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1.
Neurobiol Dis ; 127: 114-130, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30763678

RESUMO

Parkinson's disease (PD) is the second most frequent neurodegenerative disorder in the old population. Among its monogenic variants, a frequent cause is a mutation in the Parkin gene (Prkn). Deficient function of Parkin triggers ubiquitous mitochondrial dysfunction and inflammation in the brain, but it remains unclear how selective neural circuits become vulnerable and finally undergo atrophy. We attempted to go beyond previous work, mostly done in peripheral tumor cells, which identified protein targets of Parkin activity, an ubiquitin E3 ligase. Thus, we now used aged Parkin-knockout (KO) mouse brain for a global quantification of ubiquitylated peptides by mass spectrometry (MS). This approach confirmed the most abundant substrate to be VDAC3, a mitochondrial outer membrane porin that modulates calcium flux, while uncovering also >3-fold dysregulations for neuron-specific factors. Ubiquitylation decreases were prominent for Hippocalcin (HPCA), Calmodulin (CALM1/CALML3), Pyruvate Kinase (PKM2), sodium/potassium-transporting ATPases (ATP1A1/2/3/4), the Rab27A-GTPase activating protein alpha (TBC1D10A) and an ubiquitin ligase adapter (DDB1), while strong increases occurred for calcium transporter ATP2C1 and G-protein subunits G(i)/G(o)/G(Tr). Quantitative immunoblots validated elevated abundance for the electrogenic pump ATP1A2, for HPCA as neuron-specific calcium sensor, which stimulates guanylate cyclases and modifies axonal slow afterhyperpolarization (sAHP), and for the calcium-sensing G-protein GNA11. We assessed if compensatory molecular regulations become insufficient over time, leading to functional deficits. Patch clamp experiments in acute Parkin-KO brain slices indeed revealed alterations of the electrophysiological properties in aged noradrenergic locus coeruleus (LC) neurons. LC neurons of aged Parkin-KO brain showed an acceleration of the spontaneous pacemaker frequency, a reduction in sAHP and shortening of action potential duration, without modulation of KCNQ potassium currents. These findings indicate altered calcium-dependent excitability in a PARK2 model of PD, mediated by diminished turnover of potential Parkin targets such as ATP1A2 and HPCA. The data also identified further novel Parkin substrate candidates like SIRT2, OTUD7B and CUL5. Our elucidation of neuron-specific mechanisms of PD pathogenesis helps to explain the known exceptional susceptibility of noradrenergic and dopaminergic projections to alterations of calcium homeostasis and its mitochondrial buffering.


Assuntos
Neurônios Adrenérgicos/metabolismo , Encéfalo/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Hipocalcina/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Espectrometria de Massas , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Técnicas de Patch-Clamp , Ubiquitina-Proteína Ligases/genética , Canais de Ânion Dependentes de Voltagem/metabolismo
2.
Biochemistry (Mosc) ; 76(8): 890-9, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22022962

RESUMO

The regulation of neuronal excitability is complex, as ion channels and neurotransmitter receptors are underlying a large variety of modulating effects. Alterations in the expression patterns of receptors or channel subunits as well as differential splicing contribute to the regulation of neuronal excitability. RNA editing is another and more recently explored mechanism to increase protein diversity, as the genomic recoding leads to new gene products with novel functional and pharmacological properties. In humans A-to-I RNA editing targets several neuronal receptors and channels, including GluR2/5/6 subunits, the Kv1.1 channel, and the 5-HT(2C) receptor. Our review summarizes that RNA editing of these proteins does not only change protein function, but also the pharmacology and presumably the drug therapy in human diseases.


Assuntos
Adenosina/genética , Inosina/genética , Canais Iônicos/genética , Doenças do Sistema Nervoso/tratamento farmacológico , Doenças do Sistema Nervoso/genética , Neurotransmissores/farmacologia , Edição de RNA , Adenosina/metabolismo , Animais , Humanos , Inosina/metabolismo , Canais Iônicos/metabolismo , Doenças do Sistema Nervoso/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo
3.
Br J Pharmacol ; 134(7): 1467-79, 2001 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-11724753

RESUMO

1. We identified the ethacrynic-acid derivative DCPIB as a potent inhibitor of I(Cl,swell), which blocks native I(Cl,swell) of calf bovine pulmonary artery endothelial (CPAE) cells with an IC(50) of 4.1 microM. Similarly, 10 microM DCPIB almost completely inhibited the swelling-induced chloride conductance in Xenopus oocytes and in guinea-pig atrial cardiomyocytes. Block of I(Cl,swell) by DCPIB was fully reversible and voltage independent. 2. DCPIB (10 microM) showed selectivity for I(Cl,swell) and had no significant inhibitory effects on I(Cl,Ca) in CPAE cells, on chloride currents elicited by several members of the CLC-chloride channel family or on the human cystic fibrosis transmembrane conductance regulator (hCFTR) after heterologous expression in Xenopus oocytes. DCPIB (10 microM) also showed no significant inhibition of several native anion and cation currents of guinea pig heart like I(Cl,PKA), I(Kr), I(Ks), I(K1), I(Na) and I(Ca). 3. In all atrial cardiomyocytes (n=7), osmotic swelling produced an increase in chloride current and a strong shortening of the action potential duration (APD). Both swelling-induced chloride conductance and AP shortening were inhibited by treatment of swollen cells with DCPIB (10 microM). In agreement with the selectivity for I(Cl,swell), DCPIB did not affect atrial APD under isoosmotic conditions. 4. Preincubation of atrial cardiomyocytes with DCPIB (10 microM) completely prevented both the swelling-induced chloride currents and the AP shortening but not the hypotonic cell swelling. 5. We conclude that swelling-induced AP shortening in isolated atrial cells is mainly caused by activation of I(Cl,swell). DCPIB therefore is a valuable pharmacological tool to study the role of I(Cl,swell) in cardiac excitability under pathophysiological conditions leading to cell swelling.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Canais de Cloreto/antagonistas & inibidores , Ciclopentanos/farmacologia , Átrios do Coração/efeitos dos fármacos , Indanos/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Função Atrial , Tamanho Celular/fisiologia , Células Cultivadas , Canais de Cloreto/genética , Canais de Cloreto/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Relação Dose-Resposta a Droga , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Cobaias , Átrios do Coração/citologia , Potenciais da Membrana/efeitos dos fármacos , Oócitos , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/genética , Canais de Potássio/fisiologia , Artéria Pulmonar/citologia , Artéria Pulmonar/efeitos dos fármacos , Artéria Pulmonar/fisiologia , Sensibilidade e Especificidade , Canais de Potássio Shal , Fatores de Tempo , Xenopus
4.
Cardiovasc Res ; 52(2): 255-64, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11684073

RESUMO

OBJECTIVE: The Ca(2+) independent transient outward K(+) current (I(to1)) in the heart is responsible for the initial phase of repolarization. The hKv4.3 K(+) channel alpha-subunit contributes to the I(to1) current in many regions of the human heart. Consistently, downregulation of hKv4.3 transcripts in heart failure and atrial fibrillation is linked to reduction in I(to1) conductance. The recently cloned KChIP family of calcium sensors has been shown to modulate A-type potassium channels of the Kv4 K(+) channel subfamily. METHODS AND RESULTS: We describe the cloning and tissue distribution of hKChIP2, as well as its functional interaction with hKv4.3 after expression in Xenopus oocytes. Furthermore, we isolated a short splice variant of the hKChIP2 gene (hKCNIP2), which represents the major hKChIP2 transcript. Northern blot analyses revealed that hKChIP2 is expressed in the human heart and occurs in the adult atria and ventricles but not in the fetal heart. Upon coexpression with hKv4.3 both hKChIP2 isoforms increased the current amplitude, slowed the inactivation and increased the recovery from inactivation of hKv4.3 currents. For the first time we analyzed the influence of a KChIP protein on the voltage of half-maximal inactivation of Kv4 channels. We demonstrate that the hKChIP2 isoforms shifted the half-maximal inactivation to more positive potentials, but to a different extent. By elucidating the genomic structure, we provide important information for future analysis of the hKCNIP2 gene in candidate disorders. In the course of this work we mapped the hKCNIP2 gene to chromosome 10q24. CONCLUSIONS: Heteromeric hKv4.3/hKChIP2 currents more closely resemble native epicardial I(to1), suggesting that hKChIP2 is a true beta-subunit of human cardiac I(to1). As a result hKChIP2 might play a role in cardiac diseases, where a contribution of I(to1) has been shown.


Assuntos
Processamento Alternativo , Proteínas de Ligação ao Cálcio/genética , Cromossomos Humanos Par 10 , Miocárdio/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Canais de Potássio/genética , Animais , Northern Blotting/métodos , Mapeamento Cromossômico , Clonagem Molecular , Feminino , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Íntrons , Proteínas Interatuantes com Canais de Kv , Miocárdio/metabolismo , Oócitos/metabolismo , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase/métodos , Canais de Potássio/análise , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , Análise de Sequência de DNA , Canais de Potássio Shal , ATPase Trocadora de Sódio-Potássio , Xenopus laevis
5.
FEBS Lett ; 492(1-2): 84-9, 2001 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-11248242

RESUMO

We report the primary sequence of TASK-4, a novel member of the acid-sensitive subfamily of tandem pore K(+) channels. TASK-4 transcripts are widely expressed in humans, with highest levels in liver, lung, pancreas, placenta, aorta and heart. In Xenopus oocytes TASK-4 generated K(+) currents displaying a marked outward rectification which was lost by elevation of extracellular K(+). TASK-4 currents were efficiently blocked by barium (83% inhibition at 2 mM), only weakly inhibited by 1 mM concentrations of quinine, bupivacaine and lidocaine, but not blocked by tetraethylammonium, 4-aminopyridine and Cs(+). TASK-4 was sensitive to extracellular pH, but in contrast to other TASK channels, pH sensitivity was shifted to more alkaline pH. Thus, TASK-4 in concert with other TASK channels might regulate cellular membrane potential over a wide range of extracellular pH.


Assuntos
Canais de Potássio de Domínios Poros em Tandem , Canais de Potássio/genética , Sequência de Aminoácidos , Animais , Nó Atrioventricular/metabolismo , Bário/farmacologia , Clonagem Molecular , Eletrofisiologia , Átrios do Coração/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Oócitos , Filogenia , Bloqueadores dos Canais de Potássio , Canais de Potássio/metabolismo , Conformação Proteica , Homologia de Sequência de Aminoácidos , Distribuição Tecidual , Xenopus laevis
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