20 research outputs found

    Inhibitory Effects of Cannabidiol on Voltage-Dependent Sodium Currents

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    Cannabis sativa contains many related compounds known as phytocannabinoids. The main psychoactive and nonpsychoactive compounds are Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD), respectively. Much of the evidence for clinical efficacy of CBD-mediated antiepileptic effects has been from case reports or smaller surveys. The mechanisms for CBD\u27s anticonvulsant effects are unclear and likely involve noncannabinoid receptor pathways. CBD is reported to modulate several ion channels, including sodium channels (Nav). Evaluating the therapeutic mechanisms and safety of CBD demands a richer understanding of its interactions with central nervous system targets. Here, we used voltage-clamp electrophysiology of HEK-293 cells and iPSC neurons to characterize the effects of CBD on Nav channels. Our results show that CBD inhibits hNav1.1–1.7 currents, with an IC50 of 1.9–3.8 μM, suggesting that this inhibition could occur at therapeutically relevant concentrations. A steep Hill slope of ∼3 suggested multiple interactions of CBD with Nav channels. CBD exhibited resting-state blockade, became more potent at depolarized potentials, and also slowed recovery from inactivation, supporting the idea that CBD binding preferentially stabilizes inactivated Nav channel states. We also found that CBD inhibits other voltage-dependent currents from diverse channels, including bacterial homomeric Nav channel (NaChBac) and voltage-gated potassium channel subunit Kv2.1. Lastly, the CBD block of Nav was temperature-dependent, with potency increasing at lower temperatures. We conclude that CBD\u27s mode of action likely involves 1) compound partitioning in lipid membranes, which alters membrane fluidity affecting gating, and 2) undetermined direct interactions with sodium and potassium channels, whose combined effects are loss of channel excitability.&nbsp

    Caracterização eletrofisiológica das toxinas moduladoras de canais de sódio (Tst1 e Tst3) purificadas da peçonha do escorpião Tityus stigmurus

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    Tese (doutorado) — Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2022.imadamente 1,5% das espécies de aracnídeos existentes. Dentro do território brasileiro, são encontradas diversas espécies de escorpiões, estando presente em todos os estados do país. Dentre as espécies de maior importância e abundância estão os animais pertencentes ao gênero Tityus, tendo em vista o grande número de acidentes e problemas graves causados. Uma espécie de destaque é o Tityus stigmurus, espécie endêmica do Nordeste do Brasil. Essa espécie se destaca por ser a maior causadora de picadas nesta região do país, sendo responsável pelo grande número de casos acidentes graves, desencadeando altos índices de internações e morte. A peçonha destes animais é formada por diversos compostos diferentes, no qual os que mais se sobressaem são peptídeos, também conhecidos como neurotoxinas. Essas neurotoxinas são capazes de interagir e afetar o funcionamento de canais iônicos dependentes de voltagem tais como potássio (Kv), cálcio (Cav) e sódio (Nav), responsáveis pela propagação e iniciação de sinais nervosos. Neste trabalho, a peçonha do escorpião Tityus stigmurus foi extraída e submetida ao processo de fracionamento utilizando a técnica de Cromatografia Líquida de Alta Eficiência em Fase Reversa (RP-HPLC). As frações coletadas foram analisadas em Espectrômetro de Massa (MALDI-TOF) e as frações de interesse para este trabalho foram separadas e sequenciadas de forma parcial pelo método de ISD. Após o processo de purificação e identificação, as toxinas purificadas, correspondente às toxinas Tst1 e Tst3, foram testadas em canais de sódio dependentes de voltagem (subtipos Nav 1.1 a 1.7) utilizando a técnica de patchclamp no modo whole cell. A toxina Tst1 demonstrou uma atividade característica da classe das β-toxinas, alterando o potencial de abertura dos canais de sódio (Nav) e inibindo a corrente dos mesmos, tendo uma ação superior no subtipo Nav 1.3. Já a toxina Tst3 manifestou uma atividade característico das α-toxinas, alterando a inativação rápida dos canais de sódio, sendo os subtipos Nav 1.3, 1.6 e 1.7 os mais comprometidos. Assim, as toxinas Tst1 e Tst3 são as primeiras toxinas purificadas da peçonha de Tityus stigmurus amplamente caracterizadas em diferentes isoformas de canais de sódio (Nav).Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) e Fundação de Apoio à Pesquisa do Distrito Federal (FAP/DF).Belonging to the Arthropoda phylum, scorpions represent approximately 1.5% of arachnid species. In Brazil several species of scorpions are found, being present in all regions. Among them, the most important and abundant species are the animals belonging to the genus Tityus, due to their great variety and medical importance. One of the most important ones is Tityus stigmurus, an endemic species from the Northeast region of Brazil. This species stands out for the high number of accidents in the Northeast region of the country, being responsible for a large number of accidents, leading to hospitalization and cases of death. The venom of these animals is composed of several different compounds, in which the most prominent are peptides, also known as neurotoxins. These neurotoxins are able to interact and affect the functioning of ion channels such as potassium (Kv), calcium (Cav), and voltage-gated sodium channels (Nav), responsible for the propagation and initiation of nerve signals. In this work, the venom of the scorpion Tityus stigmurus was extracted and fractioned using Reverse Phase High-Performance Liquid Chromatography (RPHPLC) technique. The fractions collected were analyzed in a Mass Spectrometer (MALDI-TOF) and the fractions of interest were separated and partially sequenced using ISD method. After the purification and identification, the purified toxins, corresponding to toxins Tst1 and Tst3, were tested in voltage-gated sodium channels (Nav 1.1 to 1.7) using the patch-clamp technique in whole cell mode. The toxin Tst1 demonstrated a characteristic activity of β-toxin, altering the opening potential of sodium channels (Nav) and inhibiting current, having a greater action on the Nav 1.3 subtype. The toxin Tst3 showed a characteristic of α-toxins, altering the rapid inactivation of sodium channels, with subtypes Nav 1.3, 1.6, and 1.7 the most compromised ones. Tst1 and Tst3 toxins are the first toxins purified from Tityus stigmurus venom widely characterized in different isoforms of sodium channels (Nav)

    Fluorine-18 labelling of novel voltage-gated sodium channel ligands for applications in positron emission tomography (PET) imaging and oncology

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    Voltage-gated sodium channels (VGSCs) are essential for the initiation and propagation of neuronal impulses. Specifically, these channels initiate the rising phase of action potentials in electrically excitable cells, allowing the conduction of electrical information. There are 9 different sub-types of VGSCs, each found in different tissues, with a minimum 50% identical genetic code.1 Recently, it has been reported that some VGSC sub-types are over-expressed in highly metastatic tumours.2 Their presence allows the up-regulation of some key biochemical processes. In fact, the metastatic potential of the tumours has a positive correlation with the expression of sodium channels.2-6 This thesis focussed on developing a novel positron emission tomography (PET) tracer to target VGSCs. We hypothesised that a PET scan using a VGSC ligand may be able to highlight tumours with high metastatic potential, and assist in the selection of the most appropriate clinical treatment. At present, there is no known clinical PET tracer for imaging VGSCs. Our project involved the adaptation of the 3-(4-substituted-phenoxyphenyl) pyrazoles,7 a known group of VGSC modulators, into novel fluorine-18 labelled PET imaging agents. These were chosen due to their strong affinity for VGSCs and their structural characteristics, which allowed multiple approches to introduce a fluorine-18 label. Several compounds of interest were successfully synthesised. Optimal radiolabelling strategies were identified and methodologies developed, to produce PET agents in good synthesis time, yield and purity. The fluorine-19 standards for these compounds underwent a full VGSC isoform selectivity screen to identify the most potent and selective compounds. The fluorine-18 PET agents were also put through a series of in vitro and in vivo studies including automated and manual electrophysiology, biodistribution, metabolism and autoradiography. Ultimately, we aimed to target specific VGSC sub-types that were highly expressed by specific aggressive tumour types. Following this study, an interesting VGSC PET agent was revealed that warrants further investigation

    Voltage-Gated Sodium Channel Modulation by a New Spider Toxin Ssp1a Isolated From an Australian Theraphosid

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    Given the important role of voltage-gated sodium (NaV) channel-modulating spider toxins in elucidating the function, pharmacology, and mechanism of action of therapeutically relevant NaV channels, we screened the venom from Australian theraphosid species against the human pain target hNaV1.7. Using assay-guided fractionation, we isolated a 33-residue inhibitor cystine knot (ICK) peptide (Ssp1a) belonging to the NaSpTx1 family. Recombinant Ssp1a (rSsp1a) inhibited neuronal hNaV subtypes with a rank order of potency hNaV1.7 > 1.6 > 1.2 > 1.3 > 1.1. rSsp1a inhibited hNaV1.7, hNaV1.2 and hNaV1.3 without significantly altering the voltage-dependence of activation, inactivation, or delay in recovery from inactivation. However, rSsp1a demonstrated voltage-dependent inhibition at hNaV1.7 and rSsp1a-bound hNaV1.7 opened at extreme depolarizations, suggesting rSsp1a likely interacted with voltage-sensing domain II (VSD II) of hNaV1.7 to trap the channel in its resting state. Nuclear magnetic resonance spectroscopy revealed key structural features of Ssp1a, including an amphipathic surface with hydrophobic and charged patches shown by docking studies to comprise the interacting surface. This study provides the basis for future structure-function studies to guide the development of subtype selective inhibitors

    The association of bacterial C9-based TTX-like compounds with Prorocentrum minimum opens new uncertainties about shellfish seafood safety

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    In 2012, Tetrodotoxin (TTX) was identified in mussels and linked to the presence of Prorocentrum minimum (P. minimum) in Greece. The connexion between TTX and P. minimum was further studied in this paper. First, the presence of TTX-producer bacteria, Vibrio and Pseudomonas spp, was confirmed in Greek mussels. In addition these samples showed high activity as inhibitors of sodium currents (INa). P. minimum was before associated with neurotoxic symptoms, however, the nature and structure of toxins produced by this dinoflagellate remains unknown. Three P. minimum strains, ccmp1529, ccmp2811 and ccmp2956, growing in different conditions of temperature, salinity and light were used to study the production of toxic compounds. Electrophysiological assays showed no effect of ccmp2811 strain on INa, while ccmp1529 and ccmp2956 strains were able to significantly reduce INa in the same way as TTX. In these samples two new compounds, m/z 265 and m/z 308, were identified and characterized by liquid chromatography tandem high-resolution mass spectrometry. Besides, two TTX-related bacteria, Roseobacter and Vibrio sp, were observed. These results show for the first time that P. minimum produce TTX-like compounds with a similar ion pattern and C9-base to TTX analogues and with the same effect on INaInés Rodríguez is supported by a fellowship from Subprograma de Formación de Personal Investigador MINECO (AGL2012-40185-CO2-01), Spain. The research leading to these results has received funding from the following FEDER cofunded-grants. From CDTI and Technological Funds, supported by Ministerio de Economía y Competitividad, AGL2012-40185-CO2-01, AGL2014-58210-R, and Consellería de Cultura, Educación e Ordenación Universitaria, GRC2013-016. From CDTI under ISIP Programme, Spain, IDI-20130304 APTAFOOD. From the European Union’s Seventh Framework Programme managed by REA – Research Executive Agency (FP7/2007-2013) under grant agreement 312184 PHARMASEA.S

    Caracterização eletrofisiológica da toxina TF1a purificada da peçonha do escorpião Tityus fasciolatus

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    Dissertação (mestrado)—Fundação Universidade de Brasília, Programa de Pós-Graduação em Biologia Animal, 2018.Os escorpiões, pertencentes ao filo Arthropoda, representam aproximadamente 1,5% das espécies presentes na classe dos aracnídeos. No Brasil, existem diversas espécies de escorpiões presentes em todas as regiões, dentre eles os que mais se destacam são os pertencentes ao gênero Tityus devido à sua grande distribuição geográfica e importância médica. Dentre eles, uma espécie que se destaca é o Tityus fasciolatus mais presente na região central do Brasil. Essa espécie de escorpião, assim como as outras, possui uma peçonha extremamente complexa formada por uma série de compostos, e dentre eles estão os peptídeos conhecidos como neurotoxinas capazes de interagir e afetar o funcionamento dos canais de sódio dependentes de voltagem, responsáveis pela iniciação e propagação dos potenciais de ação. A peçonha do escorpião Tityus fasciolatus foi coletada e submetida ao fracionamento utilizando a técnica de cromatografia líquida de alta eficiência em fase reversa (RP-HPLC). As frações obtidas foram analisadas em espectrômetro de massa (MALDI-TOF) a fim de identificar o peptídeo de interesse, que teve sua sequência determinada e sua atividade testada nos sete subtipos de canais de sódio de mamíferos e em canais de sódio de inseto e aracnídeo por meio da técnica de patch-clamp em configuração voltage clamp. O peptídeo purificado denominado Tf1a foi capaz de alterar a cinética de todos os subtipos de canais de mamífero e, ainda, agir sobre os canais de inseto e de aranha modificando também seu funcionamento. O efeito observado permite classificar a toxina Tf1a como uma β-toxina escorpiônica do tipo like. Sendo assim esse trabalho foi capaz de descrever uma nova neurotoxina purificada da peçonha de um escorpião e caracterizar a sua atividade em diversos tipos de canais iônicos, colaborando assim com o entendimento da ação destes peptídeos.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).Scorpions, belonging to the Arthropoda phylum, represent approximately 1.5% of the species present in the Arachnidae class. There are several species of scorpions present in all regions of Brazil, among them the ones that stand out most are those belonging to the genus Tityus thanks to its great geographic distribution and medical importance. Between them, one species that stands out is the Tityus fasciolatus most present in the central region of Brazil. This species of scorpion, like the others, has an extremely complex venom formed by a several compounds. Among them are the peptides also known as neurotoxins capable of interacting and affect the functioning of the voltagegated sodium channels, responsible for the initiation and propagation of the action potential. The venom of the scorpion Tityus fasciolatus was collected and submitted to fractionation using reverse phase high performance liquid chromatography (RP-HPLC) technique. The fractions obtained were analyzed by mass spectrometer (MALDI-TOF) to identify the peptide of interest, which had its sequence determined and its activity tested on the seven subtypes of mammalian sodium channels and on insect and arachnid sodium channels by the use of the patch-clamp technique in voltage clamp configuration. The purified peptide named Tf1a was able to change the kinetics of all subtypes of mammalian channels and also act on the insect and arachnid channels, modifying their normal functioning. The effects caused allows us to classify the toxin Tf1a as a β-like scorpion toxin. Thus, this work was able to describe a new neurotoxin purified from the venom of a scorpion and characterize its activity in several types of ion channels, thus collaborating with the understanding of the action of these peptides

    Busca pelo alvo molecular do peptídeo Ap1a isolado da peçonha da aranha caranguejeira Acanthoscurria paulensis

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    Dissertação (mestrado)—Universidade de Brasília, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2018.Aranhas possuem uma complexa variedade de componentes em sua peçonha. Dentre esses componentes, os peptídeos neurotóxicos destacam-se por sua capacidade de paralisar a presa por meio da interação com canais iônicos dependentes de voltagem e com receptores glutamatérgicos presentes no sistema nervoso central (SNC) e no sistema nervoso periférico (SNP). Estudos prévios no laboratório de Toxinologia da Universidade de Brasília (UnB) verificaram que a peçonha da aranha caranguejeira Acanthuscurria paulensis apresenta cerca de 100 compostos, distribuídos entre intervalos de massa de 635-21.895 Da, sendo a fração com maior abundância, de massa monoisotópica [M+H]+ = 5457,79 Da, denominada Ap1a. Foi evidenciando que o peptídeo Ap1a apresenta 48 resíduos de aminoácidos e três pontes dissulfeto. O objetivo do presente trabalho é descrever o(s) alvo(s) molecular(es) do peptídeo Ap1a, isolado da aranha A. paulensis, através de ensaios eletrofisiológicos em canais de Na+ e K+ , bem como analisar seu efeito em ensaios de ligação de [3,4 3 H]- L- Glutamato. Foram realizadas 48 cromatografias da peçonha bruta de A. paulensis para obtenção da fração de interesse, denominada Ap1a, a qual eluiu, aproximadamente, aos 41% de acetonitrila. Para purificação da fração de interesse realizou- se uma recromatografia com gradiente de acetonitrila otimizado. O peptídeo Ap1a, 1 μM, apresentou efeitos em todos os canais de sódio de mamíferos testados: hNav 1.1, hNav 1.3 e hNav 1.7, sendo que, ao se avaliar a ativação dos canais testados, o canal hNav 1.1 foi o que mais deslocou o potencial de ativação para voltagens mais hiperpolarizadas (ΔV1/2 = - 6,58 mV ± 0,72) quando em presença da Ap1a. Contudo, ao se analisar as alterações na amplitude da corrente iônica, o canal hNav 1.7 apresentou maior efeito (32,43% ± 4,93). O peptídeo Ap1a à 1 μM não apresentou efeito nos canais de sódio tanto de barata (Blattella germanica) quanto de ácaro (Varroa destructor). Além disso, nenhum bloqueio ou modulação nos canais potássio de inseto do tipo Shaker, Shab, Shal, Shaw e hKv 10.1 foi evidenciado. Ensaios de ligação de [3,4 3 H]- L- Glutamato foram realizados em triplicata na presença de concentrações crescentes do peptídeo Ap1a (0,01 a 1000 μM), todavia em nenhuma das concentrações utilizadas foi observado alguma diferença estatística significativa na ligação de [3,4 3 H]-L-glutamato. Com base nisso, o presente estudo contribuiu para a ampliação do conhecimento específico do peptídeo Ap1a, isolado da peçonha da A. paulenis, uma das inúmeras aranhas caranguejeiras endêmicas do Brasil, através da realização de uma ampla investigação eletrofisiológica em diversos canais iônicos dependentes de voltagem, bem como em receptores ionotrôpicos de glutamato.Spiders have venom composed by several compounds of different chemical classes, among them potentially neurotoxic peptides, which are essencial in the prey paralysis process acting in voltage-dependent ion channels and in glutamatergic receptors present in the central nervous system (CNS) and peripheral nervous system (PNS). Previous studies in the laboratório de Toxinologia da Universidade de Brasília (UnB) found that the venom of the tarantula Acanthuscurria paulensis presents about 100 compounds, distributed between mass intervals of 635-21,895 Da, being the fraction with greater abundance, of monoisotopic mass [ M + H] + = 5457.79 Da, designated Ap1a. It was also found that the Ap1a has 48 amino acid residues and three disulfide bonds. The aim of the present work is to describe the molecular target (s) of the Ap1a, isolated from the A. paulensis tarantula, through electrophysiological recordings on Na + and K + channels, as well as to analyze its effect in binding assays of [3,4 3 H] -L-glutamate. Ap1a was purified through chromatography of the crude venom followed by rechromatography. Ap1a, 1 μM, showed effects on all the mammalian sodium channels tested: hNav 1.1, hNav 1.3 and hNav 1.7, and, when evaluating the activation of the channels tested, hNav 1.1 was the one that displaced the most activation potential for more hyperpolarized voltages (ΔV1 / 2 = -6.58 mV ± 0.72) when in the presence of Ap1a. However, when analyzing the changes in the amplitude of the ionic current, hNav 1.7 had a greater effect (32.43% ± 4.93). Ap1a, 1 μM, also showed no effect on the sodium channels of both cockroach (Blattella germanica) and mite (Varroa destructor). In addition, no blockage or modulation in the Shaker, Shab, Shal and Shaw potassium channels was found. [3,4 3 H] -L-Glutamate binding assays were performed in triplicate in the presence of increasing concentrations of Ap1a (0.01 to 1000 μM), however at any of these concentrations used was no significant statistical difference in binding of [3,4 3 H] -L-glutamate. Based on this, the present study contributed to increase the specific knowledge of Ap1a, isolated from A. paulenis venom, one of the numerous endemic tarantula in Brazil, through a broad electrophysiological investigation in several voltage dependent ion channels, as well as in ionotropic glutamate receptors

    Inhibition of voltage-dependent sodium currents by cannabidiol

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    Voltage-gated sodium channels initiate action potentials in excitable tissues. Altering these channels’ function can lead to many pathophysiological conditions. The family of voltage-gated sodium channel genes encodes 10 proteins (including Nav2.1) distributed throughout the central and peripheral nervous systems, cardiac and skeletal muscles. The SCN4A gene encodes the Nav1.4 channel, which is primarily responsible for depolarization of the skeletal muscle fibers. Many mutations in SCN4A are found and associated with the myotonic syndromes and periodic paralyses. These conditions are both considered gain-of-function and can be severely life-limiting with respect to performing everyday tasks. From a broader standpoint, hyperexcitability presents as a significant problem in other tissues besides skeletal muscles. Gain-of-function in sodium channels has been linked to a wide-range of pathophysiological conditions such as inherited erythromelalgia, epilepsy, and arrhythmias. Treating these types of pathologies requires an in-depth understanding of their underlying mechanisms. One way to gain this understanding is to investigate physiological triggers. There is also a dire need for novel ways of reducing the hyperexcitability associated with mutant sodium channels. One promising compound is the non-psychotropic component of the Cannabis sativa plant, cannabidiol. This compound has recently been shown to modulate some of the neuronal sodium channels. Although cannabidiol has shown efficacy in clinical trials, the underlying mechanism of action remains unknown. Sodium channels could be among the molecular targets for cannabidiol.In my doctoral research: 1) I studied how a single missense mutation, P1158S, in Nav1.4 causes various degrees of gain-of-function (myotonia and periodic paralysis) by using pH changes to probe P1158S gating modifications; 2) I studied the inhibitory effects of cannabidiol on voltage-dependent sodium currents; 3) I investigated the mechanism through which cannabidiol imparts inhibition. Overall, these data reveal novel insights into sodium channel hyperexcitability and pharmacologically targeting of this hyperexcitability using cannabidiol
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