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The recent development of single-cell and single-nucleus RNA sequencing has highlighted the extraordinary diversity of dorsal root ganglia neurons. However, the few available genetic tools limit our understanding of the functional significance of this heterogeneity. We generated a new mouse line expressing the flippase recombinase from the scn10a locus. By crossing Nav1.8Ires-FLPo mice with the AdvillinCre and RC::FL-hM3Dq mouse lines in an intersectional genetics approach, we were able to obtain somatodendritic expression of hM3Dq-mCherry selectively in the Nav1.8 lineage. The bath application of clozapine N-oxide triggered strong calcium responses selectively in mCherry+ neurons. The intraplantar injection of CNO caused robust flinching, shaking, and biting responses accompanied by strong cFos activation in the ipsilateral lumbar spinal cord. The Nav1.8Ires-FLPo mouse model will be a valuable tool for extending our understanding of the in vivo functional specialization of neuronal subsets of the Nav1.8 lineage for which inducible Cre lines are available.
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BACKGROUND AND PURPOSE: Voltage sensitivity is a common feature of many membrane proteins, including some G-protein coupled receptors (GPCRs). However, the functional consequences of voltage sensitivity in GPCRs are not well understood. EXPERIMENTAL APPROACH: In this study, we investigated the voltage sensitivity of the post-synaptic metabotropic glutamate receptor mGlu5 and its impact on synaptic transmission. Using biosensors and electrophysiological recordings in non-excitable HEK293T cells or neurons. KEY RESULTS: We found that mGlu5 receptor function is optimal at resting membrane potentials. We observed that membrane depolarization significantly reduced mGlu5 receptor activation, Gq-PLC/PKC stimulation, Ca2+ release and mGlu5 receptor-gated currents through transient receptor potential canonical, TRPC6, channels or glutamate ionotropic NMDA receptors. Notably, we report a previously unknown activity of the NMDA receptor at the resting potential of neurons, enabled by mGlu5. CONCLUSIONS AND IMPLICATIONS: Our findings suggest that mGlu5 receptor activity is directly regulated by membrane voltage which may have a significant impact on synaptic processes and pathophysiological functions.
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Receptor del Glutamato Metabotropico 5 , Transmisión Sináptica , Animales , Humanos , Células HEK293 , Potenciales de la Membrana , Neuronas/metabolismo , Neuronas/fisiología , Receptor del Glutamato Metabotropico 5/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transmisión Sináptica/fisiología , RatonesRESUMEN
L-type voltage-gated calcium channels are involved in multiple physiological functions. Currently available antagonists do not discriminate between L-type channel isoforms. Importantly, no selective blocker is available to dissect the role of L-type isoforms Cav1.2 and Cav1.3 that are concomitantly co-expressed in the heart, neuroendocrine and neuronal cells. Here we show that calciseptine, a snake toxin purified from mamba venom, selectively blocks Cav1.2 -mediated L-type calcium currents (ICaL) at concentrations leaving Cav1.3-mediated ICaL unaffected in both native cardiac myocytes and HEK-293T cells expressing recombinant Cav1.2 and Cav1.3 channels. Functionally, calciseptine potently inhibits cardiac contraction without altering the pacemaker activity in sino-atrial node cells, underscoring differential roles of Cav1.2- and Cav1.3 in cardiac contractility and automaticity. In summary, calciseptine is a selective L-type Cav1.2 Ca2+ channel blocker and should be a valuable tool to dissect the role of these L-channel isoforms.
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Canales de Calcio Tipo L , Dendroaspis , Animales , Canales de Calcio Tipo L/fisiología , Dendroaspis/metabolismo , Miocitos Cardíacos/metabolismo , Isoformas de Proteínas , Calcio/metabolismoRESUMEN
Herein, we demonstrate the use of an atmospheric pressure plasma with a Dielectric Barrier Discharge (DBD) for the synthesis of FeOx nanoparticles with a simultaneous formation of graphene oxide domains at low substrate temperature. For that, the interaction of the plasma to control good decomposition of the Fe precursor is essential and this is demonstrated by FTIR analyses. Thanks to a fine tuning of the plasma conditions, a homogeneous spatial distribution around 5 nm nanoparticles (NPs) was obtained, whereas without plasma, in the same configuration of the process, a heterogeneity regarding size and shape for the NPs was obtained. The Raman spectrum of the plasma deposit confirmed the presence of graphene oxide as the characteristic G and D bands were observed with I(D)/I(G) = 0.92. Thanks to optical emission spectroscopy (OES) measurements, it is proposed that the carbon deposition on FeOx nanoparticles is produced on the near plasma post discharge. XPS studies showed that the main contribution of iron was in Fe2+ form, corresponding to the FeO phase. No metallic Fe or carbide were detected. As there are many studies reporting the synergetic effect of FeOx NPs and graphene oxide, we believe that this new one-step simultaneous synthesis method may be of high interest for applications requiring direct deposition on temperature labile substrates such as polymers.
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Herein, we report the impact of plasma on gold nanoparticles synthesis. We used an atmospheric plasma torch fed with an aerosolized tetrachloroauric(iii) acid trihydrate (HAuCl4·3H2O) solution. The investigation showed that using pure ethanol as a solvent for the gold precursor enabled a better dispersion compared to a water-containing solution. We demonstrated here that the deposition parameters are easy to control, presenting the influence of solvent concentration and deposition time. The advantage of our method is that no capping agent was used. We assume that plasma creates a carbon-based matrix around the gold nanoparticles preventing them to agglomerate. The XPS results revealed the impact of using plasma. Metallic gold was detected in the plasma-treated sample, whereas the no-plasma sample revealed only Au(i) and Au(iii) contributions originating from the HAuCl4 precursor. Detailed HRTEM, EDS mapping, and SAED analyses led to more insights into the structure.
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Venom-derived peptides targeting ion channels involved in pain are regarded as a promising alternative to current, and often ineffective, chronic pain treatments. Many peptide toxins are known to specifically and potently block established therapeutic targets, among which the voltage-gated sodium and calcium channels are major contributors. Here, we report on the discovery and characterization of a novel spider toxin isolated from the crude venom of Pterinochilus murinus that shows inhibitory activity at both hNaV 1.7 and hCaV 3.2 channels, two therapeutic targets implicated in pain pathways. Bioassay-guided HPLC fractionation revealed a 36-amino acid peptide with three disulfide bridges named µ/ω-theraphotoxin-Pmu1a (Pmu1a). Following isolation and characterization, the toxin was chemically synthesized and its biological activity was further assessed using electrophysiology, revealing Pmu1a to be a toxin that potently blocks both hNaV 1.7 and hCaV 3. Nuclear magnetic resonance structure determination of Pmu1a shows an inhibitor cystine knot fold that is the characteristic of many spider peptides. Combined, these data show the potential of Pmu1a as a basis for the design of compounds with dual activity at the therapeutically relevant hCaV 3.2 and hNaV 1.7 voltage-gated channels.
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Venenos de Araña , Arañas , Animales , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Venenos de Araña/farmacología , Venenos de Araña/química , Venenos de Araña/metabolismo , Dolor , Péptidos/farmacología , Espectroscopía de Resonancia Magnética , Arañas/metabolismoRESUMEN
Gallium-doped zinc oxide (GZO) films were fabricated using RF magnetron sputtering and atomic layer deposition (ALD). The latter ones demonstrate higher electrical conductivities (up to 2700 S cm-1) and enhanced charge mobilities (18 cm2 V-1 s-1). The morphological analysis reveals differences mostly due to the very different nature of the deposition processes. The film deposited via ALD shows an increased transmittance in the visible range and a very small one in the infrared range that leads to a figure of merit of 0.009 Ω-1 (10 times higher than for the films deposited via sputtering). A benchmarking is made with an RF sputtered indium-doped tin oxide (ITO) film used conventionally in the industry. Another comparison between ZnO, Al:ZnO (AZO), and Ga:ZnO (GZO) films fabricated by ALD is presented, and the evolution of physical properties with doping is evidenced. Finally, we processed GZO thin films on a glass substrate into patterned transparent patch antennas to demonstrate an application case of short-range communication by means of the Bluetooth Low Energy (BLE) protocol. The GZO transparent antennas' performances are compared to a reference ITO antenna on a glass substrate and a conventional copper antenna on FR4 PCB. The results highlight the possibility to use the transparent GZO antenna for reliable short-range communication and the achievability of an antenna entirely processed by ALD.
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Affective touch is necessary for proper neurodevelopment and sociability. However, it remains unclear how the neurons innervating the skin detect affective and social behaviors. The C low-threshold mechanoreceptors (C-LTMRs), a specific population of somatosensory neurons in mice, appear particularly well suited, physiologically and anatomically, to perceive affective and social touch. However, their contribution to sociability has not been resolved yet. Our observations revealed that C-LTMR functional deficiency induced social isolation and reduced tactile interactions in adulthood. Conversely, transient increase in C-LTMR excitability in adults, using chemogenetics, was rewarding, promoted touch-seeking behaviors, and had prosocial influences on group dynamics. This work provides the first empirical evidence that specific peripheral inputs alone can drive complex social behaviors. It demonstrates the existence of a specialized neuronal circuit, originating in the skin, wired to promote interactions with other individuals.
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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.
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Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio Tipo T/genética , Femenino , Masculino , Fosfatos/metabolismo , Fosforilación , Ratas , Ratas Sprague-Dawley , Núcleos Talámicos/metabolismoRESUMEN
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.
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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 (â¼5 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.
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Canales de Calcio Tipo T/genética , Ataxia Cerebelosa/genética , Adolescente , Adulto , Atrofia/patología , Encéfalo/patología , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio Tipo T/metabolismo , Ataxia Cerebelosa/fisiopatología , Enfermedades Cerebelosas/complicaciones , Cerebelo/patología , Niño , Preescolar , Estudios de Cohortes , Discapacidades del Desarrollo/genética , Femenino , Mutación con Ganancia de Función/genética , Humanos , Discapacidad Intelectual/genética , Masculino , Microcefalia/genética , Mutación , Linaje , Fenotipo , Células de Purkinje/patologíaRESUMEN
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.
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Canales de Calcio Tipo T/metabolismo , Calmodulina/fisiología , Caveolina 3/metabolismo , Activación del Canal Iónico , Animales , Sitios de Unión , Encéfalo/fisiología , Calcio/metabolismo , Calmodulina/metabolismo , Corazón/fisiología , Humanos , Invertebrados , PeriodicidadRESUMEN
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.
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Canales de Calcio Tipo T/efectos de los fármacos , Lavandula/química , Extractos Vegetales/farmacología , Rosmarinus/química , Animales , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo T/fisiología , Células HEK293 , Humanos , Metanol/química , Neuronas/efectos de los fármacos , Técnicas de Placa-ClampRESUMEN
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.
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Canales de Calcio Tipo T/metabolismo , Calcio/metabolismo , Cationes Bivalentes/metabolismo , Retroalimentación Fisiológica , Animales , Células Cultivadas , Fenómenos Electrofisiológicos , Regulación Enzimológica de la Expresión Génica , Humanos , Ratones Endogámicos C57BLRESUMEN
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.
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Canales de Calcio Tipo T/genética , Estudios de Asociación Genética , Hiperaldosteronismo/diagnóstico , Hiperaldosteronismo/genética , Mutación , Potenciales de Acción , Adenoma/metabolismo , Adolescente , Adulto , Aldosterona/biosíntesis , Alelos , Biomarcadores , Canales de Calcio Tipo T/metabolismo , Línea Celular , Niño , Preescolar , Análisis Mutacional de ADN , Exoma , Femenino , Expresión Génica , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Hiperaldosteronismo/sangre , Lactante , Masculino , Persona de Mediana Edad , Linaje , ARN Mensajero/genética , Adulto JovenRESUMEN
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.
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Materiales Biomiméticos/administración & dosificación , Materiales Biomiméticos/metabolismo , Canales de Calcio Tipo L/metabolismo , Sistemas de Liberación de Medicamentos/métodos , Peptidomiméticos/administración & dosificación , Peptidomiméticos/metabolismo , Secuencia de Aminoácidos , Animales , Materiales Biomiméticos/química , Canales de Calcio Tipo L/genética , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Células Cultivadas , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Peptidomiméticos/química , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Estudios RetrospectivosRESUMEN
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.
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Bencenoacetamidas/uso terapéutico , Bloqueadores de los Canales de Calcio/uso terapéutico , Canales de Calcio Tipo T/metabolismo , Piridinas/uso terapéutico , Convulsiones/prevención & control , Análisis de Varianza , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Canales de Calcio Tipo T/genética , Convulsivantes/toxicidad , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Electrochoque/efectos adversos , Potenciales Evocados/efectos de los fármacos , Potenciales Evocados/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Pentilenotetrazol/toxicidad , Convulsiones/etiología , Convulsiones/patologíaRESUMEN
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.
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Canales de Calcio Tipo T/metabolismo , Activación del Canal Iónico , Canales de Calcio Tipo T/fisiología , Células HEK293 , Humanos , Técnicas de Placa-Clamp , FosforilaciónRESUMEN
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.
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Calcio/metabolismo , Membrana Celular/metabolismo , Canales de Sodio/biosíntesis , Sodio/metabolismo , Canales de Calcio/biosíntesis , Canales de Calcio/metabolismo , Membrana Celular/química , Células HEK293 , Humanos , Canales Iónicos , Proteínas de la Membrana , Nifedipino/farmacología , Técnicas de Placa-Clamp , Canales de Sodio/metabolismo , Verapamilo/administración & dosificaciónRESUMEN
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.