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1.
Sci Rep ; 9(1): 15642, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31666636

RESUMO

Cav3 / T-type Ca2+ channels are dynamically regulated by intracellular Ca2+ ions, which inhibit Cav3 availability. Here, we demonstrate that this inhibition becomes irreversible in the presence of non-hydrolysable ATP analogs, resulting in a strong hyperpolarizing shift in the steady-state inactivation of the residual Cav3 current. Importantly, the effect of these ATP analogs was prevented in the presence of intracellular BAPTA. Additional findings obtained using intracellular dialysis of inorganic phosphate and alkaline phosphatase or NaN3 treatment further support the involvement of a phosphorylation mechanism. Contrasting with Cav1 and Cav2 Ca2+ channels, the Ca2+-dependent modulation of Cav3 channels appears to be independent of calmodulin, calcineurin and endocytic pathways. Similar findings were obtained for the native T-type Ca2+ current recorded in rat thalamic neurons of the central medial nucleus. Overall, our data reveal a new Ca2+ sensitive phosphorylation-dependent mechanism regulating Cav3 channels, with potentially important physiological implications for the multiple cell functions controlled by T-type Ca2+ channels.

2.
Sci Rep ; 8(1): 9603, 2018 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-29941890

RESUMO

Transparent anti-fogging and self-cleaning coatings are of great interest for many applications, including solar panels, windshields and displays or lenses to be used in humid environments. In this paper, we report on the simultaneous synthesis, at atmospheric pressure, of anatase TiO2 nanoparticles and low-temperature, high-rate deposition of anatase TiO2/SiO2 nanocomposite coatings. These coatings exhibit durable super-hydrophilic and photocatalytic properties. The strategy followed relies on concomitant and separated injections of titania, i.e. titanium isopropoxide, and silica, i.e. hexamethyldisiloxane, precursors in the stream of a blown-arc discharge to form transparent anti-fogging and self-cleaning anatase TiO2/SiO2 nanocomposite coatings on polymer substrates.

3.
Brain ; 141(7): 1998-2013, 2018 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-29878067

RESUMO

Cerebellar atrophy is a key neuroradiological finding usually associated with cerebellar ataxia and cognitive development defect in children. Unlike the adult forms, early onset cerebellar atrophies are classically described as mostly autosomal recessive conditions and the exact contribution of de novo mutations to this phenotype has not been assessed. In contrast, recent studies pinpoint the high prevalence of pathogenic de novo mutations in other developmental disorders such as intellectual disability, autism spectrum disorders and epilepsy. Here, we investigated a cohort of 47 patients with early onset cerebellar atrophy and/or hypoplasia using a custom gene panel as well as whole exome sequencing. De novo mutations were identified in 35% of patients while 27% had mutations inherited in an autosomal recessive manner. Understanding if these de novo events act through a loss or a gain of function effect is critical for treatment considerations. To gain a better insight into the disease mechanisms causing these cerebellar defects, we focused on CACNA1G, a gene not yet associated with the early-onset form. This gene encodes the Cav3.1 subunit of T-type calcium channels highly expressed in Purkinje neurons and deep cerebellar nuclei. We identified four patients with de novo CACNA1G mutations. They all display severe motor and cognitive impairment, cerebellar atrophy as well as variable features such as facial dysmorphisms, digital anomalies, microcephaly and epilepsy. Three subjects share a recurrent c.2881G>A/p.Ala961Thr variant while the fourth patient has the c.4591A>G/p.Met1531Val variant. Both mutations drastically impaired channel inactivation properties with significantly slower kinetics (∼5 times) and negatively shifted potential for half-inactivation (>10 mV). In addition, these two mutations increase neuronal firing in a cerebellar nuclear neuron model and promote a larger window current fully inhibited by TTA-P2, a selective T-type channel blocker. This study highlights the prevalence of de novo mutations in early-onset cerebellar atrophy and demonstrates that A961T and M1531V are gain of function mutations. Moreover, it reveals that aberrant activity of Cav3.1 channels can markedly alter brain development and suggests that this condition could be amenable to treatment.

4.
PLoS One ; 12(10): e0186864, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29073181

RESUMO

Medicinal plants represent a significant reservoir of unexplored substances for early-stage drug discovery. Of interest, two flowering Mediterranean plants have been used for thousands of years for their beneficial effects on nervous disorders, including anxiety and mood. However, the therapeutic potential of these plants regarding their ability to target ion channels and neuronal excitability remains largely unknown. Towards this goal, we have investigated the ability of Lavender and Rosemary to modulate T-type calcium channels (TTCCs). TTCCs play important roles in neuronal excitability, neuroprotection, sensory processes and sleep. These channels are also involved in epilepsy and pain. Using the whole-cell patch-clamp technique, we have characterized how Lavender and Rosemary extracts, as well as their major active compounds Linalool and Rosmarinic acid, modulate the electrophysiological properties of recombinant TTCCs (CaV3.2) expressed in HEK-293T cells. Both the methanolic and essential oil extracts as well as the active compounds of these plants inhibit Cav3.2 current in a concentration-dependent manner. In addition, these products also induce a negative shift of the steady-state inactivation of CaV3.2 current with no change in the activation properties. Taken together, our findings reveal that TTCCs are a molecular target of the Lavender and Rosemary compounds, suggesting that inhibition of TTCCs could contribute to the anxiolytic and the neuroprotective effects of these plants.


Assuntos
Canais de Cálcio Tipo T/efeitos dos fármacos , Lavandula/química , Extratos Vegetais/farmacologia , Rosmarinus/química , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo T/fisiologia , Células HEK293 , Humanos , Metanol/química , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp
5.
J Biol Chem ; 292(49): 20010-20031, 2017 12 08.
Artigo em Inglês | MEDLINE | ID: mdl-28972185

RESUMO

Calcium (Cav1 and Cav2) and sodium channels possess homologous CaM-binding motifs, known as IQ motifs in their C termini, which associate with calmodulin (CaM), a universal calcium sensor. Cav3 T-type channels, which serve as pacemakers of the mammalian brain and heart, lack a C-terminal IQ motif. We illustrate that T-type channels associate with CaM using co-immunoprecipitation experiments and single particle cryo-electron microscopy. We demonstrate that protostome invertebrate (LCav3) and human Cav3.1, Cav3.2, and Cav3.3 T-type channels specifically associate with CaM at helix 2 of the gating brake in the I-II linker of the channels. Isothermal titration calorimetry results revealed that the gating brake and CaM bind each other with high-nanomolar affinity. We show that the gating brake assumes a helical conformation upon binding CaM, with associated conformational changes to both CaM lobes as indicated by amide chemical shifts of the amino acids of CaM in 1H-15N HSQC NMR spectra. Intact Ca2+-binding sites on CaM and an intact gating brake sequence (first 39 amino acids of the I-II linker) were required in Cav3.2 channels to prevent the runaway gating phenotype, a hyperpolarizing shift in voltage sensitivities and faster gating kinetics. We conclude that the presence of high-nanomolar affinity binding sites for CaM at its universal gating brake and its unique form of regulation via the tuning of the voltage range of activity could influence the participation of Cav3 T-type channels in heart and brain rhythms. Our findings may have implications for arrhythmia disorders arising from mutations in the gating brake or CaM.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Calmodulina/fisiologia , Caveolina 3/metabolismo , Ativação do Canal Iônico , Animais , Sítios de Ligação , Encéfalo/fisiologia , Cálcio/metabolismo , Calmodulina/metabolismo , Coração/fisiologia , Humanos , Invertebrados , Periodicidade
6.
Elife ; 62017 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-28109159

RESUMO

Voltage-gated Ca2+ channels are involved in numerous physiological functions and various mechanisms finely tune their activity, including the Ca2+ ion itself. This is well exemplified by the Ca2+-dependent inactivation of L-type Ca2+ channels, whose alteration contributes to the dramatic disease Timothy Syndrome. For T-type Ca2+ channels, a long-held view is that they are not regulated by intracellular Ca2+. Here we challenge this notion by using dedicated electrophysiological protocols on both native and expressed T-type Ca2+ channels. We demonstrate that a rise in submembrane Ca2+ induces a large decrease in T-type current amplitude due to a hyperpolarizing shift in the steady-state inactivation. Activation of most representative Ca2+-permeable ionotropic receptors similarly regulate T-type current properties. Altogether, our data clearly establish that Ca2+ entry exerts a feedback control on T-type channel activity, by modulating the channel availability, a mechanism that critically links cellular properties of T-type Ca2+ channels to their physiological roles.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Cálcio/metabolismo , Cátions Bivalentes/metabolismo , Retroalimentação Fisiológica , Animais , Células Cultivadas , Fenômenos Eletrofisiológicos , Regulação Enzimológica da Expressão Gênica , Humanos , Camundongos Endogâmicos C57BL
7.
EBioMedicine ; 13: 225-236, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27729216

RESUMO

Primary aldosteronism (PA) is the most common form of secondary hypertension. Mutations in KCNJ5, ATP1A1, ATP2B3 and CACNA1D are found in aldosterone producing adenoma (APA) and familial hyperaldosteronism (FH). A recurrent mutation in CACNA1H (coding for Cav3.2) was identified in a familial form of early onset PA. Here we performed whole exome sequencing (WES) in patients with different types of PA to identify new susceptibility genes. Four different heterozygous germline CACNA1H variants were identified. A de novo Cav3.2 p.Met1549Ile variant was found in early onset PA and multiplex developmental disorder. Cav3.2 p.Ser196Leu and p.Pro2083Leu were found in two patients with FH, and p.Val1951Glu was identified in one patient with APA. Electrophysiological analysis of mutant Cav3.2 channels revealed significant changes in the Ca2+ current properties for all mutants, suggesting a gain of function phenotype. Transfections of mutant Cav3.2 in H295R-S2 cells led to increased aldosterone production and/or expression of genes coding for steroidogenic enzymes after K+ stimulation. Identification of CACNA1H mutations associated with early onset PA, FH, and APA suggests that CACNA1H might be a susceptibility gene predisposing to PA with different phenotypic presentations, opening new perspectives for genetic diagnosis and management of patients with PA.


Assuntos
Canais de Cálcio Tipo T/genética , Estudos de Associação Genética , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Mutação , Potenciais de Ação , Adenoma/metabolismo , Adolescente , Adulto , Aldosterona/biossíntese , Alelos , Biomarcadores , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular , Criança , Pré-Escolar , Análise Mutacional de DNA , Exoma , Feminino , Expressão Gênica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hiperaldosteronismo/sangue , Lactente , Masculino , Pessoa de Meia-Idade , Linhagem , RNA Mensageiro/genética , Adulto Jovem
8.
Circulation ; 134(7): 534-46, 2016 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-27486162

RESUMO

BACKGROUND: L-type calcium channels (LTCCs) play important roles in regulating cardiomyocyte physiology, which is governed by appropriate LTCC trafficking to and density at the cell surface. Factors influencing the expression, half-life, subcellular trafficking, and gating of LTCCs are therefore critically involved in conditions of cardiac physiology and disease. METHODS: Yeast 2-hybrid screenings, biochemical and molecular evaluations, protein interaction assays, fluorescence microscopy, structural molecular modeling, and functional studies were used to investigate the molecular mechanisms through which the LTCC Cavß2 chaperone regulates channel density at the plasma membrane. RESULTS: On the basis of our previous results, we found a direct linear correlation between the total amount of the LTCC pore-forming Cavα1.2 and the Akt-dependent phosphorylation status of Cavß2 both in a mouse model of diabetic cardiac disease and in 6 diabetic and 7 nondiabetic cardiomyopathy patients with aortic stenosis undergoing aortic valve replacement. Mechanistically, we demonstrate that a conformational change in Cavß2 triggered by Akt phosphorylation increases LTCC density at the cardiac plasma membrane, and thus the inward calcium current, through a complex pathway involving reduction of Cavα1.2 retrograde trafficking and protein degradation through the prevention of dynamin-mediated LTCC endocytosis; promotion of Cavα1.2 anterograde trafficking by blocking Kir/Gem-dependent sequestration of Cavß2, thus facilitating the chaperoning of Cavα1.2; and promotion of Cavα1.2 transcription by the prevention of Kir/Gem-mediated shuttling of Cavß2 to the nucleus, where it limits the transcription of Cavα1.2 through recruitment of the heterochromatin protein 1γ epigenetic repressor to the Cacna1c promoter. On the basis of this mechanism, we developed a novel mimetic peptide that, through targeting of Cavß2, corrects LTCC life-cycle alterations, facilitating the proper function of cardiac cells. Delivery of mimetic peptide into a mouse model of diabetic cardiac disease associated with LTCC abnormalities restored impaired calcium balance and recovered cardiac function. CONCLUSIONS: We have uncovered novel mechanisms modulating LTCC trafficking and life cycle and provide proof of concept for the use of Cavß2 mimetic peptide as a novel therapeutic tool for the improvement of cardiac conditions correlated with alterations in LTCC levels and function.


Assuntos
Materiais Biomiméticos/administração & dosagem , Materiais Biomiméticos/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sistemas de Liberação de Medicamentos/métodos , Peptidomiméticos/administração & dosagem , Peptidomiméticos/metabolismo , Sequência de Aminoácidos , Animais , Materiais Biomiméticos/química , Canais de Cálcio Tipo L/genética , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Células Cultivadas , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Peptidomiméticos/química , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Estudos Retrospectivos
9.
Neuropharmacology ; 101: 320-9, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26456350

RESUMO

T-type (Cav3) calcium channels play important roles in neuronal excitability, both in normal and pathological activities of the brain. In particular, they contribute to hyper-excitability disorders such as epilepsy. Here we have characterized the anticonvulsant properties of TTA-A2, a selective T-type channel blocker, in mouse. Using the maximal electroshock seizure (MES) as a model of tonic-clonic generalized seizures, we report that mice treated with TTA-A2 (0.3 mg/kg and higher doses) were significantly protected against tonic seizures. Although no major change in Local Field Potential (LFP) pattern was observed during the MES seizure, analysis of the late post-ictal period revealed a significant increase in the delta frequency power in animals treated with TTA-A2. Similar results were obtained for Cav3.1-/- mice, which were less prone to develop tonic seizures in the MES test, but not for Cav3.2-/- mice. Analysis of extracellular signal-regulated kinase 1/2 (ERK) phosphorylation and c-Fos expression revealed a rapid and elevated neuronal activation in the hippocampus following MES clonic seizures, which was unchanged in TTA-A2 treated animals. Overall, our data indicate that TTA-A2 is a potent anticonvulsant and that the Cav3.1 isoform plays a prominent role in mediating TTA-A2 tonic seizure protection.


Assuntos
Benzenoacetamidas/uso terapêutico , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Tipo T/metabolismo , Piridinas/uso terapêutico , Convulsões/prevenção & controle , Análise de Variância , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Canais de Cálcio Tipo T/genética , Convulsivantes/toxicidade , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Eletrochoque/efeitos adversos , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pentilenotetrazol/toxicidade , Convulsões/etiologia , Convulsões/patologia
10.
Proc Natl Acad Sci U S A ; 112(44): 13705-10, 2015 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-26483470

RESUMO

Phosphorylation is a major mechanism regulating the activity of ion channels that remains poorly understood with respect to T-type calcium channels (Cav3). These channels are low voltage-activated calcium channels that play a key role in cellular excitability and various physiological functions. Their dysfunction has been linked to several neurological disorders, including absence epilepsy and neuropathic pain. Recent studies have revealed that T-type channels are modulated by a variety of serine/threonine protein kinase pathways, which indicates the need for a systematic analysis of T-type channel phosphorylation. Here, we immunopurified Cav3.2 channels from rat brain, and we used high-resolution MS to construct the first, to our knowledge, in vivo phosphorylation map of a voltage-gated calcium channel in a mammalian brain. We identified as many as 34 phosphorylation sites, and we show that the vast majority of these sites are also phosphorylated on the human Cav3.2 expressed in HEK293T cells. In patch-clamp studies, treatment of the channel with alkaline phosphatase as well as analysis of dephosphomimetic mutants revealed that phosphorylation regulates important functional properties of Cav3.2 channels, including voltage-dependent activation and inactivation and kinetics. We also identified that the phosphorylation of a locus situated in the loop I-II S442/S445/T446 is crucial for this regulation. Our data show that Cav3.2 channels are highly phosphorylated in the mammalian brain and establish phosphorylation as an important mechanism involved in the dynamic regulation of Cav3.2 channel gating properties.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Ativação do Canal Iônico , Canais de Cálcio Tipo T/fisiologia , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Fosforilação
11.
PLoS One ; 9(6): e98808, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24945283

RESUMO

The properties of leaky patch currents in whole cell recording of HEK-293T cells were examined as a means to separate these control currents from expressed sodium and calcium leak channel currents from snail NALCN leak channels possessing both sodium (EKEE) and calcium (EEEE) selectivity filters. Leak currents were generated by the weakening of gigaohm patch seals by artificial membrane rupture using the ZAP function on the patch clamp amplifier. Surprisingly, we found that leak currents generated from the weakened membrane/glass seal can be surprisingly stable and exhibit behavior that is consistent with a sodium leak current derived from an expressible channel. Leaky patch currents differing by 10 fold in size were similarly reduced in size when external sodium ions were replaced with the large monovalent ion NMDG+. Leaky patch currents increased when external Ca2+ (1.2 mM) was lowered to 0.1 mM and were inhibited (>40% to >90%) with 10 µM Gd3+, 100 µM La3+, 1 mM Co2+ or 1 mM Cd2+. Leaky patch currents were relatively insensitive (<30%) to 1 mM Ni2+ and exhibited a variable amount of block with 1 mM verapamil and were insensitive to 100 µM mibefradil or 100 µM nifedipine. We hypothesize that the rapid changes in leak current size in response to changing external cations or drugs relates to their influences on the membrane seal adherence and the electro-osmotic flow of mobile cations channeling in crevices of a particular pore size in the interface between the negatively charged patch electrode and the lipid membrane. Observed sodium leak conductance currents in weak patch seals are reproducible between the electrode glass interface with cell membranes, artificial lipid or Sylgard rubber.


Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Canais de Sódio/biossíntese , Sódio/metabolismo , Canais de Cálcio/biossíntese , Canais de Cálcio/metabolismo , Membrana Celular/química , Células HEK293 , Humanos , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Canais de Sódio/metabolismo , Verapamil/administração & dosagem
12.
Pflugers Arch ; 466(4): 689-700, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24531745

RESUMO

T-type calcium channels (T-channels/CaV3) have unique biophysical properties allowing a calcium influx at resting membrane potential of most cells. T-channels are ubiquitously expressed in many tissues and contribute to low-threshold spikes and burst firing in central neurons as well as to pacemaker activities in cardiac cells. They also emerged as potential targets to treat cancer and hypertension. Regulation of these channels appears complex, and several studies have indicated that CaV3.1, CaV3.2, and CaV3.3 currents are directly inhibited by multiple endogenous lipids independently of membrane receptors or intracellular pathways. These bioactive lipids include arachidonic acid and ω3 poly-unsaturated fatty acids; the endocannabinoid anandamide and other N-acylethanolamides; the lipoamino-acids and lipo-neurotransmitters; the P450 epoxygenase metabolite 5,6-epoxyeicosatrienoic acid; as well as similar molecules with 18-22 carbons in the alkyl chain. In this review, we summarize evidence for direct effects of these signaling molecules, the molecular mechanisms underlying the current inhibition, and the involved chemical features. The impact of this modulation in physiology and pathophysiology is discussed with a special emphasis on pain aspects and vasodilation. Overall, these data clearly indicate that T-current inhibition is an important mechanism by which bioactive lipids mediate their physiological functions.


Assuntos
Ácido Araquidônico/fisiologia , Canais de Cálcio Tipo T/fisiologia , Endocanabinoides/fisiologia , Ácidos Graxos/fisiologia , Animais , Ácido Araquidônico/química , Endocanabinoides/química , Ácidos Graxos/química , Humanos , Lipídeos/fisiologia , Neurotransmissores/química , Neurotransmissores/fisiologia
13.
Pain ; 155(4): 764-72, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24447516

RESUMO

To exert its analgesic action, paracetamol requires complex metabolism to produce a brain-specific lipoamino acid compound, AM404, which targets central transient receptor potential vanilloid receptors (TRPV1). Lipoamino acids are also known to induce analgesia through T-type calcium-channel inhibition (Ca(v)3.2). In this study we show that the antinociceptive effect of paracetamol in mice is lost when supraspinal Ca(v)3.2 channels are inhibited. Therefore, we hypothesized a relationship between supraspinal Ca(v)3.2 and TRPV1, via AM404, which mediates the analgesic effect of paracetamol. AM404 is able to activate TRPV1 and weakly inhibits Ca(v)3.2. Interestingly, activation of TRPV1 induces a strong inhibition of Ca(v)3.2 current. Supporting this, intracerebroventricular administration of AM404 or capsaicin produces antinociception that is lost in Ca(v)3.2(-/-) mice. Our study, for the first time, (1) provides a molecular mechanism for the supraspinal antinociceptive effect of paracetamol; (2) identifies the relationship between TRPV1 and the Ca(v)3.2 channel; and (3) suggests supraspinal Ca(v)3.2 inhibition as a potential pharmacological strategy to alleviate pain.


Assuntos
Acetaminofen/farmacologia , Analgésicos não Entorpecentes/farmacologia , Canais de Cálcio Tipo T/deficiência , Medula Espinal/efeitos dos fármacos , 4-Aminopiridina/farmacologia , Analgésicos Opioides/farmacologia , Analgésicos Opioides/uso terapêutico , Animais , Artrite/induzido quimicamente , Artrite/tratamento farmacológico , Canais de Cálcio Tipo T/genética , Células Cultivadas , Vias de Administração de Medicamentos , Interações de Medicamentos , Gânglios Espinais/citologia , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Knockout , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Medição da Dor/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Medula Espinal/citologia , Canais de Cátion TRPV/deficiência , Canais de Cátion TRPV/genética , Tetraetilamônio/farmacologia
14.
Mol Pharmacol ; 85(2): 218-25, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24214826

RESUMO

T-type calcium channels (T/Ca(v)3-channels) are implicated in various physiologic and pathophysiologic processes such as epilepsy, sleep disorders, hypertension, and cancer. T-channels are the target of endogenous signaling lipids including the endocannabinoid anandamide, the ω3-fatty acids, and the lipoamino-acids. However, the precise molecular mechanism by which these molecules inhibit T-current is unknown. In this study, we provided a detailed electrophysiologic and pharmacologic analysis indicating that the effects of the major N-acyl derivatives on the Ca(v)3.3 current share many similarities with those of TTA-A2 [(R)-2-(4-cyclopropylphenyl)-N-(1-(5-(2,2,2-trifluoroethoxy)pyridin-2-yl)ethyl)acetamide], a synthetic T-channel inhibitor. Using radioactive binding assays with the TTA-A2 derivative [(3)H]TTA-A1 [(R)-2-(4-(tert-butyl)phenyl)-N-(1-(5-methoxypyridin-2-yl)ethyl)acetamide], we demonstrated that polyunsaturated lipids, which inhibit the Ca(v)3.3 current, as NAGly (N-arachidonoyl glycine), NASer (N-arachidonoyl-l-serine), anandamide, NADA (N-arachidonoyl dopamine), NATau (N-arachidonoyl taurine), and NA-5HT (N-arachidonoyl serotonin), all displaced [(3)H]TTA-A1 binding to membranes prepared from cells expressing Ca(v)3.3, with Ki in a micromolar or submicromolar range. In contrast, lipids with a saturated alkyl chain, as N-arachidoyl glycine and N-arachidoyl ethanolamine, which did not inhibit the Ca(v)3.3 current, had no effect on [(3)H]TTA-A1 binding. Accordingly, bio-active lipids occluded TTA-A2 effect on Ca(v)3.3 current. In addition, TTA-Q4 [(S)-4-(6-chloro-4-cyclopropyl-3-(2,2-difluoroethyl)-2-oxo-1,2,3,4-tetrahydroquinazolin-4-yl)benzonitrile], a positive allosteric modulator of [(3)H]TTA-A1 binding and TTA-A2 functional inhibition, acted in a synergistic manner to increase lipid-induced inhibition of the Ca(v)3.3 current. Overall, our results demonstrate a common molecular mechanism for the synthetic T-channel inhibitors and the endogenous lipids, and indicate that TTA-A2 and TTA-Q4 could be important pharmacologic tools to dissect the involvement of T-current in the physiologic effects of endogenous lipids.


Assuntos
Benzenoacetamidas/farmacologia , Canais de Cálcio Tipo T/fisiologia , Lipídeos/fisiologia , Piridinas/farmacologia , Regulação Alostérica , Ácidos Araquidônicos/farmacologia , Benzenoacetamidas/metabolismo , Canais de Cálcio Tipo T/efeitos dos fármacos , Células Cultivadas , Dopamina/análogos & derivados , Dopamina/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Humanos , Piridinas/metabolismo
15.
Pflugers Arch ; 466(9): 1759-68, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24327205

RESUMO

T-type calcium channels (T-channels) are important actors in neuronal pacemaking, in heart rhythm, and in the control of the vascular tone. T-channels are regulated by several endogenous lipids including the primary eicosanoid arachidonic acid (AA), which display an important role in vasodilation via its metabolism leading to prostanoids, leukotrienes, and epoxyeicosatrienoic acids (EETs). However, the effects of these latter molecules on T-currents have not been investigated. Here, we describe the effects of the major cyclooxygenase, lipoxygenase, and cytochrome P450 epoxygenase products on the three human recombinant T-channels (Cav3.1, Cav3.2, and Cav3.3), as compared to those of AA. We identified the P450 epoxygenase product, 5,6-EET, as a potent physiological inhibitor of Cav3 currents. The effects of 5,6-EET were observed at sub-micromolar concentrations (IC50 = 0.54 µM), occurred in the minute range, and were reversible. The 5,6-EET inhibited the Cav3 currents at physiological resting membrane potentials mostly by inducing a large negative shift in their steady-state inactivation properties. Using knockout mice for Cav3.1 and Cav3.2, we demonstrated that the vasodilation of preconstricted mesenteric arteries induced by 5,6-EET was specifically impaired in Cav3.2 knockout mice. Overall, our results indicate that inhibition of Cav3 currents by 5,6-EET is an important mechanism controlling the vascular tone.


Assuntos
Ácido 8,11,14-Eicosatrienoico/análogos & derivados , Canais de Cálcio Tipo T/metabolismo , Músculo Liso Vascular/metabolismo , Ácido 8,11,14-Eicosatrienoico/metabolismo , Ácido 8,11,14-Eicosatrienoico/farmacologia , Animais , Canais de Cálcio Tipo T/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tono Muscular/efeitos dos fármacos , Tono Muscular/fisiologia , Músculo Liso Vascular/efeitos dos fármacos , Técnicas de Patch-Clamp , Transfecção
16.
J Neurosci ; 32(12): 4284-96, 2012 Mar 21.
Artigo em Inglês | MEDLINE | ID: mdl-22442090

RESUMO

Ligand-gated ion channels are prototypic oligomeric membrane proteins whose stoichiometry determines their functional properties and subcellular localization. Deciphering the quaternary structure of such protein complexes is an arduous task and usually requires the combination of multiple approaches. ATP-gated P2X receptors are formed by the association of three subunits, but the quaternary arrangement of the seven P2X subunits at the plasma membrane remains poorly characterized. By combining bioluminescence resonance energy transfer, bifunctional fluorescence complementation and protein biochemistry, we developed an experimental approach that allows precise determination of rat P2X receptor quaternary assembly. We found that P2X5 subunits associate with P2X1, P2X2, and P2X4 subunits. We demonstrate that P2X5 and P2X2 subunits interact to form as yet uncharacterized heteromeric receptors with alternate stoichiometries, both present at the plasma membrane. P2X2/5 receptors display functional properties such as pore dilatation, membrane blebbing, and phosphatidylserine exposure that were previously thought to be characteristic hallmarks of the P2X7 receptor. In mouse, P2X2 and P2X5 subunits colocalize and physically interact in specific neuronal populations suggesting that other P2X receptors might contribute to cellular responses typically attributed to P2X7 receptor.


Assuntos
Subunidades Proteicas/metabolismo , Receptores Purinérgicos P2X2/metabolismo , Receptores Purinérgicos P2X5/metabolismo , Receptores Purinérgicos P2X7/fisiologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Anexina A5/metabolismo , Benzoxazóis/metabolismo , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Encéfalo/metabolismo , Ensaio de Imunoadsorção Enzimática , Gânglios Espinais/citologia , Células HEK293 , Humanos , Imunoprecipitação , Proteínas Luminescentes/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Mutagênese Sítio-Dirigida/métodos , Mutação/genética , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Purinérgicos/farmacologia , Compostos de Quinolínio/metabolismo , Receptores Purinérgicos P2X2/genética , Receptores Purinérgicos P2X5/genética , Transfecção , Gravação em Vídeo , Xenopus laevis
17.
J Neurosci ; 29(42): 13106-14, 2009 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-19846698

RESUMO

Lipoamino acids are anandamide-related endogenous molecules that induce analgesia via unresolved mechanisms. Here, we provide evidence that the T-type/Cav3 calcium channels are important pharmacological targets underlying their physiological effects. Various lipoamino acids, including N-arachidonoyl glycine (NAGly), reversibly inhibited Cav3.1, Cav3.2, and Cav3.3 currents, with potent effects on Cav3.2 [EC(50) approximately 200 nm for N-arachidonoyl 3-OH-gamma-aminobutyric acid (NAGABA-OH)]. This inhibition involved a large shift in the Cav3.2 steady-state inactivation and persisted during fatty acid amide hydrolase (FAAH) inhibition as well as in cell-free outside-out patch. In contrast, lipoamino acids had weak effects on high-voltage-activated (HVA) Cav1.2 and Cav2.2 calcium currents, on Nav1.7 and Nav1.8 sodium currents, and on anandamide-sensitive TRPV1 and TASK1 currents. Accordingly, lipoamino acids strongly inhibited native Cav3.2 currents in sensory neurons with small effects on sodium and HVA calcium currents. In addition, we demonstrate here that lipoamino acids NAGly and NAGABA-OH produced a strong thermal analgesia and that these effects (but not those of morphine) were abolished in Cav3.2 knock-out mice. Collectively, our data revealed lipoamino acids as a family of endogenous T-type channel inhibitors, suggesting that these ligands can modulate multiple cell functions via this newly evidenced regulation.


Assuntos
Analgésicos/farmacologia , Ácidos Araquidônicos/farmacologia , Canais de Cálcio Tipo T/metabolismo , Glicina/análogos & derivados , Ácido gama-Aminobutírico/análogos & derivados , Animais , Comportamento Animal/fisiologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo T/classificação , Canais de Cálcio Tipo T/genética , Células Cultivadas , Modelos Animais de Doenças , Estimulação Elétrica/métodos , Gânglios Espinais/citologia , Glicina/farmacologia , Proteínas de Fluorescência Verde/genética , Humanos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/genética , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7 , Proteínas do Tecido Nervoso/genética , Neuroblastoma , Técnicas de Patch-Clamp/métodos , Canais de Potássio de Domínios Poros em Tandem/genética , Células Receptoras Sensoriais , Canais de Sódio/genética , Canais de Cátion TRPV/genética , Transfecção , Ácido gama-Aminobutírico/farmacologia
18.
EMBO Rep ; 10(8): 873-80, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19575010

RESUMO

A previously uncharacterized putative ion channel, NALCN (sodium leak channel, non-selective), has been recently shown to be responsible for the tetrodotoxin (TTX)-resistant sodium leak current implicated in the regulation of neuronal excitability. Here, we show that NALCN encodes a current that is activated by M3 muscarinic receptors (M3R) in a pancreatic beta-cell line. This current is primarily permeant to sodium ions, independent of intracellular calcium stores and G proteins but dependent on Src activation, and resistant to TTX. The current is recapitulated by co-expression of NALCN and M3R in human embryonic kidney-293 cells and in Xenopus oocytes. We also show that NALCN and M3R belong to the same protein complex, involving the intracellular I-II loop of NALCN and the intracellular i3 loop of M3R. Taken together, our data show the molecular basis of a muscarinic-activated inward sodium current that is independent of G-protein activation, and provide new insights into the properties of NALCN channels.


Assuntos
Células Secretoras de Insulina/metabolismo , Canais Iônicos/metabolismo , Receptor Muscarínico M3/metabolismo , Western Blotting , Linhagem Celular , Eletrofisiologia , Inibidores Enzimáticos/farmacologia , Humanos , Células Secretoras de Insulina/efeitos dos fármacos , Canais Iônicos/genética , Técnicas de Patch-Clamp , Pirazóis/farmacologia , Pirimidinas/farmacologia , Interferência de RNA , RNA Interferente Pequeno , Receptor Muscarínico M3/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
19.
J Cell Biol ; 184(6): 923-33, 2009 Mar 23.
Artigo em Inglês | MEDLINE | ID: mdl-19307602

RESUMO

The insulin IGF-1-PI3K-Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca(2+) handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca(2+) channel (LTCC) protein density. The pore-forming channel subunit Ca(v)alpha1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca(v)alpha1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca(v)beta2, the LTCC chaperone for Ca(v)alpha1, antagonizes Ca(v)alpha1 protein degradation by preventing Ca(v)alpha1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca(2+) channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca(2+) entry, Ca(2+) handling, and contractility.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio , Cardiomiopatia Dilatada/enzimologia , Miócitos Cardíacos/enzimologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases Dependentes de 3-Fosfoinositídeo , Motivos de Aminoácidos , Animais , Canais de Cálcio Tipo L/genética , Cardiomiopatia Dilatada/etiologia , Membrana Celular/enzimologia , Células Cultivadas , Sequência Conservada , Modelos Animais de Doenças , Masculino , Potenciais da Membrana , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutação , Contração Miocárdica , Fosforilação , Estabilidade Proteica , Subunidades Proteicas , Transporte Proteico , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Recombinantes de Fusão/metabolismo , Tamoxifeno , Fatores de Tempo , Transfecção
20.
Biochim Biophys Acta ; 1793(6): 947-52, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19071165

RESUMO

T-type calcium channels (T-channels) contribute to a wide variety of physiological functions, especially in the cardiovascular and nervous systems. Recent studies using knock-out mouse models have been instrumental in documenting further the role of T-channels in sleep, heartbeat, pain and epilepsy. Importantly, several novel aspects of the regulation of these channels have been identified over the last few years, providing new insights into their physiological and pathophysiological roles. Here, we review recent evidence supporting that the Cav3 subunits of T-channels are modulated by endogenous ligands such as anandamide, zinc, redox and oxidizing agents, as well as G-protein and protein kinases pathways. The study of T-channel mutations associated with childhood absence epilepsy has also revealed new aspects of Cav3 subunit trafficking. Collectively, these findings identify novel regulatory mechanisms involved in the fine tuning of T-channel expression and activity, and offer new directions for the design of novel therapeutic strategies targeting these channels.


Assuntos
Canais de Cálcio Tipo T/metabolismo , Isoformas de Proteínas/metabolismo , Transdução de Sinais/fisiologia , Animais , Transtorno Autístico/genética , Transtorno Autístico/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/genética , Canalopatias/genética , Canalopatias/metabolismo , Epilepsia/genética , Epilepsia/metabolismo , Isoformas de Proteínas/genética , Estrutura Secundária de Proteína
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