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1.
J Neurosci ; 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-35999055

RESUMO

Electrical activity in neurons is highly energy demanding and accompanied by rises in cytosolic Ca2+ Cytosolic Ca2+, in turn, secures energy supply by pushing mitochondrial metabolism either through augmented NADH transfer into mitochondria via the malate aspartate shuttle (MAS) or via direct activation of dehydrogenases of the TCA cycle after passing into the matrix through the mitochondrial Ca2+ uniporter (MCU). Another Ca2+-sensitive booster of mitochondrial ATP synthesis is the glycerol-3-phosphate shuttle (G3PS) whose role in neuronal energy supply has remained elusive. Essential components of G3PS are expressed in hippocampal neurons. Single neuron metabolic measurements in primary hippocampal cultures derived from rat pups of either sex reveal only moderate, if any, constitutive activity of G3PS. However, during electrical activity neurons fully rely on G3PS when MAS and MCU are unavailable. Under these conditions, G3PS is required for appropriate action potential firing. Accordingly, G3PS safeguards metabolic flexibility of neurons to cope with energy demands of electrical signaling.SIGNIFICANCE STATEMENT:Ca2+ ions are known to provide a link between the energy-demanding electrical activity and an adequate ATP supply in neurons. To do so, Ca2+ acts both, from outside and inside of the mitochondrial inner membrane. Neuronal function critically depend on this regulation and its defects are often found in various neurological disorders. Although interest in neuronal metabolism increases, many aspects thereof have remained unresolved. In particular, a Ca2+-sensitive NADH shuttling system, the glycerol-3-phosphate shuttle, has been largely ignored with respect to its function in neurons. Our results demonstrate that this shuttle is functional in hippocampal neurons and safeguards ATP supply and appropriate action potential firing when malate aspartate shuttle and mitochondrial Ca2+ uniporter are unavailable, thereby ensuring neuronal metabolic flexibility.

2.
Int J Mol Sci ; 24(1)2022 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-36614094

RESUMO

The mechanism of acetaminophen (APAP) analgesia is at least partially unknown. Previously, we showed that the APAP metabolite N-acetyl-p-benzoquinone imine (NAPQI) activated Kv7 channels in neurons in vitro, and this activation of Kv7 channels dampened neuronal firing. Here, the effect of the Kv7 channel blocker XE991 on APAP-induced analgesia was investigated in vivo. APAP had no effect on naive animals. Induction of inflammation with λ-carrageenan lowered mechanical and thermal thresholds. Systemic treatment with APAP reduced mechanical hyperalgesia, and co-application of XE991 reduced APAP's analgesic effect on mechanical pain. In a second experiment, the analgesic effect of systemic APAP was not antagonized by intrathecal XE991 application. Analysis of liver samples revealed APAP and glutathione-coupled APAP indicative of metabolization. However, there were no relevant levels of these metabolites in cerebrospinal fluid, suggesting no relevant APAP metabolite formation in the CNS. In summary, the results support an analgesic action of APAP by activating Kv7 channels at a peripheral site through formation of the metabolite NAPQI.


Assuntos
Acetaminofen , Analgésicos não Narcóticos , Animais , Acetaminofen/farmacologia , Analgésicos não Narcóticos/farmacologia , Iminas/farmacologia , Analgésicos/farmacologia , Fígado/metabolismo
3.
Int J Neuropsychopharmacol ; 24(10): 832-841, 2021 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-34278424

RESUMO

BACKGROUND: Among psychostimulants, the dopamine transporter ligands amphetamine and cocaine display the highest addictive potential; the adenosine receptor antagonist caffeine is most widely consumed but less addictive. Psychostimulant actions of amphetamine were correlated with its ability to orchestrate ventral tegmental dopamine neuron activity with contrasting shifts in firing after single vs repeated administration. Whether caffeine might impinge on dopamine neuron activity has remained elusive. METHODS: Population activity of ventral tegmental area dopamine neurons was determined by single-unit extracellular recordings and set in relation to mouse behavior in locomotion and conditioned place preference experiments, respectively. RESULTS: A single dose of caffeine reduced population activity as did amphetamine and the selective adenosine A2A antagonist KW-6002, but not the A1 antagonist DPCPX. Repeated administration of KW-6002 or amphetamine led to drug-conditioned place preference and to unaltered or even enhanced population activity. Recurrent injection of caffeine or DPCPX, in contrast, failed to cause conditioned place preference and persistently reduced population activity. Subsequent to repetitive drug administration, re-exposure to amphetamine or KW-6002, but not to caffeine or DPCPX, was able to reduce population activity. CONCLUSIONS: Behavioral sensitization to amphetamine is attributed to persistent activation of ventral tegmental area dopamine neurons via the ventral hippocampus. Accordingly, a switch from acute A2A receptor-mediated reduction of dopamine neuron population activity to enduring A1 receptor-mediated suppression is correlated with tolerance rather than sensitization in response to repeated caffeine intake.


Assuntos
Anfetamina/farmacologia , Cafeína/farmacologia , Estimulantes do Sistema Nervoso Central/farmacologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Dopamina , Proteínas da Membrana Plasmática de Transporte de Dopamina , Hipocampo/efeitos dos fármacos , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Xantinas
4.
Int J Mol Sci ; 20(3)2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30699993

RESUMO

Paroxysmal depolarization shifts (PDS) have been described by epileptologists for the first time several decades ago, but controversy still exists to date regarding their role in epilepsy. In addition to the initial view of a lack of such a role, seemingly opposing hypotheses on epileptogenic and anti-ictogenic effects of PDS have emerged. Hence, PDS may provide novel targets for epilepsy therapy. Evidence for the roles of PDS has often been obtained from investigations of the multi-unit correlate of PDS, an electrographic spike termed "interictal" because of its occurrence during seizure-free periods of epilepsy patients. Meanwhile, interictal spikes have been found to be associated with neuronal diseases other than epilepsy, e.g., Alzheimer's disease, which may indicate a broader implication of PDS in neuropathologies. In this article, we give an introduction to PDS and review evidence that links PDS to pro- as well as anti-epileptic mechanisms, and to other types of neuronal dysfunction. The perturbation of neuronal membrane voltage and of intracellular Ca2+ that comes with PDS offers many conceivable pathomechanisms of neuronal dysfunction. Out of these, the operation of L-type voltage-gated calcium channels, which play a major role in coupling excitation to long-lasting neuronal changes, is addressed in detail.


Assuntos
Epilepsia/metabolismo , Epilepsia/patologia , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Animais , Canais de Cálcio Tipo L/metabolismo , Eletrofisiologia , Hipocampo/citologia , Hipocampo/metabolismo , Humanos , Neurônios/citologia , Neurônios/metabolismo
5.
Int J Mol Sci ; 20(10)2019 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-31137507

RESUMO

The prime task of nociceptors is the transformation of noxious stimuli into action potentials that are propagated along the neurites of nociceptive neurons from the periphery to the spinal cord. This function of nociceptors relies on the coordinated operation of a variety of ion channels. In this review, we summarize how members of nine different families of ion channels expressed in sensory neurons contribute to nociception. Furthermore, data on 35 different types of G protein coupled receptors are presented, activation of which controls the gating of the aforementioned ion channels. These receptors are not only targeted by more than 20 separate endogenous modulators, but can also be affected by pharmacotherapeutic agents. Thereby, this review provides information on how ion channel modulation via G protein coupled receptors in nociceptors can be exploited to provide improved analgesic therapy.


Assuntos
Canais Iônicos/metabolismo , Nociceptores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Nociceptores/fisiologia , Transdução de Sinais
6.
Proteomics ; 17(19)2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28834300

RESUMO

Voltage-gated Kv7.2 potassium channels regulate neuronal excitability. The gating of these channels is tightly controlled by various mediators and neurotransmitters acting via G protein-coupled receptors; the underlying signaling cascades involve phosphatidylinositol-4,5-bisphosphate (PIP2 ), Ca2+ /calmodulin, and phosphorylation. Recent studies found that the PIP2 sensitivity of Kv7.2 channels is affected by two posttranslational modifications, phosphorylation and methylation, harboured within putative PIP2 -binding domains. In this study, we updated phosphorylation and methylation sites in Kv7.2 either heterologously expressed in mammalian cells or as GST-fusion proteins exposed to recombinant protein kinases by using LC-MS/MS. In vitro kinase assays revealed that CDK5, protein kinase C (PKC) alpha, PKA, p38 MAPK, CamKIIα, and GSK3ß could mediate phosphorylation. Taken together, we provided a comprehensive map of phosphorylation and methylation in Kv7.2 within protein-protein and protein-lipid interaction domains. This may help to interpret the functional roles of individual PTM sites in Kv7.2 channels. All MS data are available via ProteomeXchange with the identifier PXD005567.


Assuntos
Metilação de DNA , Canal de Potássio KCNQ2/metabolismo , Lipídeos/análise , Sequência de Aminoácidos , Células HEK293 , Humanos , Técnicas In Vitro , Canal de Potássio KCNQ2/genética , Fosforilação , Mapas de Interação de Proteínas , Homologia de Sequência , Transdução de Sinais , Espectrometria de Massas em Tandem
7.
J Physiol ; 595(3): 759-776, 2017 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-27621207

RESUMO

KEY POINTS: Phosphatidylinositol-4,5-bisphosphate (PIP2 ) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP2 -binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP2 . Dephosphorylation of Kv7.2 affected channel inhibition via M1 muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP2 , thereby ensuring the tight regulation of the channel via G protein-coupled receptors. ABSTRACT: The function of numerous ion channels is tightly controlled by G protein-coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol-4,5-bisphosphate (PIP2 ). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP2 and through phosphorylation. Using liquid chromatography-coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP2 -mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A5 mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP2 depletion via the voltage-sensitive phosphatase Dr-VSP than were wild-type channels. In vitro phosphorylation assays with the purified C-terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild-type but not mutant Kv7.2 channels towards PIP2 depletion via Dr-VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M1 receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP2 -binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR-mediated channel regulation.


Assuntos
Canal de Potássio KCNQ2/fisiologia , Fosfatidilinositol 4,5-Difosfato/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Neurônios/fisiologia , Fosforilação , Ratos Sprague-Dawley , Gânglio Cervical Superior/citologia
8.
Toxicol Appl Pharmacol ; 329: 309-317, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28641963

RESUMO

Retigabine, currently used as antiepileptic drug, has a wide range of potential medical uses. Administration of the drug in patients can lead to QT interval prolongation in the electrocardiogram and to cardiac arrhythmias in rare cases. This suggests that the drug may perturb the electrical properties of the heart, and the underlying mechanisms were investigated here. Effects of retigabine on currents through human cardiac ion channels, heterologously expressed in tsA-201 cells, were studied in whole-cell patch-clamp experiments. In addition, the drug's impact on the cardiac action potential was tested. This was done using ventricular cardiomyocytes isolated from Langendorff-perfused guinea pig hearts and cardiomyocytes derived from human induced pluripotent stem cells. Further, to unravel potential indirect effects of retigabine on the heart which might involve the autonomic nervous system, membrane potential and noradrenaline release from sympathetic ganglionic neurons were measured in the absence and presence of the drug. Retigabine significantly inhibited currents through hKv11.1 potassium, hNav1.5 sodium, as well as hCav1.2 calcium channels, but only in supra-therapeutic concentrations. In a similar concentration range, the drug shortened the action potential in both guinea pig and human cardiomyocytes. Therapeutic concentrations of retigabine, on the other hand, were sufficient to inhibit the activity of sympathetic ganglionic neurons. We conclude that retigabine- induced QT interval prolongation, and the reported cases of cardiac arrhythmias after application of the drug in a typical daily dose range, cannot be explained by a direct modulatory effect on cardiac ion channels. They are rather mediated by indirect actions at the level of the autonomic nervous system.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Anticonvulsivantes/toxicidade , Arritmias Cardíacas/induzido quimicamente , Carbamatos/toxicidade , Gânglios Simpáticos/efeitos dos fármacos , Bloqueadores Ganglionares/toxicidade , Sistema de Condução Cardíaco/efeitos dos fármacos , Canais Iônicos/antagonistas & inibidores , Miócitos Cardíacos/efeitos dos fármacos , Fenilenodiaminas/toxicidade , Animais , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Bloqueadores dos Canais de Cálcio/toxicidade , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo L/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Canal de Potássio ERG1/antagonistas & inibidores , Canal de Potássio ERG1/metabolismo , Gânglios Simpáticos/metabolismo , Gânglios Simpáticos/fisiopatologia , Cobaias , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca/efeitos dos fármacos , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais Iônicos/genética , Canais Iônicos/metabolismo , Preparação de Coração Isolado , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/efeitos dos fármacos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Norepinefrina/metabolismo , Bloqueadores dos Canais de Potássio/toxicidade , Ratos Sprague-Dawley , Medição de Risco , Fatores de Tempo , Transfecção , Bloqueadores do Canal de Sódio Disparado por Voltagem/toxicidade
9.
Epilepsia ; 58(5): 858-871, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28295232

RESUMO

OBJECTIVE: An increase of neuronal Cav 1.3 L-type calcium channels (LTCCs) has been observed in various animal models of epilepsy. However, LTCC inhibitors failed in clinical trials of epileptic treatment. There is compelling evidence that paroxysmal depolarization shifts (PDSs) involve Ca2+ influx through LTCCs. PDSs represent a hallmark of epileptiform activity. In recent years, a probable epileptogenic role for PDSs has been proposed. However, the implication of the two neuronal LTCC isoforms, Cav 1.2 and Cav 1.3, in PDSs remained unknown. Moreover, Ca2+ -dependent nonspecific cation (CAN) channels have also been suspected to contribute to PDSs. Nevertheless, direct experimental support of an important role of CAN channel activation in PDS formation is still lacking. METHODS: Primary neuronal networks derived from dissociated hippocampal neurons were generated from mice expressing a dihydropyridine-insensitive Cav 1.2 mutant (Cav 1.2DHP-/- mice) or from Cav 1.3-/- knockout mice. To investigate the role of Cav 1.2 and Cav 1.3, perforated patch-clamp recordings were made of epileptiform activity, which was elicited using either bicuculline or caffeine. LTCC activity was modulated using the dihydropyridines Bay K 8644 (agonist) and isradipine (antagonist). RESULTS: Distinct PDS could be elicited upon LTCC potentiation in Cav 1.2DHP-/- neurons but not in Cav 1.3-/- neurons. In contrast, when bicuculline led to long-lasting, seizure-like discharge events rather than PDS, these were prolonged in Cav 1.3-/- neurons but not in Cav 1.2DHP-/- neurons. Because only the Cav 1.2 isoform is functionally coupled to CAN channels in primary hippocampal networks, PDS formation does not require CAN channel activity. SIGNIFICANCE: Our data suggest that the LTCC requirement of PDS relates primarily to Cav 1.3 channels rather than to Cav 1.2 channels and CAN channels in hippocampal neurons. Hence, Cav 1.3 may represent a new therapeutic target for suppression of PDS development. The proposed epileptogenic role of PDSs may allow for a prophylactic rather than the unsuccessful seizure suppressing application of LTCC inhibitors.


Assuntos
Canais de Cálcio Tipo L/fisiologia , Potenciais Evocados/fisiologia , Hipocampo/fisiopatologia , Rede Nervosa/fisiopatologia , Neurônios/fisiologia , Animais , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos , Técnicas de Patch-Clamp
10.
Purinergic Signal ; 12(3): 497-507, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27172914

RESUMO

ADP and other nucleotides control ion currents in the nervous system via various P2Y receptors. In this respect, Cav2 and Kv7 channels have been investigated most frequently. The fine tuning of neuronal ion channel gating via G protein coupled receptors frequently relies on the formation of higher order protein complexes that are organized by scaffolding proteins and harbor receptors and channels together with interposed signaling components. However, ion channel complexes containing P2Y receptors have not been described. Therefore, the regulation of Cav2.2 and Kv7.2/7.3 channels via P2Y1 and P2Y12 receptors and the coordination of these ion channels and receptors in the plasma membranes of tsA 201 cells have been investigated here. ADP inhibited currents through Cav2.2 channels via both P2Y1 and P2Y12 receptors with phospholipase C and pertussis toxin-sensitive G proteins being involved, respectively. The nucleotide controlled the gating of Kv7 channels only via P2Y1 and phospholipase C. In fluorescence energy transfer assays using conventional as well as total internal reflection (TIRF) microscopy, both P2Y1 and P2Y12 receptors were found juxtaposed to Cav2.2 channels, but only P2Y1, and not P2Y12, was in close proximity to Kv7 channels. Using fluorescence recovery after photobleaching in TIRF microscopy, evidence for a physical interaction was obtained for the pair P2Y12/Cav2.2, but not for any other receptor/channel combination. These results reveal a membrane juxtaposition of P2Y receptors and ion channels in parallel with the control of neuronal ion currents by ADP. This juxtaposition may even result in apparent physical interactions between receptors and channels.


Assuntos
Difosfato de Adenosina/metabolismo , Canais Iônicos/metabolismo , Neurônios/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Receptores Purinérgicos P2Y1/metabolismo , Canais de Cálcio Tipo N/metabolismo , Linhagem Celular , Humanos , Canal de Potássio KCNQ2/metabolismo , Canal de Potássio KCNQ3/metabolismo , Microscopia Confocal , Microscopia de Fluorescência , Técnicas de Patch-Clamp
11.
Proc Natl Acad Sci U S A ; 110(28): 11642-7, 2013 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-23798435

RESUMO

Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.


Assuntos
Anfetamina/farmacologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Células HEK293 , Humanos , Sistemas do Segundo Mensageiro , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética
12.
Epilepsia ; 56(4): 647-57, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25779225

RESUMO

OBJECTIVE: Within its range of therapeutic plasma concentrations, the anticonvulsant retigabine (ezogabine) is believed to selectively act on Kv7 channels. Here, the contribution of specific γ-aminobutyric acid (GABA)A receptor subtypes to the antiseizure effects of retigabine was investigated. METHODS: Using patch-clamp recordings, seizure-like activity, tonic currents, and GABA-induced currents in hippocampal neurons were tested for their sensitivity toward retigabine, as were recombinant GABAA receptors expressed in tsA 201 cells. RESULTS: Retigabine reduced seizure-like activity elicited by low Mg(2+) in a concentration-dependent manner with half maximal inhibition at 1 µm. Seizure-like activity triggered by blocking either Kv7 channels or GABAA receptors was equally reduced by retigabine, but when these channels/receptors were blocked simultaneously, the inhibition was lost. Retigabine (10 µm) enhanced bicuculline-sensitive tonic currents in hippocampal neurons, but failed to affect GABA-evoked currents. However, when receptors involved in phasic GABAergic inhibition were blocked by penicillin, retigabine did enhance GABA-evoked currents. In tsA 201 cells expressing various combinations of GABAA receptor subunits, 10 µm retigabine enhanced currents through α1ß2δ, α4ß2δ, α4ß3δ, and α6ß2δ receptors, but left currents through α1ß2γ2S, α4ß3γ2S, α5ß3γ2S, and α6ß2γ2S receptors unaltered. With αß receptors, retigabine diminished currents through α1ß2 and α4ß3, but increased currents through α6ß2 receptors. The enhancement of currents through α1ß2δ receptors by retigabine was concentration dependent and became significant at 1 µm. SIGNIFICANCE: These results demonstrate that retigabine is a subtype selective modulator of GABAA receptors with preference for extrasynaptic δ-containing receptors; this property may contribute to its broad antiepileptic effectiveness and explain its lack of effect on absence seizures.


Assuntos
Anticonvulsivantes/farmacologia , Carbamatos/farmacologia , Moduladores GABAérgicos/farmacologia , Fenilenodiaminas/farmacologia , Receptores de GABA-A/fisiologia , Animais , Anticonvulsivantes/uso terapêutico , Carbamatos/uso terapêutico , Células Cultivadas , Relação Dose-Resposta a Droga , Moduladores GABAérgicos/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/fisiologia , Fenilenodiaminas/uso terapêutico , Ratos , Ratos Sprague-Dawley , Convulsões/tratamento farmacológico , Convulsões/fisiopatologia
13.
Pflugers Arch ; 466(12): 2289-303, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24668449

RESUMO

The slow cholinergic transmission in autonomic ganglia is known to be mediated by an inhibition of Kv7 channels via M1 muscarinic acetylcholine receptors. However, in the present experiments using primary cultures of rat superior cervical ganglion neurons, the extent of depolarisation caused by the M1 receptor agonist oxotremorine M did not correlate with the extent of Kv7 channel inhibition in the very same neuron. This observation triggered a search for additional mechanisms. As the activation of M1 receptors leads to a boost in protein kinase C (PKC) activity in sympathetic neurons, various PKC enzymes were inhibited by different means. Interference with classical PKC isoforms led to reductions in depolarisations and in noradrenaline release elicited by oxotremorine M, but left the Kv7 channel inhibition by the muscarinic agonist unchanged. M1 receptor-induced depolarisations were also altered when extra- or intracellular Cl(-) concentrations were changed, as were depolarising responses to γ-aminobutyric acid. Depolarisations and noradrenaline release triggered by oxotremorine M were reduced by the non-selective Cl(-) channel blockers 4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulfonic acid and niflumic acid. Oxotremorine M induced slowly rising inward currents at negative membrane potentials that were blocked by inhibitors of Ca(2+)-activated Cl(-) and TMEM16A channels and attenuated by PKC inhibitors. These channel blockers also reduced oxotremorine M-evoked noradrenaline release. Together, these results reveal that slow cholinergic excitation of sympathetic neurons involves the activation of classical PKCs and of Ca(2+)-activated Cl(-) channels in addition to the well-known inhibition of Kv7 channels.


Assuntos
Potenciais de Ação , Canais de Potássio KCNQ/metabolismo , Neurônios/metabolismo , Receptor Muscarínico M1/metabolismo , Gânglio Cervical Superior/metabolismo , Animais , Células Cultivadas , Canais de Cloreto/antagonistas & inibidores , Cloretos/metabolismo , Agonistas Muscarínicos/farmacologia , Neurônios/fisiologia , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Receptor Muscarínico M1/agonistas , Gânglio Cervical Superior/citologia , Gânglio Cervical Superior/fisiologia
14.
J Pharmacol Exp Ther ; 348(2): 346-58, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24307198

RESUMO

Ibogaine is a psychoactive indole alkaloid. Its use as an antiaddictive agent has been accompanied by QT prolongation and cardiac arrhythmias, which are most likely caused by human ether a go-go-related gene (hERG) potassium channel inhibition. Therefore, we studied in detail the interaction of ibogaine with hERG channels heterologously expressed in mammalian kidney tsA-201 cells. Currents through hERG channels were blocked regardless of whether ibogaine was applied via the extracellular or intracellular solution. The extent of inhibition was determined by the relative pH values. Block occurred during activation of the channels and was not observed for resting channels. With increasing depolarizations, ibogaine block grew and developed faster. Steady-state activation and inactivation of the channel were shifted to more negative potentials. Deactivation was slowed, whereas inactivation was accelerated. Mutations in the binding site reported for other hERG channel blockers (Y652A and F656A) reduced the potency of ibogaine, whereas an inactivation-deficient double mutant (G628C/S631C) was as sensitive as wild-type channels. Molecular drug docking indicated binding within the inner cavity of the channel independently of the protonation of ibogaine. Experimental current traces were fit to a kinetic model of hERG channel gating, revealing preferential binding of ibogaine to the open and inactivated state. Taken together, these findings show that ibogaine blocks hERG channels from the cytosolic side either in its charged form alone or in company with its uncharged form and alters the currents by changing the relative contribution of channel states over time.


Assuntos
Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Antagonistas de Aminoácidos Excitatórios/farmacologia , Alucinógenos/farmacologia , Ibogaína/farmacologia , Antagonistas de Entorpecentes/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Substituição de Aminoácidos , Sítios de Ligação/efeitos dos fármacos , Linhagem Celular , Citosol/metabolismo , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/química , Canais de Potássio Éter-A-Go-Go/genética , Canais de Potássio Éter-A-Go-Go/metabolismo , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Antagonistas de Aminoácidos Excitatórios/química , Alucinógenos/efeitos adversos , Alucinógenos/química , Humanos , Concentração de Íons de Hidrogênio , Ibogaína/efeitos adversos , Ibogaína/química , Ativação do Canal Iônico/efeitos dos fármacos , Cinética , Potenciais da Membrana/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Proteínas Mutantes/agonistas , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Antagonistas de Entorpecentes/efeitos adversos , Antagonistas de Entorpecentes/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo
15.
Addict Biol ; 19(2): 237-239, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22458604

RESUMO

Ibogaine, an alkaloid derived from the African shrub Tabernanthe iboga, has shown promising anti-addictive properties in animals. Anecdotal evidence suggests that ibogaine is also anti-addictive in humans. Thus, it alleviates drug craving and impedes relapse of drug use. Although not licensed as therapeutic drug, and despite evidence that ibogaine may disturb the rhythm of the heart, this alkaloid is currently used as an anti-addiction drug in alternative medicine. Here, we report that therapeutic concentrations of ibogaine reduce currents through human ether-a-go-go-related gene potassium channels. Thereby, we provide a mechanism by which ibogaine may generate life-threatening cardiac arrhythmias.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Comportamento Aditivo/tratamento farmacológico , Canais de Potássio Éter-A-Go-Go/antagonistas & inibidores , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ibogaína/farmacologia , Adulto , Animais , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/efeitos adversos , Feminino , Frequência Cardíaca/efeitos dos fármacos , Humanos , Ibogaína/efeitos adversos , Prevenção Secundária
16.
Br J Pharmacol ; 181(16): 2851-2868, 2024 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-38657956

RESUMO

BACKGROUND AND PURPOSE: The analgesic action of paracetamol involves KV7 channels, and its metabolite N-acetyl-p-benzo quinone imine (NAPQI), a cysteine modifying reagent, was shown to increase currents through such channels in nociceptors. Modification of cysteine residues by N-ethylmaleimide, H2O2, or nitric oxide has been found to modulate currents through KV7 channels. The study aims to identify whether, and if so which, cysteine residues in neuronal KV7 channels might be responsible for the effects of NAPQI. EXPERIMENTAL APPROACH: To address this question, we used a combination of perforated patch-clamp recordings, site-directed mutagenesis, and mass spectrometry applied to recombinant KV7.1 to KV7.5 channels. KEY RESULTS: Currents through the cardiac subtype KV7.1 were reduced by NAPQI. Currents through all other subtypes were increased, either by an isolated shift of the channel voltage dependence to more negative values (KV7.3) or by such a shift combined with increased maximal current levels (KV7.2, KV7.4, KV7.5). A stretch of three cysteine residues in the S2-S3 linker region of KV7.2 was necessary and sufficient to mediate these effects. CONCLUSION AND IMPLICATION: The paracetamol metabolite N-acetyl-p-benzo quinone imine (NAPQI) modifies cysteine residues of KV7 subunits and reinforces channel gating in homomeric and heteromeric KV7.2 to KV7.5, but not in KV7.1 channels. In KV7.2, a triple cysteine motif located within the S2-S3 linker region mediates this reinforcement that can be expected to reduce the excitability of nociceptors and to mediate antinociceptive actions of paracetamol.


Assuntos
Acetaminofen , Benzoquinonas , Cisteína , Iminas , Cisteína/metabolismo , Acetaminofen/farmacologia , Benzoquinonas/farmacologia , Benzoquinonas/metabolismo , Animais , Iminas/farmacologia , Iminas/química , Iminas/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Canais de Potássio KCNQ/metabolismo , Canais de Potássio KCNQ/genética , Humanos , Motivos de Aminoácidos , Analgésicos não Narcóticos/farmacologia , Células HEK293 , Ratos
17.
J Biol Chem ; 287(1): 438-445, 2012 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-22072712

RESUMO

Serotonin (5-HT) uptake by the human serotonin transporter (hSERT) is driven by ion gradients. The stoichiometry of transported 5-HT and ions is predicted to result in electroneutral charge movement. However, hSERT mediates a current when challenged with 5-HT. This discrepancy can be accounted for by an uncoupled ion flux. Here, we investigated the mechanistic basis of the uncoupled currents and its relation to the conformational cycle of hSERT. Our observations support the conclusion that the conducting state underlying the uncoupled ion flux is in equilibrium with an inward facing state of the transporter with K+ bound. We identified conditions associated with accumulation of the transporter in inward facing conformations. Manipulations that increased the abundance of inward facing states resulted in enhanced steady-state currents. We present a comprehensive kinetic model of the transport cycle, which recapitulates salient features of the recorded currents. This study provides a framework for exploring transporter-associated currents.


Assuntos
Condutividade Elétrica , Fenômenos Eletrofisiológicos , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Animais , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Cinética , Microscopia de Fluorescência , Modelos Biológicos , Potássio/metabolismo , Conformação Proteica/efeitos dos fármacos , Serotonina/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/química
18.
J Biol Chem ; 287(22): 18524-34, 2012 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-22451652

RESUMO

Ibogaine, a hallucinogenic alkaloid proposed as a treatment for opiate withdrawal, has been shown to inhibit serotonin transporter (SERT) noncompetitively, in contrast to all other known inhibitors, which are competitive with substrate. Ibogaine binding to SERT increases accessibility in the permeation pathway connecting the substrate-binding site with the cytoplasm. Because of the structural similarity between ibogaine and serotonin, it had been suggested that ibogaine binds to the substrate site of SERT. The results presented here show that ibogaine binds to a distinct site, accessible from the cell exterior, to inhibit both serotonin transport and serotonin-induced ionic currents. Ibogaine noncompetitively inhibited transport by both SERT and the homologous dopamine transporter (DAT). Ibogaine blocked substrate-induced currents also in DAT and increased accessibility of the DAT cytoplasmic permeation pathway. When present on the cell exterior, ibogaine inhibited SERT substrate-induced currents, but not when it was introduced into the cytoplasm through the patch electrode. Similar to noncompetitive transport inhibition, the current block was not reversed by increasing substrate concentration. The kinetics of inhibitor binding and dissociation, as determined by their effect on SERT currents, indicated that ibogaine does not inhibit by forming a long-lived complex with SERT, but rather binds directly to the transporter in an inward-open conformation. A kinetic model for transport describing the noncompetitive action of ibogaine and the competitive action of cocaine accounts well for the results of the present study.


Assuntos
Proteínas da Membrana Plasmática de Transporte de Dopamina/antagonistas & inibidores , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ibogaína/farmacologia , Proteínas da Membrana Plasmática de Transporte de Serotonina/efeitos dos fármacos , Animais , Sítios de Ligação , Linhagem Celular , Proteínas da Membrana Plasmática de Transporte de Dopamina/metabolismo , Antagonistas de Aminoácidos Excitatórios/metabolismo , Humanos , Ibogaína/metabolismo , Cinética , Técnicas de Patch-Clamp , Ensaio Radioligante , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Xenopus laevis
19.
Front Pharmacol ; 14: 1276100, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37881182

RESUMO

Introduction: In addition to members of the family of Na+/Cl- dependent monoamine transporters, organic cation transporters (OCTs), in particular OCT3, as well as the plasma membrane monoamine transporter (PMAT) may contribute to neuronal reuptake of according neurotransmitters. As opposed to the numerous blockers of monoamine transporters, only a very limited number of specific blockers of OCT3 and PMAT are available. In fact, decynium-22 is the only blocking agent with micromolar affinities for both transport proteins, and this molecule is frequently used to establish roles of OCT3 and/or PMAT as targets for antidepressant drugs and psychostimulants, respectively. Methods/Results: To test for a function of these transporters in the sympathetic nervous system, uptake and release of [3H]1-methyl-4-phenylpyridinium (MPP+) was investigated in primary cultures of rat superior cervical ganglia. Uptake was reduced by cocaine or desipramine, blockers of the noradrenaline transporter, by about 70% and by corticosterone or ß-estradiol, blockers of OCT3, by about 30%; decynium-22 achieved complete inhibition of uptake with half maximal effects at 3 µM. Depolarization dependent release was enhanced by corticosterone or ß-estradiol, but reduced by decynium-22. As the latter effect is unlikely to be related to actions at OCT3 and/or PMAT, electrophysiological recordings were performed to reveal that decynium-22 inhibits action potential firing and currents through voltage activated calcium channels in superior cervical ganglion neurons. Discussion: These results demonstrate that decynium-22 can impair exocytotic neurotransmitter release by interfering with several types of ion channels. Such transporter-independent effects of decynium-22 that my interfere with basic neuronal functions need to be considered when interpreting results obtained with decynium-22 as prototypic inhibitor of transmitter reuptake via OCT3 and/or PMAT.

20.
Purinergic Signal ; 8(4): 677-92, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22476939

RESUMO

Neuroblastoma SH-SY5Y (SH) cells endogenously express A(2A) adenosine receptors and can be differentiated into a sympathetic neuronal phenotype, capable of depolarisation-dependent noradrenaline release. Using differentiated SH culture, we here explored the link between A(2A)-receptor signalling and neurotransmitter release. In response to the receptor agonist CGS21680, the cells produced cyclic AMP (cAMP), and when depolarised, they released increased amounts of noradrenaline. An A(2A)-receptor antagonist, XAC, as well as an inhibitor of cAMP-dependent protein kinase A (PKA), H89, depressed agonist-dependent release. In the presence of XAC or H89, noradrenaline release was found to be below basal values. This suggested that release facilitation also owes to constitutive receptor activity. We demonstrate that even in the absence of an agonist, the native A(2A)-receptor stimulated cAMP production, leading to the activation of PKA and enhanced noradrenaline release. Ancillary, non-cAMP-dependent effects of the receptor (i.e. phosphorylation of CREB, of Rabphilin3A) were refractory to constitutive activation. PKA-dependent facilitation of noradrenaline release was recapitulated with membrane-permeable 8-Br-cAMP; in addition to facilitation, 8-Br-cAMP caused marked inhibition of release, an effect not observed upon receptor activation. Inhibition by receptor-independent cAMP was likely due to suppression of voltage-dependent calcium current (VDCC) and increased activity of Src-family kinases. Receptor-mediated release facilitation was reproduced in the presence of tetrodotoxin (blocking action potentials); hence, the signalling occurred at the active zone comprising release sites. Our findings thus support (1) presynaptic localisation of the A(2A)-receptor and (2) suggest that compartmentalised pathways transmit cAMP signalling in order to facilitate depolarisation-dependent neurotransmitter release.


Assuntos
Agonistas do Receptor A2 de Adenosina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Norepinefrina/metabolismo , Receptor A2A de Adenosina/metabolismo , Transdução de Sinais , Adenosina/análogos & derivados , Adenosina/farmacologia , Diferenciação Celular/fisiologia , Linhagem Celular , Humanos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fenetilaminas/farmacologia , Transmissão Sináptica/efeitos dos fármacos
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