's disease, but how its action on transmembrane proteins other than the amyloid precursor protein affects the nervous system is only beginning to be understood. We report here that BACE1 regulates neuronal excitability through an unorthodox, nonenzymatic interaction with members of the KCNQ (Kv7) family that give rise to the M-current, a noninactivating potassium current with slow kinetics. In hippocampal neurons from BACE1(-/-) mice, loss of M-current enhanced neuronal excitability. We relate the diminished M-current to the previously reported epileptic phenotype of BACE1-deficient mice. In HEK293T cells, BACE1 amplified reconstituted M-currents, altered their voltage dependence, accelerated activation, and slowed deactivation. Biochemical evidence strongly suggested that BACE1 physically associates with channel proteins in a ß-subunit-like fashion. Our results establish BACE1 as a physiologically essential constituent of regular M-current function and elucidate a striking new feature of how BACE1 impacts on neuronal activity in the intact and diseased brain.
Assuntos
Potenciais de Ação , Secretases da Proteína Precursora do Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Hipocampo/metabolismo , Canais de Potássio KCNQ/metabolismo , Secretases da Proteína Precursora do Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Células Cultivadas , Feminino , Células HEK293 , Hipocampo/citologia , Hipocampo/fisiologia , Humanos , Canais de Potássio KCNQ/genética , Masculino , Camundongos , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Células Piramidais/metabolismo , Células Piramidais/fisiologiaRESUMO
Mutations in KCNQ2 and KCNQ3 were originally described in infants with benign familial neonatal seizures (BFNS). Recently, KCNQ2 mutations have also been shown to cause epileptic encephalopathy. This report describes three infants carrying abnormalities of KCNQ2 and one infant with a KCNQ3 mutation. The different KCNQ2 abnormalities led to different phenotypes and included a novel intragenic duplication, c.419_430dup, in an infant with BFNS, a 0.761Mb 20q13.3 contiguous gene deletion in an infant with seizures at 3 months, and a recurrent de novo missense mutation c.881C>T in a neonate with "KCNQ2-encephalopathy." The mutation in KCNQ3, c.989G>A, was novel and occurred in an infant with BFNS. KCNQ-related seizures often present with tonic/clonic manifestations, cyanosis, or apnea. Certain genotype-phenotype correlations help predict outcome. Similarly affected family members suggests benign familial "KCNQ-related" epilepsy, whereas neonatal seizures with unexplained multifocal epileptiform discharges or burst suppression on electroencephalography, and acute abnormalities of the basal ganglia/thalami are suggestive of KCNQ2-encephalopathy, which is often sporadic. 20q13.33 contiguous gene deletion encompassing KCNQ2 may harbor atypical features depending on deletion size. Although the phenotype often guides direct targeted gene testing in these conditions, array CGH should also be considered in suspected sporadic or atypical familial cases to diagnose 20q13.33 deletion.
Assuntos
Epilepsia/genética , Canais de Potássio KCNQ/genética , Mutação/genética , Gânglios da Base/patologia , Deleção Cromossômica , Cromossomos Humanos Par 22/genética , Eletroencefalografia , Feminino , Estudos de Associação Genética , Humanos , Lactente , Recém-Nascido , Imageamento por Ressonância Magnética , Masculino , Fenótipo , Tálamo/patologiaRESUMO
In humans, KCNQ2/3 channels form an M-current that regulates neuronal excitability, with mutations in these channels causing benign neonatal familial convulsions. The M-current is important in mechanisms of neural plasticity underlying associative memory and in the response to ethanol, with KCNQ controlling the release of dopamine after ethanol exposure. We show that dKCNQ is broadly expressed in the nervous system, with targeted reduction in neuronal KCNQ increasing neural excitability and KCNQ overexpression decreasing excitability and calcium signalling, consistent with KCNQ regulating the resting membrane potential and neural release as in mammalian neurons. We show that the single KCNQ channel in Drosophila (dKCNQ) has similar electrophysiological properties to neuronal KCNQ2/3, including conserved acute sensitivity to ethanol block, with the fly channel (IC(50)â=â19.8 mM) being more sensitive than its mammalian ortholog (IC(50)â=â42.1 mM). This suggests that the role of KCNQ in alcohol behaviour can be determined for the first time by using Drosophila. We present evidence that loss of KCNQ function in Drosophila increased sensitivity and tolerance to the sedative effects of ethanol. Acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression produced ethanol hypersensitivity, suggesting that both act via a common mechanism involving membrane depolarisation and increased dopamine signalling leading to ethanol sedation.
Assuntos
Etanol/farmacologia , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Animais , Cálcio/metabolismo , Cruzamentos Genéticos , DNA Complementar/metabolismo , Drosophila melanogaster , Eletrofisiologia/métodos , Deleção de Genes , Células HEK293 , Humanos , Concentração Inibidora 50 , Mutação , Neurônios/metabolismo , Técnicas de Patch-Clamp , Interferência de RNARESUMO
OBJECTIVE: Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester. METHOD: Twenty-three fetal cells were isolated from maternal blood by removing the red blood cells by lysis or combining this with removal of large proportions of maternal white blood cells by magnetic-activated cell sorting. Fetal cells identified by XY fluorescence in situ hybridization and confirmed by reverse-color fluorescence in situ hybridization were shot off microscope slides by laser capture microdissection. The expression pattern of a subset of expressed genes was compared between fetal cells and maternal blood cells using stem cell microarray analysis. RESULTS: Twenty-eight genes were identified as fetal cell marker candidates. CONCLUSION: Of the 28 fetal marker candidate genes, five coded for proteins, which are located on the outer surface of the cell membrane and not expressed in blood. The protein product of these five genes, MMP14, MCAM, KCNQ4, CLDN6, and F3, may be used as markers for fetal cell enrichment.
Assuntos
Biomarcadores/sangue , Feto/citologia , Genes , Análise de Sequência com Séries de Oligonucleotídeos , Antígeno CD146/genética , Claudinas/genética , DNA Complementar/análise , Feminino , Humanos , Canais de Potássio KCNQ/genética , Microdissecção e Captura a Laser , Masculino , Metaloproteinase 14 da Matriz/genética , Gravidez , Análise para Determinação do SexoRESUMO
Sensorineural hearing loss (SNHL) comprises hearing disorders with diverse pathologies of the inner ear and the auditory nerve. To date, an unambiguous phenotypical characterization of the specific pathologies in an affected individual remains impossible. Here, we evaluated the use of scalp-recorded auditory steady-state responses (ASSR) and transient auditory brainstem responses (ABR) for differentiating the disease mechanisms underlying sensorineural hearing loss in well-characterized mouse models. We first characterized the ASSR evoked by sinusoidally amplitude-modulated tones in wild-type mice. ASSR were robustly elicited within three ranges of modulation frequencies below 200 Hz, from 200 to 600 Hz and beyond 600 Hz in most recordings. Using phase information we estimated the apparent ASSR latency to be about 3 ms, suggesting generation in the auditory brainstem. Auditory thresholds obtained by automated and visual analysis of ASSR recordings were comparable to those found with tone-burst evoked ABR in the same mice. We then recorded ASSR and ABR from mouse mutants bearing defects of either outer hair cell amplification (KCNQ4-knockout) or inner hair cell synaptic transmission (Bassoon-mutant). Both mutants showed an increase of ASSR and ABR thresholds of approximately 40 dB versus wild-type when investigated at 8 weeks of age. Mice with defective amplification displayed a steep rise of ASSR and ABR amplitudes with increasing sound intensity, presumably reflecting a strong recruitment of synchronously activated neural elements beyond threshold. In contrast, the amplitudes of ASSR and ABR responses of mice with impaired synaptic transmission grew very little with sound intensity. In summary, ASSR allow for a rapid, objective and frequency-specific hearing assessment and together with ABR and otoacoustic emissions can contribute to the differential diagnosis of SNHL.
Assuntos
Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Potenciais Evocados Auditivos/fisiologia , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/fisiopatologia , Estimulação Acústica , Animais , Diagnóstico Diferencial , Perda Auditiva Neurossensorial/genética , Canais de Potássio KCNQ/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Emissões Otoacústicas Espontâneas/fisiologia , FenótipoRESUMO
The family of Kv7 (KCNQ) potassium channels consists of five members. Kv7.2 and 3 are the primary molecular correlates of the M-current, but also Kv7.4 and Kv7.5 display M-current characteristics. M-channel modulators include blockers (e.g., linopirdine) for cognition enhancement and openers (e.g., retigabine) for treatment of epilepsy and neuropathic pain. We investigated the effect of a Bristol-Myers Squibb compound (S)-N-[1-(3-morpholin-4-yl-phenyl)-ethyl]-3-phenyl-acrylamide [(S)-1] on cloned human Kv7.1-5 potassium channels expressed in Xenopus laevis oocytes. Using two-electrode voltage-clamp recordings we found that (S)-1 blocks Kv7.1 and Kv7.1/KCNE1 currents. In contrast, (S)-1 produced a hyperpolarizing shift of the activation curve for Kv7.2, Kv7.2/Kv7.3, Kv7.4 and Kv7.5. Further, the compound enhanced the maximal current amplitude at all potentials for Kv7.4 and Kv7.5 whereas the combined activation/block of Kv7.2 and Kv7.2/3 was strongly voltage-dependent. The tryptophan residue 242 in S5, known to be crucial for the effect of retigabine, was also shown to be critical for the enhancing effect of (S)-1 and BMS204352. Furthermore, no additive effect on Kv7.4 current amplitude was observed when both retigabine and (S)-1 or BMS204352 were applied simultaneously. In conclusion, (S)-1 differentially affects the Kv7 channel subtypes and is dependent on a single tryptophan for the current enhancing effect in Kv7.4.