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1.
Stem Cell Res ; 60: 102688, 2022 04.
Artículo en Inglés | MEDLINE | ID: mdl-35101670

RESUMEN

Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT) is an exercise and emotional stress-induced life-threatening inherited heart rhythm disorder, characterized by an abnormal cellular calcium homeostasis. Most reported cases have been linked to mutations in the gene encoding the type 2 ryanodine receptor gene, RYR2. We generated induced pluripotent stem cells (hiPSCs) from peripheral blood mononuclear cells (PBMC) from three CPVT-affected patients, two of them carrying p.R4959Q mutation and one carrying p.Y2476D mutation. These generated hiPSC lines are a useful model to study pathophysiological consequences of RYR2 dysfunction in humans and the molecular basis of CPVT.


Asunto(s)
Células Madre Pluripotentes Inducidas , Calcio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Leucocitos Mononucleares/metabolismo , Mutación/genética , Canal Liberador de Calcio Receptor de Rianodina/genética , Taquicardia Ventricular
2.
Toxins (Basel) ; 14(2)2022 01 21.
Artículo en Inglés | MEDLINE | ID: mdl-35202107

RESUMEN

α-bungarotoxin is a large, 74 amino acid toxin containing five disulphide bridges, initially identified in the venom of Bungarus multicinctus snake. Like most large toxins, chemical synthesis of α-bungarotoxin is challenging, explaining why all previous reports use purified or recombinant α-bungarotoxin. However, only chemical synthesis allows easy insertion of non-natural amino acids or new chemical functionalities. Herein, we describe a procedure for the chemical synthesis of a fluorescent-tagged α-bungarotoxin. The full-length peptide was designed to include an alkyne function at the amino-terminus through the addition of a pentynoic acid linker. Chemical synthesis of α-bungarotoxin requires hydrazide-based coupling of three peptide fragments in successive steps. After completion of the oxidative folding, an azide-modified Cy5 fluorophore was coupled by click chemistry onto the toxin. Next, we determined the efficacy of the fluorescent-tagged α-bungarotoxin to block acetylcholine (ACh)-mediated currents in response to muscle nicotinic receptor activation in TE671 cells. Using automated patch-clamp recordings, we demonstrate that fluorescent synthetic α-bungarotoxin has the expected nanomolar affinity for the nicotinic receptor. The blocking effect of fluorescent α-bungarotoxin could be displaced by incubation with a 20-mer peptide mimicking the α-bungarotoxin binding site. In addition, TE671 cells could be labelled with fluorescent toxin, as witnessed by confocal microscopy, and this labelling was partially displaced by the 20-mer competitive peptide. We thus demonstrate that synthetic fluorescent-tagged α-bungarotoxin preserves excellent properties for binding onto muscle nicotinic receptors.


Asunto(s)
Bungarotoxinas/síntesis química , Bungarotoxinas/farmacología , Inhibidores de la Colinesterasa/síntesis química , Inhibidores de la Colinesterasa/farmacología , Colorantes Fluorescentes/química , Acetilcolina , Línea Celular , Química Clic , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Conformación Proteica
3.
Int J Mol Sci ; 21(19)2020 Sep 28.
Artículo en Inglés | MEDLINE | ID: mdl-32998413

RESUMEN

IKr current, a major component of cardiac repolarization, is mediated by human Ether-à-go-go-Related Gene (hERG, Kv11.1) potassium channels. The blockage of these channels by pharmacological compounds is associated to drug-induced long QT syndrome (LQTS), which is a life-threatening disorder characterized by ventricular arrhythmias and defects in cardiac repolarization that can be illustrated using cardiomyocytes derived from human-induced pluripotent stem cells (hiPS-CMs). This study was meant to assess the modification in hiPS-CMs excitability and contractile properties by BeKm-1, a natural scorpion venom peptide that selectively interacts with the extracellular face of hERG, by opposition to reference compounds that act onto the intracellular face. Using an automated patch-clamp system, we compared the affinity of BeKm-1 for hERG channels with some reference compounds. We fully assessed its effects on the electrophysiological, calcium handling, and beating properties of hiPS-CMs. By delaying cardiomyocyte repolarization, the peptide induces early afterdepolarizations and reduces spontaneous action potentials, calcium transients, and contraction frequencies, therefore recapitulating several of the critical phenotype features associated with arrhythmic risk in drug-induced LQTS. BeKm-1 exemplifies an interesting reference compound in the integrated hiPS-CMs cell model for all drugs that may block the hERG channel from the outer face. Being a peptide that is easily modifiable, it will serve as an ideal molecular platform for the design of new hERG modulators displaying additional functionalities.


Asunto(s)
Calcio/metabolismo , Canal de Potasio ERG1/antagonistas & inhibidores , Miocitos Cardíacos/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Potasio/metabolismo , Venenos de Escorpión/farmacología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Antiarrítmicos/farmacología , Canales de Calcio/metabolismo , Diferenciación Celular , Canal de Potasio ERG1/metabolismo , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Transporte Iónico , Síndrome de QT Prolongado/metabolismo , Síndrome de QT Prolongado/fisiopatología , Modelos Biológicos , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp , Fenetilaminas/farmacología , Piperidinas/farmacología , Piridinas/farmacología , Sulfonamidas/farmacología
4.
Biochem J ; 477(20): 3985-3999, 2020 10 30.
Artículo en Inglés | MEDLINE | ID: mdl-33034621

RESUMEN

Ryanodine receptors are responsible for the massive release of calcium from the sarcoplasmic reticulum that triggers heart muscle contraction. Maurocalcin (MCa) is a 33 amino acid peptide toxin known to target skeletal ryanodine receptor. We investigated the effect of MCa and its analog MCaE12A on isolated cardiac ryanodine receptor (RyR2), and showed that they increase RyR2 sensitivity to cytoplasmic calcium concentrations promoting channel opening and decreases its sensitivity to inhibiting calcium concentrations. By measuring intracellular Ca2+ transients, calcium sparks and contraction on cardiomyocytes isolated from adult rats or differentiated from human-induced pluripotent stem cells, we demonstrated that MCaE12A passively penetrates cardiomyocytes and promotes the abnormal opening of RyR2. We also investigated the effect of MCaE12A on the pacemaker activity of sinus node cells from different mice lines and showed that, MCaE12A improves pacemaker activity of sinus node cells obtained from mice lacking L-type Cav1.3 channel, or following selective pharmacologic inhibition of calcium influx via Cav1.3. Our results identify MCaE12A as a high-affinity modulator of RyR2 and make it an important tool for RyR2 structure-to-function studies as well as for manipulating Ca2+ homeostasis and dynamic of cardiac cells.


Asunto(s)
Calcio/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Venenos de Escorpión/farmacología , Nodo Sinoatrial/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Señalización del Calcio/efectos de los fármacos , Citoplasma/efectos de los fármacos , Citoplasma/metabolismo , Homeostasis , Humanos , Masculino , Ratones , Ratones Noqueados , Miocitos Cardíacos/metabolismo , Células Madre Pluripotentes , Ratas , Ratas Wistar , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismo , Venenos de Escorpión/química , Nodo Sinoatrial/citología , Nodo Sinoatrial/fisiología , Porcinos
5.
Sci Rep ; 10(1): 9835, 2020 06 17.
Artículo en Inglés | MEDLINE | ID: mdl-32555258

RESUMEN

Sinus node (SAN) dysfunction (SND) manifests as low heart rate (HR) and is often accompanied by atrial tachycardia or atrioventricular (AV) block. The only currently available therapy for chronic SND is the implantation of an electronic pacemaker. Because of the growing burden of SND in the population, new pharmacological therapies of chronic SND and heart block are desirable. We developed a collection of genetically modified mouse strains recapitulating human primary SND associated with different degrees of AV block. These mice were generated with genetic ablation of L-type Cav1.3 (Cav1.3-/-), T-type Cav3.1 (Cav3.1-/-), or both (Cav1.3-/-/Cav3.1-/-). We also studied mice haplo-insufficient for the Na+ channel Nav1.5 (Nav1.5+/) and mice in which the cAMP-dependent regulation of hyperpolarization-activated f-(HCN4) channels has been abolished (HCN4-CNBD). We analysed, by telemetric ECG recording, whether pharmacological inhibition of the G-protein-activated K+ current (IKACh) by the peptide tertiapin-Q could improve HR and AV conduction in these mouse strains. Tertiapin-Q significantly improved the HR of Cav1.3-/- (19%), Cav1.3-/-/Cav3.1-/- (23%) and HCN4-CNBD (14%) mice. Tertiapin-Q also improved cardiac conduction of Nav1.5+/- mice by 24%. Our data suggest that the development of pharmacological IKACh inhibitors for the management of SND and conduction disease is a viable approach.


Asunto(s)
Venenos de Abeja/farmacología , Bradicardia/fisiopatología , Proteínas de Unión al GTP/metabolismo , Sistema de Conducción Cardíaco/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Nodo Sinoatrial/efectos de los fármacos , Animales , Bradicardia/metabolismo , Canales de Calcio Tipo L/metabolismo , Modelos Animales de Enfermedad , Frecuencia Cardíaca/efectos de los fármacos , Ratones , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Nodo Sinoatrial/fisiopatología
6.
Molecules ; 24(2)2019 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-30634526

RESUMEN

The medical staff is often powerless to treat patients affected by drug abuse or misuse and poisoning. In the case of envenomation, the treatment of choice remains horse sera administration that poses a wealth of other medical conditions and threats. Previously, we have demonstrated that DNA-based aptamers represent powerful neutralizing tools for lethal animal toxins of venomous origin. Herein, we further pursued our investigations in order to understand whether all toxin-interacting aptamers possessed equivalent potencies to neutralize αC-conotoxin PrXA in vitro and in vivo. We confirmed the high lethality in mice produced by αC-conotoxin PrXA regardless of the mode of injection and further characterized myoclonus produced by the toxin. We used high-throughput patch-clamp technology to assess the effect of αC-conotoxin PrXA on ACh-mediated responses in TE671 cells, responses that are carried by muscle-type nicotinic receptors. We show that 2 out of 4 aptamers reduce the affinity of the toxin for its receptor, most likely by interfering with the pharmacophore. In vivo, more complex responses on myoclonus and mice lethality are observed depending on the type of aptamer and mode of administration (concomitant or differed). Concomitant administration always works better than differed administration indicating the stability of the complex in vivo. The most remarkable conclusion is that an aptamer that has no or a limited efficacy in vitro may nevertheless be functional in vivo probably owing to an impact on the biodistribution or pharmacokinetics of the toxin in vivo. Overall, the results highlight that a blind selection of aptamers against toxins leads to efficient neutralizing compounds in vivo regardless of the mode of action. This opens the door to the use of aptamer mixtures as substitutes to horse sera for the neutralization of life-threatening animal venoms, an important WHO concern in tropical areas.


Asunto(s)
Aptámeros de Nucleótidos/administración & dosificación , Conotoxinas/toxicidad , Mioclonía/prevención & control , Animales , Aptámeros de Nucleótidos/farmacología , Línea Celular , Modelos Animales de Enfermedad , Femenino , Masculino , Ratones , Mioclonía/mortalidad , Receptores Nicotínicos/metabolismo , Técnica SELEX de Producción de Aptámeros
7.
Brain Behav ; 8(8): e00978, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29934975

RESUMEN

BACKGROUND: Autism spectrum disorder (ASD) comprises a group of neurodevelopmental psychiatric disorders characterized by deficits in social interactions, interpersonal communication, repetitive and stereotyped behaviors and may be associated with intellectual disabilities. The description of ASD as a synaptopathology highlights the importance of the synapse and the implication of ion channels in the etiology of these disorders. METHODS: A narrative and critical review of the relevant papers from 1982 to 2017 known by the authors was conducted. RESULTS: Genome-wide linkages, association studies, and genetic analyses of patients with ASD have led to the identification of several candidate genes and mutations linked to ASD. Many of the candidate genes encode for proteins involved in neuronal development and regulation of synaptic function including ion channels and actors implicated in synapse formation. The involvement of ion channels in ASD is of great interest as they represent attractive therapeutic targets. In agreement with this view, recent findings have shown that drugs modulating ion channel function are effective for the treatment of certain types of patients with ASD. CONCLUSION: This review describes the genetic aspects of ASD with a focus on genes encoding ion channels and highlights the therapeutic implications of ion channels in the treatment of ASD.


Asunto(s)
Trastorno del Espectro Autista , Canales Iónicos/genética , Terapia Molecular Dirigida , Transmisión Sináptica/efectos de los fármacos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/terapia , Estudio de Asociación del Genoma Completo , Humanos , Transmisión Sináptica/genética
8.
Mol Biol Cell ; 28(25): 3699-3708, 2017 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-29021340

RESUMEN

The ß4 isoform of the ß-subunits of voltage-gated calcium channel regulates cell proliferation and cell cycle progression. Herein we show that coexpression of the ß4-subunit with actors of the canonical Wnt/ß-catenin signaling pathway in a hepatoma cell line inhibits Wnt-responsive gene transcription and decreases cell division, in agreement with the role of the Wnt pathway in cell proliferation. ß4-subunit-mediated inhibition of Wnt signaling is observed in the presence of LiCl, an inhibitor of glycogen synthase kinase (GSK3) that promotes ß-catenin translocation to the nucleus. Expression of ß4-subunit mutants that lost the ability to translocate to the nucleus has no effect on Wnt signaling, suggesting that ß4-subunit inhibition of Wnt signaling occurs downstream from GSK3 and requires targeting of ß4-subunit to the nucleus. ß4-subunit coimmunoprecipitates with the TCF4 transcription factor and overexpression of TCF4 reverses the effect of ß4-subunit on the Wnt pathway. We thus propose that the interaction of nuclear ß4-subunit with TCF4 prevents ß-catenin binding to TCF4 and leads to the inhibition of the Wnt-responsive gene transcription. Thereby, our results show that ß4-subunit is a TCF4 repressor and therefore appears as an interesting candidate for the regulation of this pathway in neurons where ß4-subunit is specifically expressed.


Asunto(s)
Canales de Calcio/metabolismo , Glucógeno Sintasa Quinasa 3/metabolismo , Vía de Señalización Wnt , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Células CHO , Canales de Calcio/fisiología , Línea Celular , Línea Celular Tumoral , Proliferación Celular/fisiología , Cricetulus , Regulación hacia Abajo , Humanos , Regiones Promotoras Genéticas , Unión Proteica , Transducción de Señal , Factor de Transcripción 4/metabolismo , Factores de Transcripción/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , beta Catenina/fisiología
9.
Sci Rep ; 7(1): 7202, 2017 08 03.
Artículo en Inglés | MEDLINE | ID: mdl-28775316

RESUMEN

Medical means to save the life of human patients affected by drug abuse, envenomation or critical poisoning are currently limited. While the compounds at risks are most often well identified, particularly for bioterrorism, chemical intervention to counteract the toxic effects of the ingested/injected compound(s) is restricted to the use of antibodies. Herein, we illustrate that DNA aptamers, targeted to block the pharmacophore of a poisonous compound, represent a fast-acting and reliable method of neutralization in vivo that possesses efficient and long-lasting life-saving properties. For this proof of concept, we used one putative bioweapon, αC-conotoxin PrXA, a marine snail ultrafast-killing paralytic toxin, to identify peptide-binding DNA aptamers. We illustrate that they can efficiently neutralize the toxin-induced (i) displacement of [125I]-α-bungarotoxin binding onto nicotinic receptors, (ii) inhibition of diaphragm muscle contraction, and (iii) lethality in mice. Our results demonstrate the preclinical value of DNA aptamers as fast-acting, safe and cheap antidotes to lethal toxins at risk of misuse in bioterrorism and offer hope for an alternative method than donor sera to treat cases of envenomation.


Asunto(s)
Oligonucleótidos/administración & dosificación , Péptidos/antagonistas & inhibidores , Toxinas Biológicas/antagonistas & inhibidores , Animales , Aptámeros de Nucleótidos/administración & dosificación , Conotoxinas/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Femenino , Dosificación Letal Mediana , Masculino , Ratones , Pruebas de Neutralización , Péptidos/toxicidad , Toxinas Biológicas/toxicidad
10.
Int J Biochem Cell Biol ; 89: 57-70, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28587927

RESUMEN

The ß subunits of Voltage-Gated Calcium Channel (VGCC) are cytosolic proteins that interact with the VGCC pore -forming subunit and participate in the trafficking of the channel to the cell membrane and in ion influx regulation. ß subunits also exert functions independently of their binding to VGCC by translocation to the cell nucleus including the control of gene expression. Mutations of the neuronal Cacnb4 (ß4) subunit are linked to human neuropsychiatric disorders including epilepsy and intellectual disabilities. It is believed that the pathogenic phenotype induced by these mutations is associated with channel-independent functions of the ß4 subunit. In this report, we investigated the role of ß4 subunit in cell proliferation and cell cycle progression and examined whether these functions could be altered by a pathogenic mutation. To this end, stably transfected Chinese Hamster Ovary (CHO-K1) cells expressing either rat full-length ß4 or the rat C-terminally truncated epileptic mutant variant ß1-481 were generated. The subcellular localization of both proteins differed significantly. Full-length ß4 localizes almost exclusively in the cell nucleus and concentrates into the nucleolar compartment, while the C-terminal-truncated ß1-481 subunit was less concentrated within the nucleus and absent from the nucleoli. Cell proliferation was found to be reduced by the expression of ß4, while it was unaffected by the epileptic mutant. Also, full-length ß4 interfered with cell cycle progression by presumably preventing cells from entering the S-phase via a mechanism that partially involves endogenous B56δ, a regulatory subunit of the phosphatase 2A (PP2A) that binds ß4 but not ß1-481. Analysis of ß4 subcellular distribution during the cell cycle revealed that the protein is highly expressed in the nucleus at the G1/S transition phase and that it is translocated out of the nucleus during chromatin condensation and cell division. These results suggest that nuclear accumulation of ß4 at the G1/S transition phase affects the progression into S-phase resulting in a decrease in the rate of cell proliferation. Regulation of the cell cycle exit is a critical step in determining the number of neuronal precursors and neuronal differentiation suggesting that mutations of the ß4 subunit could affect neural development and formation of the mature central nervous system.


Asunto(s)
Canales de Calcio/metabolismo , Animales , Células CHO , Canales de Calcio/genética , Ciclo Celular , Nucléolo Celular/metabolismo , Proliferación Celular , Cricetinae , Cricetulus , Regulación de la Expresión Génica , Ratones , Mutación , Transporte de Proteínas
11.
J Biol Chem ; 292(22): 9365-9381, 2017 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-28377503

RESUMEN

Voltage-dependent Ca2+ channels (VDCCs) mediate neurotransmitter release controlled by presynaptic proteins such as the scaffolding proteins Rab3-interacting molecules (RIMs). RIMs confer sustained activity and anchoring of synaptic vesicles to the VDCCs. Multiple sites on the VDCC α1 and ß subunits have been reported to mediate the RIMs-VDCC interaction, but their significance is unclear. Because alternative splicing of exons 44 and 47 in the P/Q-type VDCC α1 subunit CaV2.1 gene generates major variants of the CaV2.1 C-terminal region, known for associating with presynaptic proteins, we focused here on the protein regions encoded by these two exons. Co-immunoprecipitation experiments indicated that the C-terminal domain (CTD) encoded by CaV2.1 exons 40-47 interacts with the α-RIMs, RIM1α and RIM2α, and this interaction was abolished by alternative splicing that deletes the protein regions encoded by exons 44 and 47. Electrophysiological characterization of VDCC currents revealed that the suppressive effect of RIM2α on voltage-dependent inactivation (VDI) was stronger than that of RIM1α for the CaV2.1 variant containing the region encoded by exons 44 and 47. Importantly, in the CaV2.1 variant in which exons 44 and 47 were deleted, strong RIM2α-mediated VDI suppression was attenuated to a level comparable with that of RIM1α-mediated VDI suppression, which was unaffected by the exclusion of exons 44 and 47. Studies of deletion mutants of the exon 47 region identified 17 amino acid residues on the C-terminal side of a polyglutamine stretch as being essential for the potentiated VDI suppression characteristic of RIM2α. These results suggest that the interactions of the CaV2.1 CTD with RIMs enable CaV2.1 proteins to distinguish α-RIM isoforms in VDI suppression of P/Q-type VDCC currents.


Asunto(s)
Canales de Calcio Tipo N/metabolismo , Canales de Calcio/metabolismo , Proteínas de Unión al GTP/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Canales de Calcio/genética , Canales de Calcio Tipo N/genética , Proteínas de Unión al GTP/genética , Células HEK293 , Humanos , Ratones , Proteínas del Tejido Nervioso/genética , Dominios Proteicos
12.
Cell Calcium ; 60(4): 245-55, 2016 10.
Artículo en Inglés | MEDLINE | ID: mdl-27422341

RESUMEN

The mechanism underlying fatigue in skeletal muscle (SM) related to the redox-potential hypothesis, ranges from a direct effect of oxygen reactive species, to a number of other free radical intermediates targeting specific amino acids in the Ca(2+)-regulatory proteins of the sarcoplasmic reticulum (SR). In the present study, we investigate the selective oxidation/reduction of the protein motif Cys-(Xn=2-6)-Cys, known as a vicinal thiol group (VTG), present in the SR Ca(2+)-ATPase (SERCA) and in the Ca(2+)-channel ryanodine receptor (RyR) which are modified during muscle fatigue in SM. Selective oxidation of VTG with phenyl arsine oxide (PAO) increases fatigue in rat isolated SM and fatigue is prevented when muscle is previously incubated with a VTG selective reducing agent, 2,3-dimercaptopropanol (British anti-Lewisite (BAL)). In isolated SR membranes, PAO [<0.1mM] modifies SERCA conformation and inhibits ATPase activity but does not affect Ca(2+)-release. However, PAO at [>0.1mM] inhibits SERCA and RyR activities in a reversible manner by selectively reducing them. Interestingly, as observed by differential scanning calorimetry, the conformation of SERCA from fatigued muscle changed in a similar manner as when SERCA VTG where oxidized. The addition of BAL to fatigued muscle restored the structural conformation and activity of SERCA with full recovery of muscle force production after fatigue. We conclude that VTG reversible oxidation of SR Ca(2+) regulatory proteins are involved in muscle contraction/relaxation and are a molecular mechanism to be considered for muscle fatigue.


Asunto(s)
Calcio/metabolismo , Cisteína/metabolismo , Fatiga Muscular , Músculo Esquelético/metabolismo , Retículo Sarcoplasmático/metabolismo , Compuestos de Sulfhidrilo/metabolismo , Animales , Masculino , Oxidación-Reducción , Ratas , Ratas Wistar
13.
Biochem J ; 473(13): 1831-44, 2016 07 01.
Artículo en Inglés | MEDLINE | ID: mdl-27354560

RESUMEN

Calcium plays a key role in cell signalling by its intervention in a wide range of physiological processes. Its entry into cells occurs mainly via voltage-gated calcium channels (VGCC), which are found not only in the plasma membrane of excitable cells but also in cells insensitive to electrical signals. VGCC are composed of different subunits, α1, ß, α2δ and γ, among which the cytosolic ß subunit (Cavß) controls the trafficking of the channel to the plasma membrane, its regulation and its gating properties. For many years, these were the main functions associated with Cavß. However, a growing number of proteins have been found to interact with Cavß, emphasizing the multifunctional role of this versatile protein. Interestingly, some of the newly assigned functions of Cavß are independent of its role in the regulation of VGCC, and thus further increase its functional roles. Based on the identity of Cavß protein partners, this review emphasizes the diverse cellular functions of Cavß and summarizes both past findings as well as recent progress in the understanding of VGCC.


Asunto(s)
Canales de Calcio/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/química , Membrana Celular/metabolismo , Humanos , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo
14.
Proc Natl Acad Sci U S A ; 113(17): E2460-8, 2016 Apr 26.
Artículo en Inglés | MEDLINE | ID: mdl-27071086

RESUMEN

The venom peptide maurocalcin (MCa) is atypical among toxins because of its ability to rapidly translocate into cells and potently activate the intracellular calcium channel type 1 ryanodine receptor (RyR1). Therefore, MCa is potentially subjected to posttranslational modifications within recipient cells. Here, we report that MCa Thr(26) belongs to a consensus PKA phosphorylation site and can be phosphorylated by PKA both in vitro and after cell penetration in cellulo. Unexpectedly, phosphorylation converts MCa from positive to negative RyR1 allosteric modulator. Thr(26) phosphorylation leads to charge neutralization of Arg(24), a residue crucial for MCa agonist activity. The functional effect of Thr(26) phosphorylation is partially mimicked by aspartyl mutation. This represents the first case, to our knowledge, of both ex situ posttranslational modification and pharmacological reprogramming of a small natural cystine-rich peptide by target cells. So far, phosphorylated MCa is the first specific negative allosteric modulator of RyR1, to our knowledge, and represents a lead compound for further development of phosphatase-resistant analogs.


Asunto(s)
Venenos de Escorpión/metabolismo , Calcio/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Homeostasis , Humanos , Fosforilación , Procesamiento Proteico-Postraduccional , Canal Liberador de Calcio Receptor de Rianodina/efectos de los fármacos , Venenos de Escorpión/farmacología
15.
Biochim Biophys Acta ; 1843(10): 2356-64, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24667409

RESUMEN

L-Maurocalcine (L-MCa) is the first reported animal cell-penetrating toxin. Characterizing its cell penetration properties is crucial considering its potential as a vector for the intracellular delivery of drugs. Radiolabeling is a sensitive and quantitative method to follow the cell accumulation of a molecule of interest. An L-MCa analog containing an additional N-terminal tyrosine residue (Tyr-L-MCa) was synthesized, shown to fold and oxidize properly, and successfully radioiodinated to (125)I-Tyr-L-MCa. Using various microscopy techniques, the average volume of the rat line F98 glioma cells was evaluated at 8.9 to 18.9×10(-7)µl. (125)I-Tyr-L-MCa accumulates within cells with a dose-dependency similar to the one previously published using 5,6-carboxyfluorescein-L-MCa. According to subcellular fractionation of F98 cells, plasma membranes keep less than 3% of the peptide, regardless of the extracellular concentration, while the nucleus accumulates over 75% and the cytosol around 20% of the radioactive material. Taking into account both nuclear and cytosolic fractions, cells accumulate intracellular concentrations of the peptide that are equal to the extracellular concentrations. Estimation of (125)I-Tyr-L-MCa cell entry kinetics indicate a first rapid phase with a 5min time constant for the plasma membrane followed by slower processes for the cytoplasm and the nucleus. Once inside cells, the labeled material no longer escapes from the intracellular environment since 90% of the radioactivity remains 24h after washout. Dead cells were found to have a lower uptake than live ones. The quantitative information gained herein will be useful for better framing the use of L-MCa in biotechnological applications. This article is part of a Special Issue entitled: Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.


Asunto(s)
Membrana Celular/metabolismo , Péptidos de Penetración Celular/metabolismo , Venenos de Escorpión/metabolismo , Tirosina/química , Secuencia de Aminoácidos , Animales , Transporte Biológico , Línea Celular Tumoral , Permeabilidad de la Membrana Celular , Núcleo Celular/metabolismo , Tamaño de la Célula , Péptidos de Penetración Celular/síntesis química , Citosol/metabolismo , Portadores de Fármacos , Radioisótopos de Yodo , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Ratas , Venenos de Escorpión/síntesis química , Técnicas de Síntesis en Fase Sólida
16.
Pharmaceuticals (Basel) ; 6(3): 320-39, 2013 Mar 18.
Artículo en Inglés | MEDLINE | ID: mdl-24276021

RESUMEN

Maurocalcine is a highly potent cell-penetrating peptide isolated from the Tunisian scorpion Maurus palmatus. Many cell-penetrating peptide analogues have been derived from the full-length maurocalcine by internal cysteine substitutions and sequence truncation. Herein we have further characterized the cell-penetrating properties of one such peptide, MCaUF1-9, whose sequence matches that of the hydrophobic face of maurocalcine. This peptide shows very favorable cell-penetration efficacy compared to Tat, penetratin or polyarginine. The peptide appears so specialized in cell penetration that it seems hard to improve by site directed mutagenesis. A comparative analysis of the efficacies of similar peptides isolated from other toxin members of the same family leads to the identification of hadrucalcin's hydrophobic face as an even better CPP. Protonation of the histidine residue at position 6 renders the cell penetration of MCaUF1-9 pH-sensitive. Greater cell penetration at acidic pH suggests that MCaUF1-9 can be used to specifically target cancer cells in vivo where tumor masses grow in more acidic environments.

17.
Biosci Rep ; 33(3)2013 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-23721518

RESUMEN

The ICK (inhibitor cystine knot) defines a large superfamily of polypeptides with high structural stability and functional diversity. Here, we describe a new scorpion venom-derived K+ channel toxin (named λ-MeuKTx-1) with an ICK fold through gene cloning, chemical synthesis, nuclear magnetic resonance spectroscopy, Ca2+ release measurements and electrophysiological recordings. λ-MeuKTx-1 was found to adopt an ICK fold that contains a three-strand anti-parallel ß-sheet and a 310-helix. Functionally, this peptide selectively inhibits the Drosophila Shaker K+ channel but is not capable of activating skeletal-type Ca2+ release channels/ryanodine receptors, which is remarkably different from the previously known scorpion venom ICK peptides. The removal of two C-terminal residues of λ-MeuKTx-1 led to the loss of the inhibitory activity on the channel, whereas the C-terminal amidation resulted in the emergence of activity on four mammalian K+ channels accompanied by the loss of activity on the Shaker channel. A combination of structural and pharmacological data allows the recognition of three putative functional sites involved in channel blockade of λ-MeuKTx-1. The presence of a functional dyad in λ-MeuKTx-1 supports functional convergence among scorpion venom peptides with different folds. Furthermore, similarities in precursor organization, exon-intron structure, 3D-fold and function suggest that scorpion venom ICK-type K+ channel inhibitors and Ca2+ release channel activators share a common ancestor and their divergence occurs after speciation between buthidae and non-buthids. The structural and functional characterizations of the first scorpion venom ICK toxin with K+ channel-blocking activity sheds light on functionally divergent and convergent evolution of this conserved scaffold of ancient origin.


Asunto(s)
Péptidos/química , Péptidos/genética , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/metabolismo , Venenos de Escorpión/química , Venenos de Escorpión/genética , Escorpiones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Calcio/metabolismo , Clonación Molecular , Cistina/química , Cistina/genética , Cistina/metabolismo , Evolución Molecular , Modelos Moleculares , Datos de Secuencia Molecular , Péptidos/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Replegamiento Proteico , Estructura Secundaria de Proteína , Venenos de Escorpión/farmacología , Escorpiones/química , Xenopus
18.
Cell Calcium ; 53(5-6): 357-65, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23623374

RESUMEN

The scorpion toxin maurocalcine acts as a high affinity agonist of the type-1 ryanodine receptor expressed in skeletal muscle. Here, we investigated the effects of the reducing agent dithiothreitol or the oxidizing reagent thimerosal on type-1 ryanodine receptor stimulation by maurocalcine. Maurocalcine addition to sarcoplasmic reticulum vesicles actively loaded with calcium elicited Ca²âº release from native vesicles and from vesicles pre-incubated with dithiothreitol; thimerosal addition to native vesicles after Ca²âº uptake completion prevented this response. Maurocalcine enhanced equilibrium [³H]-ryanodine binding to native and to dithiothreitol-treated reticulum vesicles, and increased 5-fold the apparent Ki for Mg²âº inhibition of [³H]-ryanodine binding to native vesicles. Single calcium release channels incorporated in planar lipid bilayers displayed a long-lived open sub-conductance state after maurocalcine addition. The fractional time spent in this sub-conductance state decreased when lowering cytoplasmic [Ca²âº] from 10 µM to 0.1 µM or at cytoplasmic [Mg²âº]≥30 µM. At 0.1 µM [Ca²âº], only channels that displayed poor activation by Ca²âº were readily activated by 5 nM maurocalcine; subsequent incubation with thimerosal abolished the sub-conductance state induced by maurocalcine. We interpret these results as an indication that maurocalcine acts as a more effective type-1 ryanodine receptor channel agonist under reducing conditions.


Asunto(s)
Vesículas Citoplasmáticas/metabolismo , Membrana Dobles de Lípidos/metabolismo , Músculo Esquelético/fisiología , Canal Liberador de Calcio Receptor de Rianodina/metabolismo , Rianodina/metabolismo , Retículo Sarcoplasmático/metabolismo , Venenos de Escorpión/farmacología , Animales , Calcio/metabolismo , Ditiotreitol/farmacología , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Oxidación-Reducción , Unión Proteica/efectos de los fármacos , Conejos , Timerosal/farmacología
19.
Channels (Austin) ; 7(2): 119-25, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23511121

RESUMEN

The pore-forming subunit of voltage-gated calcium channels is associated to auxiliary subunits among which the cytoplasmic ß subunit. The different isoforms of this subunit control both the plasma membrane targeting and the biophysical properties of the channel moiety. In a recent study, we demonstrated that the Cacnb4 (ß 4) isoform is at the center of a new signaling pathway that connects neuronal excitability and gene transcription. This mechanism relies on nuclear targeting of ß 4 triggered by neuronal electrical stimulation. This re-localization of ß 4 is promoted by its interaction with Ppp2r5d a regulatory subunit of PP2A in complex with PP2A itself. The formation, as well as the nuclear translocation, of the ß 4/ Ppp2r5d/ PP2A complex is totally impaired by the premature R482X stops mutation of ß 4 that has been previously associated with juvenile epilepsy. Taking as a case study the tyrosine hydroxylase gene that is strongly upregulated in brain of lethargic mice, deficient for ß 4 expression, we deciphered the molecular steps presiding to this signaling pathway. Here we show that expression of wild-type ß 4 in HEK293 cells results in the regulation of several genes, while expression of the mutated ß 4 (ß 1-481) produces a different set of gene regulation. Several genes regulated by ß 4 in HEK293 cells were also regulated upon neuronal differentiation of NG108-15 cells that induces nuclear translocation of ß 4 suggesting a link between ß 4 nuclear targeting and gene regulation.


Asunto(s)
Canales de Calcio/metabolismo , Núcleo Celular/metabolismo , Regulación de la Expresión Génica , Transcripción Genética , Transporte Activo de Núcleo Celular , Animales , Canales de Calcio/genética , Redes Reguladoras de Genes , Células HEK293 , Humanos , Ratones , Mutación , Neurogénesis/genética , Neuronas/citología , Neuronas/metabolismo , Proteína Fosfatasa 2/metabolismo , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Transducción de Señal , Tirosina 3-Monooxigenasa/metabolismo
20.
EMBO J ; 31(18): 3730-44, 2012 Sep 12.
Artículo en Inglés | MEDLINE | ID: mdl-22892567

RESUMEN

Calcium current through voltage-gated calcium channels (VGCC) controls gene expression. Here, we describe a novel signalling pathway in which the VGCC Cacnb4 subunit directly couples neuronal excitability to transcription. Electrical activity induces Cacnb4 association to Ppp2r5d, a regulatory subunit of PP2A phosphatase, followed by (i) nuclear translocation of Cacnb4/Ppp2r5d/PP2A, (ii) association with the tyrosine hydroxylase (TH) gene promoter through the nuclear transcription factor thyroid hormone receptor alpha (TRα), and (iii) histone binding through association of Cacnb4 with HP1γ concomitantly with Ser(10) histone H3 dephosphorylation by PP2A. This signalling cascade leads to TH gene repression by Cacnb4 and is controlled by the state of interaction between the SH3 and guanylate kinase (GK) modules of Cacnb4. The human R482X CACNB4 mutation, responsible for a form of juvenile myoclonic epilepsy, prevents association with Ppp2r5 and nuclear targeting of the complex by altering Cacnb4 conformation. These findings demonstrate that an intact VGCC subunit acts as a repressor recruiting platform to control neuronal gene expression.


Asunto(s)
Canales de Calcio/biosíntesis , Canales de Calcio/genética , Epilepsias Mioclónicas/metabolismo , Regulación de la Expresión Génica , Transporte Activo de Núcleo Celular , Animales , Biofisica/métodos , Canales de Calcio/metabolismo , Electrofisiología/métodos , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Histonas/metabolismo , Humanos , Ratones , Mutación , Proteína Fosfatasa 2/metabolismo , Transducción de Señal , Receptores alfa de Hormona Tiroidea/metabolismo , Transcripción Genética
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