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1.
Mol Pharmacol ; 97(3): 145-158, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31871302

RESUMO

Smooth muscle cells express Kv7.4 and Kv7.5 voltage-dependent potassium channels, which have each been implicated as regulators of smooth muscle contractility, though they display different sensitivities to signaling via cAMP/protein kinase A (PKA) and protein kinase C (PKC). We expressed chimeric channels composed of different components of the Kv7.4 and Kv7.5 α-subunits in vascular smooth muscle cells to determine which components are essential for enhancement or inhibition of channel activity. Forskolin, an activator of the cAMP/PKA pathway, increased wild-type Kv7.5 but not wild-type Kv7.4 current amplitude. Replacing the amino terminus of Kv7.4 with the amino terminus of Kv7.5 conferred partial responsiveness to forskolin. In contrast, swapping carboxy-terminal phosphatidylinositol 4,5-bisphosphate (PIP2) binding domains, or the entire C terminus, was without effect on the forskolin response, but the latter conferred responsiveness to arginine-vasopressin (an inhibitory PKC-dependent response). Serine-to-alanine mutation at position 53 of the Kv7.5 amino terminus abrogated its ability to confer forskolin sensitivity to Kv7.4. Forskolin treatment reduced the sensitivity of Kv7.5 channels to Ciona intestinalis voltage-sensing phosphatase (Ci-VSP)-induced PIP2 depletion, whereas activation of PKC with phorbol-12-myristate-13-acetate potentiated the Ci-VSP-induced decline in Kv7.5 current amplitude. Our findings suggest that PKA-dependent phosphorylation of serine 53 on the amino terminus of Kv7.5 increases its affinity for PIP2, whereas PKC-dependent phosphorylation of the Kv7.5 carboxy terminus is associated with a reduction in PIP2 affinity; these changes in PIP2 affinity have corresponding effects on channel activity. Resting affinities for PIP2 differ for Kv7.4 and Kv7.5 based on differential responsiveness to Ci-VSP activation and different rates of current rundown in ruptured patch recordings. SIGNIFICANCE STATEMENT: Kv7.4 and Kv7.5 channels are known signal transduction intermediates and drug targets for regulation of smooth muscle tone. The present studies identify distinct functional domains that confer differential sensitivities of Kv7.4 and Kv7.5 to stimulatory and inhibitory signaling and reveal structural features of the channel subunits that determine their biophysical properties. These findings may improve our understanding of the roles of these channels in smooth muscle physiology and disease, particularly in conditions where Kv7.4 and Kv7.5 are differentially expressed.


Assuntos
Canais de Potássio KCNQ/química , Canais de Potássio KCNQ/metabolismo , Miócitos de Músculo Liso/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animais , Cardiotônicos/farmacologia , Linhagem Celular , Colforsina/farmacologia , Humanos , Canais de Potássio KCNQ/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia
2.
Commun Biol ; 2: 401, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31701029

RESUMO

Voltage-gated potassium (Kv) channel dysfunction causes a variety of inherited disorders, but developing small molecules that activate Kv channels has proven challenging. We recently discovered that the inhibitory neurotransmitter γ-aminobutyric acid (GABA) directly activates Kv channels KCNQ3 and KCNQ5. Here, finding that inhibitory neurotransmitter glycine does not activate KCNQs, we re-engineered it in silico to introduce predicted KCNQ-opening properties, screened by in silico docking, then validated the hits in vitro. Attaching a fluorophenyl ring to glycine optimized its electrostatic potential, converting it to a low-nM affinity KCNQ channel activator. Repositioning the phenyl ring fluorine and/or adding a methylsulfonyl group increased the efficacy of the re-engineered glycines and switched their target KCNQs. Combining KCNQ2- and KCNQ3-specific glycine derivatives synergistically potentiated KCNQ2/3 activation by exploiting heteromeric channel composition. Thus, in silico optimization and docking, combined with functional screening of only three compounds, facilitated re-engineering of glycine to develop several potent KCNQ activators.


Assuntos
Canais de Potássio KCNQ/química , Canais de Potássio KCNQ/metabolismo , Animais , Simulação por Computador , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Glicinérgicos/química , Glicinérgicos/metabolismo , Canais de Potássio KCNQ/genética , Canal de Potássio KCNQ2/química , Canal de Potássio KCNQ2/genética , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/química , Canal de Potássio KCNQ3/genética , Canal de Potássio KCNQ3/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Engenharia de Proteínas/métodos , Receptores da Glicina/antagonistas & inibidores , Eletricidade Estática , Biologia Sintética , Proteínas de Xenopus/química , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
3.
Proc Natl Acad Sci U S A ; 116(42): 21236-21245, 2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31570602

RESUMO

Botanical folk medicines have been used throughout human history to treat common disorders such as hypertension, often with unknown underlying mechanisms. Here, we discovered that hypotensive folk medicines from a genetically diverse range of plant species each selectively activated the vascular-expressed KCNQ5 potassium channel, a feature lacking in the modern synthetic pharmacopeia, whereas nonhypotensive plant extracts did not. Analyzing constituents of the hypotensive Sophora flavescens root, we found that the quinolizidine alkaloid aloperine is a KCNQ-dependent vasorelaxant that potently and isoform-selectively activates KCNQ5 by binding near the foot of the channel voltage sensor. Our findings reveal that KCNQ5-selective activation is a defining molecular mechanistic signature of genetically diverse traditional botanical hypotensives, transcending plant genus and human cultural boundaries. Discovery of botanical KCNQ5-selective potassium channel openers may enable future targeted therapies for diseases including hypertension and KCNQ5 loss-of-function encephalopathy.


Assuntos
Canais de Potássio KCNQ/metabolismo , Animais , Masculino , Medicina Tradicional/métodos , Raízes de Plantas/química , Ratos , Ratos Wistar
4.
Int J Mol Sci ; 20(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487785

RESUMO

Kv7.2-Kv7.5 channels mediate the M-current (IKM), a K+-selective current regulating neuronal excitability and representing an attractive target for pharmacological therapy against hyperexcitability diseases such as pain. Kv7 channels interact functionally with transient receptor potential vanilloid 1 (TRPV1) channels activated by endogenous and/or exogenous pain-inducing substances, such as bradykinin (BK) or capsaicin (CAP), respectively; however, whether Kv7 channels of specific molecular composition provide a dominant contribution in BK- or CAP-evoked responses is yet unknown. To this aim, Kv7 transcripts expression and function were assessed in F11 immortalized sensorial neurons, a cellular model widely used to assess nociceptive molecular mechanisms. In these cells, the effects of the pan-Kv7 activator retigabine were investigated, as well as the effects of ICA-27243 and (S)-1, two Kv7 activators acting preferentially on Kv7.2/Kv7.3 and Kv7.4/Kv7.5 channels, respectively, on BK- and CAP-induced changes in intracellular Ca2+ concentrations ([Ca2+]i). The results obtained revealed the expression of transcripts of all Kv7 genes, leading to an IKM-like current. Moreover, all tested Kv7 openers inhibited BK- and CAP-induced responses by a similar extent (~60%); at least for BK-induced Ca2+ responses, the potency of retigabine (IC50~1 µM) was higher than that of ICA-27243 (IC50~5 µM) and (S)-1 (IC50~7 µM). Altogether, these results suggest that IKM activation effectively counteracts the cellular processes triggered by TRPV1-mediated pain-inducing stimuli, and highlight a possible critical contribution of Kv7.4 subunits.


Assuntos
Sinalização do Cálcio , Capsaicina/farmacologia , Canais de Potássio KCNQ/metabolismo , Células Receptoras Sensoriais/metabolismo , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/metabolismo , Animais , Bradicinina/farmacologia , Cálcio/metabolismo , Carbamatos/farmacologia , Linhagem Celular , Canais de Potássio Ativados por Cálcio de Condutância Alta/metabolismo , Moduladores de Transporte de Membrana/farmacologia , Fenilenodiaminas/farmacologia , Ratos , Células Receptoras Sensoriais/efeitos dos fármacos
5.
Int J Mol Sci ; 20(18)2019 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-31500374

RESUMO

Alcohol causes diverse acute and chronic symptoms that often lead to critical health problems. Exposure to ethanol alters the activities of sympathetic neurons that control the muscles, eyes, and blood vessels in the brain. Although recent studies have revealed the cellular targets of ethanol, such as ion channels, the molecular mechanism by which alcohol modulates the excitability of sympathetic neurons has not been determined. Here, we demonstrated that ethanol increased the discharge of membrane potentials in sympathetic neurons by inhibiting the M-type or Kv7 channel consisting of the Kv7.2/7.3 subunits, which were involved in determining the membrane potential and excitability of neurons. Three types of sympathetic neurons, classified by their threshold of activation and firing patterns, displayed distinct sensitivities to ethanol, which were negatively correlated with the size of the Kv7 current that differs depending on the type of neuron. Using a heterologous expression system, we further revealed that the inhibitory effects of ethanol on Kv7.2/7.3 currents were facilitated or diminished by adjusting the amount of plasma membrane phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). These results suggested that ethanol and PI(4,5)P2 modulated gating of the Kv7 channel in superior cervical ganglion neurons in an antagonistic manner, leading to regulation of the membrane potential and neuronal excitability, as well as the physiological functions mediated by sympathetic neurons.


Assuntos
Potenciais de Ação , Etanol/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Gânglio Cervical Superior/citologia , Biomarcadores , Membrana Celular/metabolismo , Células Cultivadas , Etanol/farmacologia , Expressão Gênica , Canais de Potássio KCNQ/antagonistas & inibidores , Canais de Potássio KCNQ/genética
6.
Methods Mol Biol ; 2007: 79-87, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31148107

RESUMO

Hydrogen sulfide (H2S) evokes vascular effects through several mechanisms including in wide part the activation of some ion channels such as ATP-sensitive potassium (KATP) channels and voltage-gated Kv7 potassium channels. Electrophysiological methods are very accurate, but they require high expertise and high specialized equipment. A more manageable fluorimetric technique which allows to record the membrane potential variations by the employment of an anionic bis-oxonol dye named DiBac4(3) with the administration of different blockers of several potassium channels could be useful to discover the targets of H2S-induced vascular hyperpolarization. Coupled with this technique, a fluorimetric detection (by the use of WSP-1 dye) of H2S generation in human vascular smooth muscle cells after H2S-donor administration could confirm the ability of these molecules to evoke the hyperpolarizing effect through the H2S release.


Assuntos
Aorta/metabolismo , Fluorometria , Sulfeto de Hidrogênio , Canais de Potássio KCNQ/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Aorta/citologia , Barbitúricos/química , Linhagem Celular , Humanos , Sulfeto de Hidrogênio/metabolismo , Sulfeto de Hidrogênio/farmacologia , Isoxazóis/química , Canais de Potássio KCNQ/antagonistas & inibidores , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Bloqueadores dos Canais de Potássio/farmacologia
7.
J Pharmacol Exp Ther ; 370(3): 472-479, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31235533

RESUMO

Celecoxib is a nonsteroidal anti-inflammatory drug (NSAID) commonly used to treat pain conditions in humans. In addition to its blocking activity on cyclooxygenase (COX) enzymes, several other targets could contribute to its analgesic activity. Here we explore the spinal antinociceptive actions of celecoxib and the potential implication of Kv7 channels in mediating its effects. Spinal cord in vitro preparations from hind paw-inflamed animals were used to assess the segmental sensory-motor and the early sensory processing of nociceptive information. Electrophysiological recordings of ventral roots and dorsal horn neurones were obtained, and the effects of celecoxib and Kv7 modulators on responses to repetitive dorsal root stimulation at C-fiber intensity were assessed. Celecoxib applied at clinically relevant concentrations produced depressant effects on responses to dorsal root stimulation recorded from both ventral roots and individual dorsal horn neurones; by contrast, the non-nociceptive monosynaptic reflex was unaffected. The NSAID indomethacin had no effect on spinal reflexes, but further coapplication of celecoxib still produced depressant effects. The depressant actions of celecoxib were abolished after Kv7 channel blockade and mimicked by its structural analog dimethyl-celecoxib, which lacks COX-blocking activity. The present results identify Kv7 channels as novel central targets for celecoxib, which may be relevant to its analgesic effect. This finding contributes to better understand the pharmacology of celecoxib and reinforces both the role of Kv7 channels in modulating the excitability of central pain pathways and its validity as target for the design of analgesics.


Assuntos
Analgésicos/farmacologia , Celecoxib/farmacologia , Canais de Potássio KCNQ/metabolismo , Medula Espinal/efeitos dos fármacos , Animais , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Células do Corno Posterior/citologia , Células do Corno Posterior/efeitos dos fármacos , Medula Espinal/citologia , Medula Espinal/fisiologia
8.
Commun Biol ; 2: 145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31044170

RESUMO

Little is known about the properties and function of ion channels that affect synaptic terminal-resting properties. One particular subthreshold-active ion channel, the Kv7 potassium channel, is highly localized to axons, but its role in regulating synaptic terminal intrinsic excitability and release is largely unexplored. Using electrophysiological recordings together with computational modeling, we found that the KV7 current was active at rest in adult hippocampal mossy fiber synaptic terminals and enhanced their membrane conductance. The current also restrained action potential-induced Ca2+ influx via N- and P/Q-type Ca2+ channels in boutons. This was associated with a substantial reduction in the spike half-width and afterdepolarization following presynaptic spikes. Further, by constraining spike-induced Ca2+ influx, the presynaptic KV7 current decreased neurotransmission onto CA3 pyramidal neurons and short-term synaptic plasticity at the mossy fiber-CA3 synapse. This is a distinctive mechanism by which KV7 channels influence hippocampal neuronal excitability and synaptic plasticity.


Assuntos
Potenciais de Ação/fisiologia , Cálcio/metabolismo , Potenciais Pós-Sinápticos Excitadores/fisiologia , Canais de Potássio KCNQ/metabolismo , Fibras Musgosas Hipocampais/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transmissão Sináptica/fisiologia , Animais , Região CA3 Hipocampal/metabolismo , Biologia Computacional/métodos , Masculino , Plasticidade Neuronal/fisiologia , Células Piramidais/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/metabolismo
9.
Cell Death Dis ; 10(4): 310, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30952836

RESUMO

Amyotrophic lateral sclerosis (ALS) is a type of motor neuron disease (MND) in which humans lose motor functions due to progressive loss of motoneurons in the cortex, brainstem, and spinal cord. In patients and in animal models of MND it has been observed that there is a change in the properties of motoneurons, termed neuronal hyperexcitability, which is an exaggerated response of the neurons to a stimulus. Previous studies suggested neuronal excitability is one of the leading causes for neuronal loss, however the factors that instigate excitability in neurons over the course of disease onset and progression are not well understood, as these studies have looked mainly at embryonic or early postnatal stages (pre-symptomatic). As hyperexcitability is not a static phenomenon, the aim of this study was to assess the overall excitability of upper motoneurons during disease progression, specifically focusing on their oscillatory behavior and capabilities to fire repetitively. Our results suggest that increases in the intrinsic excitability of motoneurons are a global phenomenon of aging, however the cellular mechanisms that underlie this hyperexcitability are distinct in SOD1G93A ALS mice compared with wild-type controls. The ionic mechanism driving increased excitability involves alterations of the expression levels of HCN and KCNQ channel genes leading to a complex dynamic of H-current and M-current activation. Moreover, we show a negative correlation between the disease onset and disease progression, which correlates with a decrease in the expression level of HCN and KCNQ channels. These findings provide a potential explanation for the increased vulnerability of motoneurons to ALS with aging.


Assuntos
Envelhecimento , Esclerose Amiotrófica Lateral/fisiopatologia , Excitabilidade Cortical , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios Motores/fisiologia , Superóxido Dismutase-1/genética , Envelhecimento/metabolismo , Envelhecimento/patologia , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Excitabilidade Cortical/efeitos dos fármacos , Excitabilidade Cortical/genética , Modelos Animais de Doenças , Progressão da Doença , Feminino , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais de Potássio KCNQ/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Potenciais da Membrana/fisiologia , Camundongos , Camundongos Transgênicos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Superóxido Dismutase-1/metabolismo
10.
J Vasc Res ; 56(1): 11-15, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30763932

RESUMO

Peripartum cardiomyopathy (PPCM) is a rare form of congestive heart failure characterized by left ventricular dysfunction that develops towards the end of pregnancy or during the early postpartum phase. Even though the majority of PPCM patients show partial or complete recovery of their heart functions, the mortality rate of PPCM remains high. Previous research has suggested that vascular dysfunction triggered by late-gestational hormones and potent anti-angiogenic factors play key roles in the pathogenesis of PPCM; however, the exact mechanisms remain elusive due to limited patient tissues for characterization. Here, we report a case of PPCM where the coronary vessels from the patient's explanted heart showed marked vascular dysfunction with impaired nitric oxide response. Importantly, these vessels exhibited deficient adenosine-mediated vasorelaxation when subjected to myograph studies, suggesting impaired Kv7 ion channels. Results from this work may lead to new therapeutic strategies for improving Kv7 function in PPCM patients.


Assuntos
Cardiomiopatias/etiologia , Doença da Artéria Coronariana/etiologia , Vasos Coronários/fisiopatologia , Período Periparto , Vasodilatação , Disfunção Ventricular Esquerda/etiologia , Função Ventricular Esquerda , Cardiomiopatias/diagnóstico por imagem , Cardiomiopatias/fisiopatologia , Cardiomiopatias/cirurgia , Doença da Artéria Coronariana/diagnóstico por imagem , Doença da Artéria Coronariana/fisiopatologia , Doença da Artéria Coronariana/cirurgia , Vasos Coronários/diagnóstico por imagem , Vasos Coronários/metabolismo , Progressão da Doença , Feminino , Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/cirurgia , Transplante de Coração , Humanos , Canais de Potássio KCNQ/metabolismo , Pessoa de Meia-Idade , Gravidez , Disfunção Ventricular Esquerda/diagnóstico por imagem , Disfunção Ventricular Esquerda/fisiopatologia , Disfunção Ventricular Esquerda/cirurgia
11.
J Biol Chem ; 294(15): 6094-6112, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30808708

RESUMO

Calmodulin (CaM) conveys intracellular Ca2+ signals to KCNQ (Kv7, "M-type") K+ channels and many other ion channels. Whether this "calmodulation" involves a dramatic structural rearrangement or only slight perturbations of the CaM/KCNQ complex is as yet unclear. A consensus structural model of conformational shifts occurring between low nanomolar and physiologically high intracellular [Ca2+] is still under debate. Here, we used various techniques of biophysical chemical analyses to investigate the interactions between CaM and synthetic peptides corresponding to the A and B domains of the KCNQ4 subtype. We found that in the absence of CaM, the peptides are disordered, whereas Ca2+/CaM imposed helical structure on both KCNQ A and B domains. Isothermal titration calorimetry revealed that Ca2+/CaM has higher affinity for the B domain than for the A domain of KCNQ2-4 and much higher affinity for the B domain when prebound with the A domain. X-ray crystallography confirmed that these discrete peptides spontaneously form a complex with Ca2+/CaM, similar to previous reports of CaM binding KCNQ-AB domains that are linked together. Microscale thermophoresis and heteronuclear single-quantum coherence NMR spectroscopy indicated the C-lobe of Ca2+-free CaM to interact with the KCNQ4 B domain (Kd ∼10-20 µm), with increasing Ca2+ molar ratios shifting the CaM-B domain interactions via only the CaM C-lobe to also include the N-lobe. Our findings suggest that in response to increased Ca2+, CaM undergoes lobe switching that imposes a dramatic mutually induced conformational fit to both the proximal C terminus of KCNQ4 channels and CaM, likely underlying Ca2+-dependent regulation of KCNQ gating.


Assuntos
Cálcio/química , Calmodulina/química , Canais de Potássio KCNQ/química , Animais , Células CHO , Cálcio/metabolismo , Calmodulina/genética , Calmodulina/metabolismo , Cricetulus , Cristalografia por Raios X , Humanos , Ativação do Canal Iônico , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína
12.
J Cell Physiol ; 234(8): 12714-12726, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30523632

RESUMO

KCNQ/M potassium channels play a vital role in neuronal excitability; however, it is required to explore their pharmacological modulation on N-Methyl- d-aspartic acid receptors (NMDARs)-mediated glutamatergic transmission of neurons upon ischemic insults. In the current study, both presynaptic glutamatergic release and activities of NMDARs were measured by NMDAR-induced miniature excitatory postsynaptic currents (mEPSCs) in cultured cortical neurons of C57 mice undergoing oxygen and glucose deprivation (OGD) or OGD/reperfusion (OGD/R). The KCNQ/M-channel opener, retigabine (RTG), suppressed the overactivation of postsynaptic NMDARs induced by OGD and then NO transient; RTG also decreased OGD-induced neuronal death measured with MTT assay, suggesting the beneficial role of KCNQ/M-channels for the neurons exposed to ischemic insults. However, when the neurons exposed to the subsequent reperfusion, KCNQ/M-channels played a differential role from its protective effect. OGD/R increased presynaptic glutamatergic release, which was further augmented by RTG or decreased by KCNQ/M-channel blocker, XE991. Reactive oxygen species (ROS) were produced partly in a NO-dependent manner. In addition, XE991 decreased neuronal injuries upon reperfusion measured with DCF and PI staining. Meanwhile, the addition of RTG upon OGD or XE991 upon reperfusion can reverse OGD or OGD/R-reduced mitochondrial membrane potential. Our present study indicates the dual role of KCNQ/M-channels in OGD and OGD/R, which will decide the fate of neurons. Provided that activation of KCNQ/M-channels has differential effects on neuronal injuries during OGD or OGD/R, we propose that therapy targeting KCNQ/M-channels may be effective for ischemic injuries but the proper timing is so crucial for the corresponding treatment.


Assuntos
Glucose/metabolismo , Canais de Potássio KCNQ/metabolismo , Neurônios/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Carbamatos/farmacologia , Feminino , Ácido Glutâmico/metabolismo , Masculino , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/efeitos dos fármacos , Fenilenodiaminas/farmacologia
13.
J Neurosci ; 39(3): 382-392, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30459225

RESUMO

Pain is a complex process that involves both detection in the peripheral nervous system and perception in the CNS. Individual-to-individual differences in pain are well documented, but not well understood. Here we capitalized on inherited erythromelalgia (IEM), a well characterized human genetic model of chronic pain, and studied a unique family containing related IEM subjects with the same disease-causing NaV1.7 mutation, which is known to make dorsal root ganglion (DRG) neurons hyperexcitable, but different pain profiles (affected son with severe pain, affected mother with moderate pain, and an unaffected father). We show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell (iPSC)-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing (WES) and dynamic clamp, we show that it is possible to pinpoint a specific variant of another gene, KCNQ in this particular kindred, that modulates the excitability of iPSC-derived sensory neurons in this family. While different gene variants may modulate DRG neuron excitability and thereby contribute to interindividual differences in pain in other families, this study shows that subject-specific iPSCs can be used to model interindividual differences in pain. We further provide proof-of-principle that iPSCs, WES, and dynamic clamp can be used to investigate peripheral mechanisms and pinpoint specific gene variants that modulate pain signaling and contribute to interindividual differences in pain.SIGNIFICANCE STATEMENT Individual-to-individual differences in pain are well documented, but not well understood. In this study, we show, first, that, at least in some cases, relative sensitivity to pain can be modeled in subject-specific induced pluripotent stem cell-derived sensory neurons in vitro; second, that, in some cases, mechanisms operating in peripheral sensory neurons contribute to interindividual differences in pain; and third, using whole exome sequencing and dynamic clamp, we show that it is possible to pinpoint a specific gene variant that modulates pain signaling and contributes to interindividual differences in pain.


Assuntos
Dor Crônica/genética , Células-Tronco Pluripotentes Induzidas , Resiliência Psicológica , Adulto , Criança , Dor Crônica/fisiopatologia , Eritromelalgia/genética , Eritromelalgia/fisiopatologia , Potenciais Pós-Sinápticos Excitadores , Exoma/genética , Feminino , Gânglios Espinais/citologia , Gânglios Espinais/fisiopatologia , Humanos , Imuno-Histoquímica , Individualidade , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Masculino , Potenciais da Membrana , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Medição da Dor , Técnicas de Patch-Clamp , Células Receptoras Sensoriais
14.
Pflugers Arch ; 471(2): 313-327, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30250967

RESUMO

Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are synthesized from dietary carbohydrates by colonic bacterial fermentation. These SCFAs supply energy, suppress cancer, and affect ion transport. However, their roles in ion transport and regulation in the intracellular environment remain unknown. In order to elucidate the roles of SCFAs, we measured short-circuit currents (ISC) and performed RT-PCR and immunohistochemical analyses of ion transporters in rat rectal colon. The application of 30 mM butyrate shifted ISC in a negative direction, but did not attenuate the activity of epithelial Na+ channels (ENaC). The application of bumetanide, a Na+-K+-2Cl- cotransporter inhibitor, to the basolateral side reduced the negative ISC shift induced by butyrate. The application of XE991, a KCNQ-type K+ channel inhibitor, to the apical side decreased the ISC shift induced by butyrate in a dose-dependent manner. The ISC shift was independent of HCO3- and insensitive to ibuprofen, an SMCT1 inhibitor. The mucosa from rat rectal colon expressed mRNAs of H+-coupled monocarboxylate transporters (MCT1, MCT4, and MCT5, also referred to as SLC16A1, SLC16A3, and SLC16A4, respectively). RT-PCR and immunofluorescence analyses demonstrated that KCNQ2 and KCNQ4 localized to the apical membrane of surface cells in rat rectal colon. These results indicate that butyrate, which may be transported by H+-coupled monocarboxylate transporters, activates K+ secretion through KCNQ-type K+ channels on the apical membrane in rat rectal colon. KCNQ-type K+ channels may play a role in intestinal secretion and defense mechanisms in the gastrointestinal tract.


Assuntos
Butiratos/metabolismo , Colo/metabolismo , Secreções Intestinais/metabolismo , Potássio/metabolismo , Reto/metabolismo , Animais , Antracenos/farmacologia , Bumetanida/farmacologia , Cloretos/metabolismo , Colo/efeitos dos fármacos , Ácidos Graxos Voláteis/metabolismo , Absorção Intestinal/efeitos dos fármacos , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Secreções Intestinais/efeitos dos fármacos , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Canais de Potássio KCNQ/metabolismo , Masculino , Transportadores de Ácidos Monocarboxílicos/metabolismo , Propionatos/farmacologia , Ratos , Ratos Sprague-Dawley , Sódio/metabolismo , Canais de Sódio/metabolismo
15.
Physiol Rep ; 6(23): e13920, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30548427

RESUMO

In tauopathies, such as Alzheimer's disease with or without concomitant amyloid ß plaques, cerebral arteries display pathological remodeling, leading to reduced brain tissue oxygenation and cognitive impairment. The precise mechanisms that underlie this vascular dysfunction remain unclear. Kv7 voltage-dependent K+ channels contribute to the development of myogenic tone in rat cerebral arteries. Thus, we hypothesized that Kv7 channel function would be impaired in the cerebral arteries of a tauopathy mouse model (rTg4510), which might underlie cerebral hypoperfusion associated with the development of neurofibrillary tangles in tauopathies. To test our hypothesis we performed wire myography and quantitative PCR on cerebral arteries, mesenteric arteries and the inferior frontotemporal region of the brain surrounding the middle cerebral artery from tau transgenic mice (rTg4510) and aged-matched controls. We also performed whole-cell patch clamp experiments on HEK293 cells stably expressing Kv7.4. Here, we show that Kv7 channels are functionally impaired in the cerebral arteries of rTg4510 mice, but not in mesenteric arteries from the same mice. The quantitative PCR analysis of the cerebral arteries found no change in the expression of the genes encoding the Kv7 channel α-subunits, however, we found reduced expression of the ancillary subunit, KCNE5 (also termed KCNE1L), in the cerebral arteries of rTg4510 mice. In the brain, rTg4510 mice showed reduced expression of Kv7.3, Kv7.5, and Kv2.1. Co-expression of KCNE5 with Kv7.4 in HEK293 cells produced larger currents at voltages >0 mV and increased the deactivation time for the Kv7.4 channel. Thus, our results demonstrate that Kv7 channel function is attenuated in the cerebral arteries of Tg4510 mice, which may result from decreased KCNE5 expression. Reduced Kv7 channel function might contribute to cerebral hypoperfusion in tauopathies, such as Alzheimer's disease.


Assuntos
Artérias Cerebrais/metabolismo , Canais de Potássio KCNQ/metabolismo , Tauopatias/metabolismo , Potenciais de Ação , Animais , Artérias Cerebrais/fisiopatologia , Células HEK293 , Humanos , Canais de Potássio KCNQ/genética , Masculino , Camundongos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo
16.
Sci Rep ; 8(1): 16659, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30413759

RESUMO

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to a type of nonsyndromic hearing loss, deafness nonsyndromic autosomal dominant 2 (DFNA2). We performed whole-exome sequencing for 98 families with hearing loss and found mutations in KCNQ4 in five families. In this study, we characterized two novel mutations in KCNQ4: a missense mutation (c.796G>T; p.Asp266Tyr) and an in-frame deletion mutation (c.259_267del; p.Val87_Asn89del). p.Asp266Tyr located in the channel pore region resulted in early onset and moderate hearing loss, whereas p.Val87_Asn89del located in the N-terminal cytoplasmic region resulted in late onset and high frequency-specific hearing loss. When heterologously expressed in HEK 293 T cells, both mutant proteins did not show defects in protein trafficking to the plasma membrane or in interactions with wild-type (WT) KCNQ4 channels. Patch-clamp analysis demonstrated that both p.Asp266Tyr and p.Val87_Asn89del mutant channels lost conductance and were completely unresponsive to KCNQ activators, such as retigabine, zinc pyrithione, and ML213. Channels assembled from WT-p.Asp266Tyr concatemers, like those from WT-WT concatemers, exhibited conductance and responsiveness to KCNQ activators. However, channels assembled from WT-p.Val87_Asn89del concatemers showed impaired conductance, suggesting that p.Val87_Asn89del caused complete loss-of-function with a strong dominant-negative effect on functional WT channels. Therefore, the main pathological mechanism may be related to loss of K+ channel activity, not defects in trafficking.


Assuntos
Surdez/genética , Canais de Potássio KCNQ/genética , Canais de Potássio KCNQ/metabolismo , Mutação , Sequenciamento Completo do Exoma/métodos , Adulto , Sequência de Aminoácidos , Criança , Análise Mutacional de DNA , Surdez/patologia , Feminino , Células HEK293 , Células HeLa , Humanos , Masculino , Linhagem
17.
Bioorg Med Chem Lett ; 28(23-24): 3793-3797, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30327146

RESUMO

Neuronal voltage-gated potassium channels, KV7s, are the molecular mediators of the M current and regulate membrane excitability in the central and peripheral neuronal systems. Herein, we report novel small molecule KV7 openers that demonstrate anti-seizure activities in electroshock and pentylenetetrazol-induced seizure models without influencing Rotarod readouts in mice. The anti-seizure activity was determined to be proportional to the unbound concentration in the brain. KV7 channels are also expressed in the bladder smooth muscle (detrusor) and activation of these channels may cause localized undesired effects. Therefore, the impact of individual KV7 isoforms was investigated in human detrusor tissue using a panel of KV7 openers with distinct activity profiles among KV7 isoforms. KCNQ4 and KCNQ5 mRNA were highly expressed in detrusor tissue, yet a compound that has significantly reduced activity on homomeric KV7.4 did not reduce detrusor contraction. This may suggest that the homomeric KV7.4 channel plays a less significant role in bladder contraction and further investigation is needed.


Assuntos
Anticonvulsivantes/química , Anticonvulsivantes/farmacologia , Epilepsia/tratamento farmacológico , Canais de Potássio KCNQ/metabolismo , Convulsões/tratamento farmacológico , Animais , Anticonvulsivantes/uso terapêutico , Epilepsia/metabolismo , Humanos , Camundongos , Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Músculo Liso/metabolismo , Isoformas de Proteínas/metabolismo , Convulsões/metabolismo , Bexiga Urinária/efeitos dos fármacos , Bexiga Urinária/metabolismo
18.
Bioorg Med Chem Lett ; 28(17): 3004-3008, 2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-30061030

RESUMO

KCNQ (Kv7) has emerged as a validated target for the development of novel anti-epileptic drugs. In this paper, a series of novel N-phenylbutanamide derivatives were designed, synthesized and evaluated as KCNQ openers for the treatment of epilepsy. These compounds were evaluated for their KCNQ opening activity in vitro and in vivo. Several compounds were found to be potent KCNQ openers. Compound 1 with favorable in vitro activity was submitted to evaluation in vivo. Results showed that compound 1 owned significant anti-convulsant activity with no adverse effects. It was also found to posses favorable pharmacokinetic profiles in rat. This research may provide novel potent compounds for the discovery of KCNQ openers in treating epilepsy.


Assuntos
Desenho de Fármacos , Epilepsia/tratamento farmacológico , Canais de Potássio KCNQ/antagonistas & inibidores , Fenilbutiratos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Animais , Relação Dose-Resposta a Droga , Epilepsia/metabolismo , Teste de Esforço , Canais de Potássio KCNQ/metabolismo , Camundongos , Estrutura Molecular , Fenilbutiratos/síntese química , Fenilbutiratos/química , Bloqueadores dos Canais de Potássio/síntese química , Bloqueadores dos Canais de Potássio/química , Ratos , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição Tecidual
19.
Int J Mol Sci ; 19(8)2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-30061510

RESUMO

ß-adrenergic receptor (ßAR) activation promotes relaxation of both vascular and airway smooth muscle cells (VSMCs and ASMCs, respectively), though the signaling mechanisms have not been fully elucidated. We previously found that the activity of Kv7.5 voltage-activated potassium channels in VSMCs is robustly enhanced by activation of ßARs via a mechanism involving protein kinase A (PKA)-dependent phosphorylation. We also found that enhancement of Kv7 channel activity in ASMCs promotes airway relaxation. Here we provide evidence that Kv7.5 channels are natively expressed in primary cultures of human ASMCs and that they conduct currents which are robustly enhanced in response to activation of the ßAR/cyclic adenosine monophosphate (cAMP)/PKA pathway. MIT Scansite software analysis of putative PKA phosphorylation sites on Kv7.5 identified 8 candidate serine or threonine residues. Each residue was individually mutated to an alanine to prevent its phosphorylation and then tested for responses to ßAR activation or to stimuli that elevate cAMP levels. Only the mutation of serine 53 (S53A), located on the amino terminus of Kv7.5, significantly reduced the increase in Kv7.5 current in response to these stimuli. A phospho-mimic mutation (S53D) exhibited characteristics of ßAR-activated Kv7.5. Serine-to-alanine mutations of 6 putative PKA phosphorylation sites on the Kv7.5 C-terminus, individually or in combination, did not significantly reduce the enhancement of the currents in response to forskolin treatment (to elevate cAMP levels). We conclude that phosphorylation of S53 on the amino terminus of Kv7.5 is essential for PKA-dependent enhancement of channel activity in response to ßAR activation in vascular and airway smooth muscle cells.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio KCNQ/metabolismo , Miócitos de Músculo Liso/citologia , Transdução de Sinais , Traqueia/citologia , Células Cultivadas , AMP Cíclico/metabolismo , Humanos , Miócitos de Músculo Liso/metabolismo , Fosforilação , Receptores Adrenérgicos beta/metabolismo , Traqueia/metabolismo
20.
Mol Pain ; 14: 1744806918793229, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30027794

RESUMO

Background Diabetic neuropathic pain is poorly controlled by analgesics, and the precise molecular mechanisms underlying hyperalgesia remain unclear. The KCNQ2/3/5 channels expressed in dorsal root ganglion neurons are important in pain transmission. The expression and activity of KCNQ2/3/5 channels in dorsal root ganglion neurons in rats with diabetic neuropathic pain were investigated in this study. Methods The mRNA levels of KCNQ2/3/5 channels were analyzed by real-time polymerase chain reaction. The protein levels of KCNQ2/3/5 channels were evaluated by Western blot assay. KCNQ2/3/5 channel expression in situ in dorsal root ganglion neurons was detected by double fluorescent labeling technique. M current (IM) density and neuronal excitability were determined by whole-cell voltage and current clamp recordings. Mechanical allodynia and thermal hyperalgesia were assessed by von Frey filaments and plantar analgesia tester, respectively. Results The mRNA and protein levels of KCNQ2/3/5 channels significantly decreased, followed by the reduction of IM density and elevation of neuronal excitability of dorsal root ganglion neurons from diabetic rats. Activation of KCNQ channels with retigabine reduced the hyperexcitability and inhibition of KCNQ channels with XE991 enhanced the hyperexcitability. Administration of retigabine alleviated both mechanical allodynia and thermal hyperalgesia, while XE991 augmented both mechanical allodynia and thermal hyperalgesia in diabetic neuropathic pain in rats. Conclusion The findings elucidate the mechanisms by which downregulation of the expression and reduction of the activity of KCNQ2/3/5 channels in diabetic rat dorsal root ganglion neurons contribute to neuronal hyperexcitability, which results in hyperalgesia. These data provide intriguing evidence that activation of KCNQ2/3/5 channels might be the potential new targets for alleviating diabetic neuropathic pain symptoms.


Assuntos
Neuropatias Diabéticas/patologia , Gânglios Espinais/patologia , Canais de Potássio KCNQ/metabolismo , Neurônios/metabolismo , Animais , Antracenos/farmacologia , Carbamatos/farmacologia , Carbamatos/uso terapêutico , Células Cultivadas , Neuropatias Diabéticas/induzido quimicamente , Neuropatias Diabéticas/tratamento farmacológico , Modelos Animais de Doenças , Regulação da Expressão Gênica/efeitos dos fármacos , Canais de Potássio KCNQ/genética , Moduladores de Transporte de Membrana/farmacologia , Moduladores de Transporte de Membrana/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Neurônios/efeitos dos fármacos , Limiar da Dor/efeitos dos fármacos , Limiar da Dor/fisiologia , Técnicas de Patch-Clamp , Fenilenodiaminas/farmacologia , Fenilenodiaminas/uso terapêutico , Bloqueadores dos Canais de Potássio/farmacologia , RNA Mensageiro/metabolismo , Ratos , Estreptozocina/toxicidade , Canais de Cátion TRPV/metabolismo
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