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1.
Behav Brain Res ; 413: 113446, 2021 09 10.
Artículo en Inglés | MEDLINE | ID: mdl-34224765

RESUMEN

TREK-1 channels are expressed in small nociceptive dorsal root ganglion (DRG) neurons where they participate in acute inflammatory and neuropathic pain. However, the role of TREK-1 in persistent pain is not well understood. The aim of this study was to investigate the local peripheral and spinal participation of TREK-1 in formalin-induced acute and long-lasting nociceptive hypersensitivity. Local peripheral or intrathecal pre-treatment with spadin, selective blocker of TREK-1, increased acute flinching behavior and secondary mechanical allodynia and hyperalgesia behavior observed 6 days after formalin injection. Local peripheral or intrathecal pre-treatment with BL-1249, selective opener of TREK-1, decreased long-lasting secondary mechanical allodynia and hyperalgesia induced by formalin. Pre-treatment with BL-1249 prevented the pro-nociceptive effect of spadin on acute nociception and long-lasting mechanical allodynia and hyperalgesia in rats. Pre-treatment with two recombinant channels that produce a high TREK-1 current, S300A and S333A (non-phosphorylated state of TREK-1), reduced formalin-induced acute pain and long-lasting mechanical allodynia and hyperalgesia. Besides, post-treatment with S300A, S333A or BL-1249 reversed long-lasting mechanical allodynia and hyperalgesia induced by formalin. Formalin increased TREK-1 expression at 1 and 6 days in DRG and dorsal spinal cord in rats, whereas that it increased c-fos expression at the DRG. Intrathecal repeated transfection of rats with S300A and S333A or injection with BL-1249 reduced formalin-induced enhanced c-fos expression. Data suggest that TREK-1 activity at peripheral and spinal sites reduces neuronal excitability in the process of acute and long-lasting nociception induced by formalin in rats.


Asunto(s)
Desinfectantes/farmacología , Formaldehído/farmacología , Ganglios Espinales , Hiperalgesia , Dolor Nociceptivo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Médula Espinal , Animales , Modelos Animales de Enfermedad , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Hiperalgesia/inducido químicamente , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/metabolismo , Masculino , Dolor Nociceptivo/inducido químicamente , Dolor Nociceptivo/tratamiento farmacológico , Dolor Nociceptivo/metabolismo , Péptidos/farmacología , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Ratas , Ratas Wistar , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Tetrahidronaftalenos/farmacología , Tetrazoles/farmacología
2.
Elife ; 92020 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-33345771

RESUMEN

K2P potassium channels are known to be modulated by volatile anesthetic (VA) drugs and play important roles in clinically relevant effects that accompany general anesthesia. Here, we utilize a photoaffinity analog of the VA isoflurane to identify a VA-binding site in the TREK1 K2P channel. The functional importance of the identified site was validated by mutagenesis and biochemical modification. Molecular dynamics simulations of TREK1 in the presence of VA found multiple neighboring residues on TREK1 TM2, TM3, and TM4 that contribute to anesthetic binding. The identified VA-binding region contains residues that play roles in the mechanisms by which heat, mechanical stretch, and pharmacological modulators alter TREK1 channel activity and overlaps with positions found to modulate TASK K2P channel VA sensitivity. Our findings define molecular contacts that mediate VA binding to TREK1 channels and suggest a mechanistic basis to explain how K2P channels are modulated by VAs.


Asunto(s)
Anestésicos por Inhalación/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Anestésicos por Inhalación/metabolismo , Animales , Sitios de Unión , Humanos , Isoflurano/farmacología , Ratones , Simulación del Acoplamiento Molecular , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Xenopus laevis , Pez Cebra
3.
J Immunol Methods ; 487: 112875, 2020 12.
Artículo en Inglés | MEDLINE | ID: mdl-33031794

RESUMEN

The expression of Kv1.3 and KCa channels in human T cells is essential for maintaining cell activation, proliferation and migration during an inflammatory response. Recently, an additional residual current, sensitive to anandamide and A293, compounds specifically inhibiting currents mediated by TASK channels, was observed after complete pharmacological blockade of Kv1.3 and KCa channels. This finding was not consistently observed throughout different studies and, an in-depth review of the different recording conditions used for the electrophysiological analysis of K+ currents in T cells revealed fluoride as major anionic component of the pipette intracellular solutions in the initial studies. While fluoride is frequently used to stabilize electrophysiological recordings, it is known as G-protein activator and to influence the intracellular Ca2+ concentration, which are mechanisms known to modulate TASK channel functioning. Therefore, we systemically addressed different fluoride- and chloride-based pipette solutions in whole-cell patch-clamp experiments in human T cells and used specific blockers to identify membrane currents carried by TASK and Kv1.3 channels. We found that fluoride increased the decay time constant of K+ outward currents, reduced the degree of the sustained current component and diminished the effect of the specific TASK channels blocker A293. These findings indicate that the use of fluoride-based pipette solutions may hinder the identification of a functional TASK channel component in electrophysiological experiments.


Asunto(s)
Fluoruros/farmacología , Potenciales de la Membrana/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Compuestos de Potasio/farmacología , Linfocitos T/efectos de los fármacos , Células Cultivadas , Fluoruros/metabolismo , Humanos , Canal de Potasio Kv1.3/efectos de los fármacos , Canal de Potasio Kv1.3/metabolismo , Cloruro de Magnesio/metabolismo , Cloruro de Magnesio/farmacología , Técnicas de Placa-Clamp , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Compuestos de Potasio/metabolismo , Linfocitos T/metabolismo , Factores de Tiempo
4.
Int J Biochem Cell Biol ; 125: 105792, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32574707

RESUMEN

In this review, we describe key signaling pathways regulating potassium channels present in the inner mitochondrial membrane. The signaling cascades covered here include phosphorylation, redox reactions, modulation by calcium ions and nucleotides. The following types of potassium channels have been identified in the inner mitochondrial membrane of various tissues: ATP-sensitive, Ca2+-activated, voltage-gated and two-pore domain potassium channels. The direct roles of these channels involve regulation of mitochondrial respiration, membrane potential and synthesis of reactive oxygen species (ROS). Changes in channel activity lead to diverse pro-life and pro-death responses in different cell types. Hence, characterizing the signaling pathways regulating mitochondrial potassium channels will facilitate understanding the physiological role of these proteins. Additionally, we describe in this paper certain regulatory mechanisms, which are unique to mitochondrial potassium channels.


Asunto(s)
Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Canales de Potasio/metabolismo , Transducción de Señal/genética , Adenosina Trifosfato , Animales , Calcio/metabolismo , Humanos , Oxidación-Reducción , Canales de Potasio/efectos de los fármacos , Canales de Potasio Calcio-Activados/efectos de los fármacos , Canales de Potasio Calcio-Activados/metabolismo , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología
5.
ACS Chem Neurosci ; 10(6): 2786-2793, 2019 06 19.
Artículo en Inglés | MEDLINE | ID: mdl-30935201

RESUMEN

K2P potassium channels stabilize the resting membrane potential in nearly all cells and have been implicated in several neuronal, cardiovascular, and immune diseases. DCPIB, a known specific and potent inhibitor of volume-regulated anion channels (VRAC), has been reported to activate TREK1 and TREK2 in astrocytes and in vitro recently. In the present study, we demonstrated DCPIB also voltage dependently activated TRAAK besides TREK1/TREK2, showing DCPIB activated all TREK subfamily members. In contrast, the compound potently inhibited several other K2P channels with no voltage dependence, including TRESK, TASK1, and TASK3. DCPIB displayed superior selectivity toward TRESK with an IC50 of 0.14 µM, demonstrating at least 100-fold higher affinity over TREK1/TRAAK channels. Furthermore, the impaired ion selectivity filter region greatly impaired the activating effect of DCPIB on TREK1 but not the inhibitory effect of DCPIB on TRESK. This indicates distinct molecular determinants underlying the effect of DCPIB on TREK1 or TRESK channels. Our results showed that DCPIB played diverse effects on K2P channels and could be a useful tool for further investigating structure-function studies of K2P channels.


Asunto(s)
Ciclopentanos/farmacología , Indanos/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Células COS , Chlorocebus aethiops , Humanos
6.
ACS Chem Neurosci ; 9(12): 3153-3165, 2018 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-30089357

RESUMEN

K2P potassium channels generate leak currents that stabilize the resting membrane potential of excitable cells. Various K2P channels are implicated in pain, ischemia, depression, migraine, and anesthetic responses, making this family an attractive target for small molecule modulator development efforts. BL-1249, a compound from the fenamate class of nonsteroidal anti-inflammatory drugs is known to activate K2P2.1(TREK-1), the founding member of the thermo- and mechanosensitive TREK subfamily; however, its mechanism of action and effects on other K2P channels are not well-defined. Here, we demonstrate that BL-1249 extracellular application activates all TREK subfamily members but has no effect on other K2P subfamilies. Patch clamp experiments demonstrate that, similar to the diverse range of other chemical and physical TREK subfamily gating cues, BL-1249 stimulates the selectivity filter "C-type" gate that controls K2P function. BL-1249 displays selectivity among the TREK subfamily, activating K2P2.1(TREK-1) and K2P10.1(TREK-2) ∼10-fold more potently than K2P4.1(TRAAK). Investigation of mutants and K2P2.1(TREK-1)/K2P4.1(TRAAK) chimeras highlight the key roles of the C-terminal tail in BL-1249 action and identify the M2/M3 transmembrane helix interface as a key site of BL-1249 selectivity. Synthesis and characterization of a set of BL-1249 analogs demonstrates that both the tetrazole and opposing tetralin moieties are critical for function, whereas the conformational mobility between the two ring systems impacts selectivity. Together, our findings underscore the landscape of modes by which small molecules can affect K2P channels and provide crucial information for the development of better and more selective K2P modulators of the TREK subfamily.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Tetrahidronaftalenos/farmacología , Tetrazoles/farmacología , Animales , Células HEK293 , Humanos , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/metabolismo , Oocitos , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Xenopus laevis
7.
ACS Chem Neurosci ; 9(12): 2886-2891, 2018 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-30001098

RESUMEN

Photoswitchable blockers of potassium channels can be used to optically control neuronal excitability and hold great promise for vision restoration. Here, we report a series of improved photoswitchable blockers that are furnished with a new pharmacophore based on the local anesthetic bupivacaine. These azobupivacaines (ABs) enable optical control over the delayed rectifier channel Kv2.1. and target the two-pore domain potassium channel TREK-1. For the first time, we have identified a compound that blocks conductance in the dark and potentiates it upon illumination. Using light as a trigger, ABs efficiently and reversibly silence action potential firing of hippocampal neurons in acute mouse brain slices.


Asunto(s)
Potenciales de Acción/efectos de los fármacos , Compuestos Azo/farmacología , Bupivacaína/análogos & derivados , Luz , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio Shab/efectos de los fármacos , Animales , Compuestos Azo/síntesis química , Células HEK293 , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Fenómenos Ópticos , Bloqueadores de los Canales de Potasio/síntesis química , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio Shab/antagonistas & inhibidores , Canales de Potasio Shab/metabolismo
8.
Eur J Pharmacol ; 831: 94-102, 2018 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-29753045

RESUMEN

Human K2P17.1 (TASK-4, TALK-2) two-pore-domain potassium (K2P) channels have recently been implicated in heart rhythm disorders including atrial fibrillation and conduction disease. The functional in vivo significance of K2P17.1 currents in cardiac electrophysiology remains incompletely understood. Danio rerio (zebrafish) may be utilized to elucidate the role of cardiac K2P channels in vivo. The aim of this work was to identify and characterize the zebrafish ortholog of K2P17.1 in comparison to its human counterpart. The zkcnk17 coding sequence was amplified from zebrafish cDNA. Zebrafish kcnk17 mRNA expression was assessed by polymerase chain reaction. Human and zebrafish K2P17.1 currents were analyzed using two-electrode voltage clamp electrophysiology and the Xenopus oocyte expression system. Kcnk17 mRNA was detected in zebrafish brain. Human and zebrafish K2P17.1 proteins exhibited 33.4% identity. Zebrafish K2P17.1 channels conducted K+ selective currents with open rectification properties. Both human and zebrafish K2P17.1 were inhibited by barium. In contrast to human K2P17.1, zK2P17.1 currents were not sensitive to extracellular alkalization, likely due to the lack of a lysine residue involved in pH sensing of hK2P17.1. In conclusion, zebrafish and human K2P17.1 channels display similar structural and regulatory properties. Zebrafish may serve as an in vivo model to study neuronal K2P17.1 function but does not appear appropriate for cardiac electrophysiology studies. Differences in pH sensitivity of zK2P17.1 currents need to be considered when zebrafish data are extrapolated to human physiology.


Asunto(s)
Canales de Potasio de Dominio Poro en Tándem/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Compuestos de Bario/farmacología , Femenino , Regulación de la Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Potenciales de la Membrana , Oocitos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/genética , Conformación Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Relación Estructura-Actividad , Xenopus laevis/genética , Xenopus laevis/metabolismo , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genética
9.
J Neurochem ; 138(2): 265-81, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27062641

RESUMEN

Brain ischaemia is a highly debilitating condition where shortage of oxygen and glucose leads to profuse cell death. Lactate is a neuroprotective metabolite whose concentrations increase up to 15-30 mmol/L during ischaemia and TREK1 is a neuroprotective potassium channel which is upregulated during ischaemia. The aim of this study was to investigate the effect of l-lactate on TREK1 expression and to evaluate the role of l-lactate-TREK1 interaction in conferring neuroprotection in ischaemia-prone hippocampus. We show that 15-30 mmol/L l-lactate increases functional TREK1 protein expression by 1.5-3-fold in hippocampal astrocytes using immunostaining and electrophysiology. Studies with transcription blocker actinomycin-D and quantitative PCR indicate that the increase in TREK1 expression is due to enhanced TREK1 mRNA transcription. We further report that l-lactate-mediated increase in TREK1 expression is via protein kinase A (PKA)-dependent pathway. This is the first report of an ischaemic metabolite affecting functional expression of an ion channel. Our studies in an in vitro model of ischaemia using oxygen glucose deprivation show that 30 mmol/L l-lactate fails to reduce cell death in rat hippocampal slices treated with TREK1 blockers, PKA inhibitors and gliotoxin. The above effects were specific to l-lactate as pyruvate failed to increase TREK1 expression and reduce cell death. l-Lactate-induced TREK1 upregulation is a novel finding of physiological significance as TREK1 channels contribute to neuroprotection by enhancing potassium buffering and glutamate clearance capacity of astrocytes. We propose that l-lactate promotes neuronal survival in hippocampus by increasing TREK1 channel expression via PKA pathway in astrocytes during ischaemia. Insufficient blood supply to the brain leads to cerebral ischaemia and increase in extracellular lactate concentrations. We incubated hippocampal astrocytes in lactate and observed increase in TREK1 channel expression via protein kinase A (PKA). Inhibition of TREK1, PKA and metabolic impairment of astrocytes prevented lactate from reducing cell death in ischaemic hippocampus. This pathway serves as an alternate mechanism of neuroprotection. Cover image for this issue: doi: 10.1111/jnc.13326.


Asunto(s)
Astrocitos/metabolismo , Isquemia Encefálica/metabolismo , Hipocampo/metabolismo , Ácido Láctico/farmacología , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Hipoxia de la Célula/fisiología , Supervivencia Celular/efectos de los fármacos , Ácido Glutámico/metabolismo , Hipocampo/efectos de los fármacos , Ácido Láctico/metabolismo , Masculino , Neuronas/efectos de los fármacos , Neuroprotección/fisiología , Oxígeno/metabolismo , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Ratas Wistar
10.
Expert Opin Ther Targets ; 20(8): 947-58, 2016 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-26918581

RESUMEN

INTRODUCTION: Atrial fibrillation (AF) is the most common arrhythmia in humans. It is progressive and the development of electrical and structural remodeling makes early intervention desirable. Existing antiarrhythmic pharmacological approaches are not always effective and can produce unwanted side effects. Additional atrial-selective antiarrhythmic strategies are therefore desirable. AREAS COVERED: Evidence for three novel ion channel atrial-selective therapeutic targets is evaluated: atrial-selective fast sodium channel current (INa) inhibition; small conductance calcium-activated potassium (SK) channels; and two-pore (K2P) potassium channels. EXPERT OPINION: Data from animal models support atrial-ventricular differences in INa kinetics and also suggest atrial-ventricular differences in sodium channel ß subunit expression. Further work is required to determine whether intrinsic atrial-ventricular differences in human INa exist or whether functional differences occur due to distinct atrial and ventricular action and resting potentials. SK and K2P channels (particularly K2P 3.1) offer potentially attractive atrial-selective targets. Work is needed to identify the underlying basis of SK current that contributes to (patho)physiological atrial repolarization and settings in which SK inhibition is anti- versus pro-arrhythmic. Although K2P3.1 appears to be a promising target with comparatively selective drugs for experimental use, a lack of selective pharmacology hinders evaluation of other K2P channels as potential atrial-selective targets.


Asunto(s)
Antiarrítmicos/farmacología , Fibrilación Atrial/tratamiento farmacológico , Terapia Molecular Dirigida , Animales , Antiarrítmicos/efectos adversos , Fibrilación Atrial/patología , Modelos Animales de Enfermedad , Diseño de Fármacos , Humanos , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/metabolismo , Canales de Sodio/efectos de los fármacos , Canales de Sodio/metabolismo
11.
Neurogastroenterol Motil ; 27(6): 865-74, 2015 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-25846134

RESUMEN

BACKGROUND: Gastroesophageal reflux can cause high acidity in the esophagus and trigger heartburn and pain. However, because of the esophageal mucosal barrier, the acidity at the nerve terminals of pain-mediating C-fibers in esophageal mucosa is predicted to be substantially lower. We hypothesized that the esophageal dorsal root ganglia (DRG) C-fibers are activated by mild acid (compared to acidic reflux), and express receptors and ion channels highly sensitive to acid. METHODS: Extracellular single unit recordings of activity originating in esophageal DRG C-fiber nerve terminals were performed in the innervated esophagus preparation ex vivo. Acid was delivered in a manner that bypassed the esophageal mucosal barrier. The expression of mRNA for selected receptors in esophagus-specific DRG neurons was evaluated using single cell RT-PCR. KEY RESULTS: Mild acid (pH = 6.5-5.5) activated esophageal DRG C-fibers in a pH-dependent manner. The response to mild acid at pH = 6 was not affected by the TRPV1 selective antagonist iodo-resiniferatoxin. The majority (70-95%) of esophageal DRG C-fiber neurons (TRPV1-positive) expressed mRNA for acid sensing ion channels (ASIC1a, ASIC1b, ASIC2b, and/or ASIC3), two-pore-domain (K2P) potassium channel TASK1, and the proton-sensing G-protein coupled receptor OGR1. Other evaluated targets (PKD2L1, TRPV4, TASK3, TALK1, G2A, GPR4, and TDAG8) were expressed rarely. CONCLUSIONS & INFERENCES: Guinea pig esophageal DRG C-fibers are activated by mild acid via a TRPV1-independent mechanism, and express mRNA for several receptors and ion channels highly sensitive to acid. The high acid sensitivity of esophageal C-fibers may contribute to heartburn and pain in conditions of reduced mucosal barrier function.


Asunto(s)
Canales Iónicos Sensibles al Ácido/efectos de los fármacos , Esófago/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ácido Clorhídrico/farmacología , Fibras Nerviosas Amielínicas/efectos de los fármacos , Neuronas/efectos de los fármacos , Nociceptores/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , Canales Iónicos Sensibles al Ácido/genética , Canales Iónicos Sensibles al Ácido/metabolismo , Animales , Esófago/inervación , Ganglios Espinales/metabolismo , Cobayas , Concentración de Iones de Hidrógeno , Membrana Mucosa , Fibras Nerviosas Amielínicas/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Nociceptores/metabolismo , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/efectos de los fármacos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Soluciones/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores
12.
Biochem Biophys Res Commun ; 451(3): 415-20, 2014 Aug 29.
Artículo en Inglés | MEDLINE | ID: mdl-25108155

RESUMEN

Atrial fibrillation (AF) contributes significantly to cardiovascular morbidity and mortality. The growing epidemic is associated with cardiac repolarization abnormalities and requires the development of more effective antiarrhythmic strategies. Two-pore-domain K(+) channels stabilize the resting membrane potential and repolarize action potentials. Recently discovered K2P17.1 channels are expressed in human atrium and represent potential targets for AF therapy. However, cardiac electropharmacology of K2P17.1 channels remains to be investigated. This study was designed to elucidate human K2P17.1 regulation by antiarrhythmic drugs. Two-electrode voltage clamp and whole-cell patch clamp electrophysiology was used to record K2P currents from Xenopus oocytes and Chinese hamster ovary (CHO) cells. The class III antiarrhythmic compound vernakalant activated K2P17.1 currents in oocytes an in mammalian cells (EC50,CHO=40 µM) in frequency-dependent manner. K2P17.1 channel activation by vernakalant was specific among K2P channel family members. By contrast, vernakalant reduced K2P4.1 and K2P10.1 currents, in line with K2P2.1 blockade reported earlier. K2P17.1 open rectification characteristics and current-voltage relationships were not affected by vernakalant. The class I drug flecainide did not significantly modulate K2P currents. In conclusion, vernakalant activates K2P17.1 background potassium channels. Pharmacologic K2P channel activation by cardiovascular drugs has not been reported previously and may be employed for personalized rhythm control in patients with AF-associated reduction of K(+) channel function.


Asunto(s)
Anisoles/farmacología , Antiarrítmicos/farmacología , Fibrilación Atrial/tratamiento farmacológico , Oocitos/fisiología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/fisiología , Pirrolidinas/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Células CHO , Cricetinae , Cricetulus , Flecainida/farmacología , Humanos , Potenciales de la Membrana/efectos de los fármacos , Oocitos/efectos de los fármacos , Técnicas de Placa-Clamp , Xenopus laevis
13.
Anesthesiology ; 119(5): 1137-48, 2013 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-23867231

RESUMEN

BACKGROUND: Xenon, the inert anesthetic gas, is neuroprotective in models of brain injury. The authors investigate the neuroprotective mechanisms of the inert gases such as xenon, argon, krypton, neon, and helium in an in vitro model of traumatic brain injury. METHODS: The authors use an in vitro model using mouse organotypic hippocampal brain slices, subjected to a focal mechanical trauma, with injury quantified by propidium iodide fluorescence. Patch clamp electrophysiology is used to investigate the effect of the inert gases on N-methyl-D-aspartate receptors and TREK-1 channels, two molecular targets likely to play a role in neuroprotection. RESULTS: Xenon (50%) and, to a lesser extent, argon (50%) are neuroprotective against traumatic injury when applied after injury (xenon 43±1% protection at 72 h after injury [N=104]; argon 30±6% protection [N=44]; mean±SEM). Helium, neon, and krypton are devoid of neuroprotective effect. Xenon (50%) prevents development of secondary injury up to 48 h after trauma. Argon (50%) attenuates secondary injury, but is less effective than xenon (xenon 50±5% reduction in secondary injury at 72 h after injury [N=104]; argon 34±8% reduction [N=44]; mean±SEM). Glycine reverses the neuroprotective effect of xenon, but not argon, consistent with competitive inhibition at the N-methyl-D-aspartate receptor glycine site mediating xenon neuroprotection against traumatic brain injury. Xenon inhibits N-methyl-D-aspartate receptors and activates TREK-1 channels, whereas argon, krypton, neon, and helium have no effect on these ion channels. CONCLUSIONS: Xenon neuroprotection against traumatic brain injury can be reversed by increasing the glycine concentration, consistent with inhibition at the N-methyl-D-aspartate receptor glycine site playing a significant role in xenon neuroprotection. Argon and xenon do not act via the same mechanism.


Asunto(s)
Anestésicos por Inhalación/farmacología , Argón/farmacología , Glicina/efectos de los fármacos , Fármacos Neuroprotectores , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Xenón/farmacología , Presión del Aire , Animales , Cámaras de Exposición Atmosférica , Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/patología , Región CA1 Hipocampal/lesiones , Región CA1 Hipocampal/patología , Recuento de Células , Línea Celular , Fenómenos Electrofisiológicos , Glicina/farmacología , Hipocampo/efectos de los fármacos , Humanos , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Ratas
14.
Am J Physiol Regul Integr Comp Physiol ; 304(12): R1070-84, 2013 Jun 15.
Artículo en Inglés | MEDLINE | ID: mdl-23594614

RESUMEN

Consistent with a critical role in respiratory and autonomic stress responses, the carotid bodies are strongly excited by pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide implicated in stress responses throughout the sympathetic nervous system. PACAP excites isolated carotid body glomus cells via activation of PAC1 receptors, with one study suggesting PAC1-induced excitation is due entirely to protein kinase A (PKA)-mediated inhibition of TASK channels. However, in other systems, PAC1 is known to be coupled to multiple intracellular signaling pathways, including PKA, phospholipase C (PLC), phospholipase D (PLD), and protein kinase C (PKC), that trigger multiple downstream effectors including increased Ca²âº mobilization, inhibition of various K⁺ channels, and activation of nonselective cation channels. This study tests if non-PKA/TASK channel signaling helps mediate the stimulatory effects of PACAP on the carotid body. Using an ex vivo arterially perfused rat carotid body preparation, we show that PACAP-38 stimulates carotid sinus nerve activity in a biphasic manner (peak response, falling to plateau). PKA blocker H-89 only reduced the plateau response (~41%), whereas the TASK-1-like K⁺ channel blocker/transient receptor potential vanilloid 1 channel agonist anandamide only inhibited the peak response (~48%), suggesting involvement of additional pathways. The PLD blocker CAY10594 significantly inhibited both peak and plateau responses. The PLC blocker U73122 decimated both peak and plateau responses. Brefeldin A, a blocker of Epac (cAMP-activated guanine exchange factor, reported to link Gs-coupled receptors with PLC/PLD), also reduced both phases of the response, as did blocking signaling downstream of PLC/PLD with the PKC inhibitors chelerythrine chloride and GF109203X. Suggesting the involvement of non-TASK ion channels in the effects of PACAP, the A-type K⁺ channel blocker 4-aminopyridine, and the putative transient receptor potential channel (TRPC)/T-type calcium channel blocker SKF96365 each significantly inhibited the peak and steady-state responses. These data suggest the stimulatory effect of PACAP-38 on carotid body sensory activity is mediated through multiple signaling pathways: the PLC-PKC pathways predominates, with TRPC and/or T-type channel activation and Kv channel inactivation; only partial involvement is attributable to PKA and PLD activation.


Asunto(s)
Cuerpo Carotídeo/fisiología , Neuronas Aferentes/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/fisiología , Fenómenos Fisiológicos Respiratorios , Transducción de Señal/fisiología , Estrés Fisiológico/fisiología , Sistema Nervioso Simpático/fisiología , Animales , Ácidos Araquidónicos/farmacología , Cuerpo Carotídeo/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Endocannabinoides/farmacología , Inhibidores Enzimáticos/farmacología , Estrenos/farmacología , Masculino , Modelos Animales , Proteínas del Tejido Nervioso , Neuronas Aferentes/efectos de los fármacos , Inhibidores de Fosfodiesterasa/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Alcamidas Poliinsaturadas/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/fisiología , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/efectos de los fármacos , Pirrolidinonas/farmacología , Ratas , Ratas Sprague-Dawley , Fenómenos Fisiológicos Respiratorios/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos
15.
Dtsch Med Wochenschr ; 137(33): 1654-8, 2012 Aug.
Artículo en Alemán | MEDLINE | ID: mdl-22875694

RESUMEN

Uncontrolled electrical activity caused by ion channel dysfunction produces arrhythmia in the heart. Despite recent advances in pharmaceutical research and development, effective and safe pharmacological management of cardiac arrhythmia still remains an unmet medical need. The emerging family of two-pore-domain potassium (K2P) channels stabilizes the resting membrane potential and facilitates action potential repolarization. In the heart, genetic inactivation or inhibition of two-pore-domain K + (K2P) currents by class III antiarrhythmic drugs results in action potential prolongation. In particular, human K2P3.1 channels are selectively expressed in the atria and represent targets for the pharmacological management of atrial fibrillation. Furthermore, stretch-sensitive K2P2.1 channels are implicated in mechanoelectrical feedback and arrhythmogenesis. The current knowledge on function, regulation, and cardiac significance of K2P channels is summarized in this work, and potential therapeutic implications are highlighted.


Asunto(s)
Arritmias Cardíacas/fisiopatología , Electrocardiografía , Canales de Potasio de Dominio Poro en Tándem/fisiología , Animales , Antiarrítmicos/efectos adversos , Antiarrítmicos/uso terapéutico , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/genética , Fibrilación Atrial/tratamiento farmacológico , Fibrilación Atrial/genética , Fibrilación Atrial/fisiopatología , Electrocardiografía/efectos de los fármacos , Silenciador del Gen , Atrios Cardíacos/efectos de los fármacos , Atrios Cardíacos/fisiopatología , Sistema de Conducción Cardíaco/efectos de los fármacos , Sistema de Conducción Cardíaco/fisiopatología , Ventrículos Cardíacos/efectos de los fármacos , Ventrículos Cardíacos/fisiopatología , Humanos , Ratones , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/genética , Ratas
16.
Am J Physiol Gastrointest Liver Physiol ; 303(3): G412-23, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22636169

RESUMEN

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 µM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 µM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 µM), ODQ (10 µM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 µM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 µM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 µM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.


Asunto(s)
Colon/fisiología , Péptidos/farmacología , Canales de Potasio de Dominio Poro en Tándem/fisiología , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/fisiología , Animales , Apamina/farmacología , Cisteína/farmacología , Masculino , Metionina/farmacología , Relajación Muscular/efectos de los fármacos , Nitroarginina/farmacología , Nitroprusiato/farmacología , Oxadiazoles/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Quinoxalinas/farmacología , Ratas , Ratas Sprague-Dawley , Canales de Potasio de Pequeña Conductancia Activados por el Calcio/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología
17.
Am J Physiol Lung Cell Mol Physiol ; 302(1): L4-L12, 2012 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-21964404

RESUMEN

Mucociliary clearance is the primary innate physical defense mechanism against inhaled pathogens and toxins. Vectorial ion transport, primarily sodium absorption and anion secretion, by airway epithelial cells supports mucociliary clearance. This is evidenced by diseases of abnormal ion transport such as cystic fibrosis and pseudohypoaldosteronism that are characterized by changes in mucociliary clearance. Sodium absorption and chloride secretion in human bronchial epithelial cells depend on potassium channel activity, which creates a favorable electrochemical gradient for both by hyperpolarizing the apical plasma membrane. Although the role of basolateral membrane potassium channels is firmly established and extensively studied, a role for apical membrane potassium channels has also been described. Here, we demonstrate that bupivacaine and quinidine, blockers of four-transmembrane domain, two-pore potassium (K2P) channels, inhibit both amiloride-sensitive sodium absorption and forskolin-stimulated anion secretion in polarized, normal human bronchial epithelial cells at lower concentrations when applied to the mucosal surface than when applied to the serosal surface. Transcripts from four genes, KCNK1 (TWIK-1), KCNK2 (TREK-1), KCNK5 (TASK-2), and KCNK6 (TWIK-2), encoding K2P channels were identified by RT-PCR. Protein expression at the apical membrane was confirmed by immunofluorescence. Our data provide further evidence that potassium channels, in particular K2P channels, are expressed and functional in the apical membrane of airway epithelial cells where they may be targets for therapeutic manipulation.


Asunto(s)
Polaridad Celular , Cloruros/metabolismo , Canales de Potasio de Dominio Poro en Tándem , Mucosa Respiratoria/metabolismo , Sodio/metabolismo , Absorción , Amilorida/farmacología , Bronquios/citología , Bupivacaína/farmacología , Línea Celular , Polaridad Celular/efectos de los fármacos , Polaridad Celular/fisiología , Colforsina/farmacología , Células Epiteliales/metabolismo , Humanos , Inmunohistoquímica/métodos , Transporte Iónico , Bloqueadores de los Canales de Potasio/metabolismo , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Quinidina/farmacología
18.
Cardiovasc Ther ; 30(1): e23-9, 2012 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20946320

RESUMEN

Potassium (K(+) ) channels are important in cardiovascular disease both as drug targets and as a cause of underlying pathology. Voltage-dependent K(+) (K(V) ) channels are inhibited by the class III antiarrhythmic agents. Certain vasodilators work by opening K(+) channels in vascular smooth muscle cells (VSMCs), and K(+) channel activation may also be a route to improving endothelial function. The two-pore domain K(+) (K(2P) ) channels form a group of 15 known channels with an expanding list of functions in the cardiovascular system. One of these K(2P) channels, TREK-1, is the focus of this review. TREK-1 channel activity is tightly regulated by intracellular and extracellular pH, membrane stretch, polyunsaturated fatty acids (PUFAs), temperature, and receptor-coupled second messenger systems. TREK-1 channels are also activated by volatile anesthetics and some neuroprotectant agents, and they are inhibited by selective serotonin reuptake inhibitors (SSRIs) as well as amide local anesthetics. Some of the clinical cardiovascular effects and side effects of these drugs may be through their actions on TREK-1 channels. It has recently been suggested that TREK-1 channels have a role in mechano-electrical coupling in the heart. They also seem important in the vascular responses to PUFAs, and this may underlie some of the beneficial cardiovascular effects of the essential dietary fatty acids. Development of selective TREK-1 openers and inhibitors may provide promising routes for intervention in cardiovascular diseases.


Asunto(s)
Vasos Sanguíneos/efectos de los fármacos , Fármacos Cardiovasculares/uso terapéutico , Enfermedades Cardiovasculares/tratamiento farmacológico , Corazón/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Animales , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/fisiopatología , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Corazón/fisiopatología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Miocardio/metabolismo , Permeabilidad , Canales de Potasio de Dominio Poro en Tándem/metabolismo
19.
Am J Respir Cell Mol Biol ; 46(3): 372-9, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-22021335

RESUMEN

As powerful vasodilators, prostacyclin analogues are presently the mainstay in the treatment of severe pulmonary arterial hypertension. Although the hemodynamic effects of prostacyclin analogues are well known, the molecular mechanism of their acute effects on pulmonary vascular tone and systemic vascular tone remains poorly understood. Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) was previously identified as a putative receptor responsible for the modulation of target gene expression in response to prostacyclin analogues. The present study investigated the signaling pathway of prostacyclin in human pulmonary arterial smooth muscle cells (PASMCs), and sought to define the role of PPARß/δ in the acute vasodilating effect. In human PASMCs, prostacyclin rapidly activated TWIK-related acid-sensitive K channel 1 (TASK-1) and calcium-dependent potassium channels (K(Ca)). This pathway was mediated via the prostanoid I receptor-protein kinase A pathway. The silencing of PPARß/δ demonstrated that the downstream K(Ca) activation was exclusively dependent on PPARß/δ signaling, whereas the activation of TASK-1 was not. In addition, the PPARß/δ-induced activation of K(Ca) was independent of NO. The acute prostacyclin-induced K(Ca) activation is critically dependent on PPARß/δ as a rapid signaling factor. This accounts in part for the vasodilating effect of prostacyclin in pulmonary arteries, and provides insights into a new molecular explanation for the effects of prostanoids.


Asunto(s)
Epoprostenol/análogos & derivados , Iloprost/farmacología , Músculo Liso Vascular/efectos de los fármacos , Miocitos del Músculo Liso/metabolismo , PPAR delta/agonistas , PPAR gamma/agonistas , Canales de Potasio Calcio-Activados/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacología , Animales , Células Cultivadas , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Epoprostenol/farmacología , Silenciador del Gen , Humanos , Masculino , Potenciales de la Membrana , Músculo Liso Vascular/metabolismo , Proteínas del Tejido Nervioso/efectos de los fármacos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , PPAR delta/genética , PPAR delta/metabolismo , PPAR gamma/genética , PPAR gamma/metabolismo , Canales de Potasio Calcio-Activados/genética , Canales de Potasio Calcio-Activados/metabolismo , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/genética , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Arteria Pulmonar/efectos de los fármacos , Arteria Pulmonar/metabolismo , Ratas , Ratas Wistar , Receptores de Epoprostenol , Receptores de Prostaglandina/efectos de los fármacos , Receptores de Prostaglandina/metabolismo
20.
Channels (Austin) ; 5(1): 34-42, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21057213

RESUMEN

TREK-1, a mechanosensitive K channel from the two-pore family (K(2)P), is involved in protective regulation of the resting potential in CNS neurons and other tissues. The structure of TREK-1 and the basis of its sensitivity to stretch and variety of lipid-soluble factors remain unknown. Using existing K channel structures as modeling templates, TREK-1 was envisioned as a two-fold symmetrical complex with the gate formed primarily by the centrally positioned TM2b helices of the second homologous repeat. Opening was modeled as a conical expansion of the barrel separating TM2b's accompanied by extension of TM2a helices with the cytoplasmic TM2a-TM1b connector. Seeking first experimental support to the models we have accomplished thermodynamic analysis of mouse TREK-1 gating and functional testing of several deletion mutants. The predicted increase of the channel in-plane area (ΔA) of ~5 nm(2) in models was supported by the experimental ΔA of ~4 nm(2) derived from the slope of open probability versus membrane tension in HEK-293T cells and their cytoskeleton-depleted blebs. In response to steps of suction, wild-type channel produced transient currents in cell-attached patches and mostly sustained currents upon patch excision. TREK-1 motifs not present in canonical K channels include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. Deletion of the extracellular loop (Δ76-124) reduced the average current density in patches, increased spontaneous activity and generated a larger sub-population of high-conductance channels, while activation by tension augmented by arachidonic acid was fully retained. Further deletion of the C-terminal end (Δ76-124/Δ334-411) removed voltage dependency but otherwise produced no additional effect. In an attempt to generate a cysteine-free version of the channel, we mutated two remaining cysteines 159 and 219 in the transmembrane region. C219A did not compromise channel activity, whereas the C159A/S mutants were essentially inactive. Treatment with ß-mercaptoethanol suggested that none of these cysteines form functionally-important disulfides.


Asunto(s)
Activación del Canal Iónico , Mecanotransducción Celular , Modelos Moleculares , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Eliminación de Secuencia , Termodinámica , Animales , Cisteína , Células HEK293 , Humanos , Potenciales de la Membrana , Mercaptoetanol/farmacología , Ratones , Mutagénesis Sitio-Dirigida , Técnicas de Placa-Clamp , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio de Dominio Poro en Tándem/genética , Presión , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Transfección
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