RESUMO
Phosphate (Pi) is an essential nutrient, and its plasma levels are under tight hormonal control. Uphill transport of Pi into cells is mediated by the two Na-dependent Pi transporter families SLC34 and SLC20. The molecular identity of a potential Pi export pathway is controversial, though XPR1 has recently been suggested by Giovannini and coworkers to mediate Pi export. We expressed XPR1 in Xenopus oocytes to determine its functional characteristics. Xenopus isoforms of proteins were used to avoid species incompatibility. Protein tagging confirmed the localization of XPR1 at the plasma membrane. Efflux experiments, however, failed to detect translocation of Pi attributable to XPR1. We tested various counter ions and export medium compositions (pH, plasma) as well as potential protein co-factors that could stimulate the activity of XPR1, though without success. Expression of truncated XPR1 constructs and individual domains of XPR1 (SPX, transmembrane core, C-terminus) demonstrated downregulation of the uptake of Pi mediated by the C-terminal domain of XPR1. Tethering the C-terminus to the transmembrane core changed the kinetics of the inhibition and the presence of the SPX domain blunted the inhibitory effect. Our observations suggest a regulatory role of XPR1 in cellular Pi handling rather than a function as Pi exporter. Accordingly, XPR1 senses intracellular Pi levels via its SPX domain and downregulates cellular Pi uptake via the C-terminal domain. The molecular identity of a potential Pi export protein remains therefore elusive.
Assuntos
Homeostase , Fosfatos , Animais , Humanos , Membrana Celular/metabolismo , Homeostase/fisiologia , Oócitos/metabolismo , Fosfatos/metabolismo , Xenopus laevis , Receptor do Retrovírus Politrópico e XenotrópicoRESUMO
G protein-coupled receptors are known to play a key role in many cellular signal transduction processes, including those mediating serotonergic signaling in the nervous system. Several factors have been shown to regulate the activity of these receptors, including membrane potential and the concentration of sodium ions. Whether voltage and sodium regulate the activity of serotonergic receptors is unknown. Here, we used Xenopus oocytes as an expression system to examine the effects of voltage and of sodium ions on the potency of one subtype of serotonin (5-hydroxytryptamine [5-HT]) receptor, the 5-HT1A receptor. We found that the potency of 5-HT in activating the receptor is voltage dependent and that it is higher at resting potential than under depolarized conditions. Furthermore, we found that removal of extracellular Na+ resulted in a decrease of 5-HT potency toward the 5-HT1A receptor and that a conserved aspartate in transmembrane domain 2 is crucial for this effect. Our results suggest that this allosteric effect of Na+ does not underlie the voltage dependence of this receptor. We propose that the characterization of modulatory factors that regulate this receptor may contribute to our future understanding of various physiological functions mediated by serotonergic transmission.
Assuntos
Receptor 5-HT1A de Serotonina , Sódio/química , Animais , Potenciais da Membrana , Oócitos , Receptor 5-HT1A de Serotonina/genética , Receptores Acoplados a Proteínas G , Serotonina/metabolismo , Serotonina/farmacologia , Xenopus laevisRESUMO
Cav 3.1 T-type Ca2+ channels play pivotal roles in neuronal low-threshold spikes, visceral pain, and pacemaker activity. Phosphorylation has been reported to potently regulate the activity and gating properties of Cav 3.1 channels. However, systematic identification of phosphorylation sites (phosphosites) in Cav 3.1 channel has been poorly investigated. In this work, we analyzed rat Cav 3.1 protein expressed in HEK-293 cells by mass spectrometry, identified 30 phosphosites located at the cytoplasmic regions, and illustrated them as a Cav 3.1 phosphorylation map which includes the reported mouse Cav 3.1 phosphosites. Site-directed mutagenesis of the phosphosites to Ala residues and functional analysis of the phospho-silent Cav 3.1 mutants expressed in Xenopus oocytes showed that the phospho-silent mutation of the N-terminal Ser18 reduced its current amplitude with accelerated current kinetics and negatively shifted channel availability. Remarkably, the phospho-silent mutations of the C-terminal Ser residues (Ser1924, Ser2001, Ser2163, Ser2166, or Ser2189) greatly reduced their current amplitude without altering the voltage-dependent gating properties. In contrast, the phosphomimetic Asp mutations of Cav 3.1 on the N- and C-terminal Ser residues reversed the effects of the phospho-silent mutations. Collectively, these findings demonstrate that the multiple phosphosites of Cav 3.1 at the N- and C-terminal regions play crucial roles in the regulation of the channel activity and voltage-dependent gating properties.
Assuntos
Canais de Cálcio Tipo T , Fosforilação , Animais , Humanos , Camundongos , Ratos , Células HEK293 , Cinética , Mutação , Canais de Cálcio Tipo T/metabolismo , Xenopus , Mutagênese Sítio-DirigidaRESUMO
Epithelial barriers constitute a fundamental requirement in every organism, as they allow the separation of different environments and set boundaries against noxious and other adverse effectors. In many inflammatory and degenerative diseases, epithelial barrier function is impaired because of a disturbance of the paracellular seal. Recently, the Xenopus laevis oocyte has been established as a heterologous expression model for the analysis of transmembrane tight junction protein interactions and is currently considered to be a suitable screening model for barrier effectors. A prerequisite for this application is a physiological anchoring of claudins to the cytoskeleton via the major scaffolding protein tjp1 (tight junction protein 1, ZO-1). We have analyzed the oocyte model with regard to the interaction of heterologously expressed claudins and tjp1. Our experiments have revealed endogenous tjp1 expression in protein and mRNA analyses of unfertilized Xenopus laevis oocytes expressing human claudin 1 (CLDN1) to claudin 5 (CLDN5). The amphibian cell model can therefore be used for the analysis of claudin interactions.
Assuntos
Claudinas , Oócitos , Animais , Humanos , Claudinas/genética , Claudinas/metabolismo , Xenopus laevis/metabolismo , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , Claudina-1/genética , Claudina-1/metabolismo , Oócitos/metabolismo , Junções Íntimas/metabolismoRESUMO
Aedes aegypti is responsible for transmitting a variety of arboviral infectious diseases such as dengue and chikungunya. Insecticides, particularly pyrethroids, are used widely for mosquito control. However, intensive used of pyrethroids has led to the selection of kdr mutations on sodium channels. L982W, locating in the PyR1 (Pyrethroid receptor site 1), was first reported in Ae. aegypti populations collected from Vietnam. Recently, the high frequency of L982W was detected in pyrethroid-resistant populations of Vietnam and Cambodia, and also concomitant mutations L982W + F1534C was detected in both countries. However, the role of L982W in pyrethroid resistance remains unclear. In this study, we examined the effects of L982W on gating properties and pyrethroid sensitivity in Xenopus oocytes. We found that mutations L982W and L982W + F1534C shifted the voltage dependence of activation in the depolarizing direction, however, neither mutations altered the voltage dependence of inactivation. L982W significantly reduced channel sensitivity to Type I pyrethroids, permethrin and bifenthrin, and Type II pyrethroids, deltamethrin and cypermethrin. No enhancement was observed when synergized with F1534C. In addition, L982W and L982W + F1534C mutations reduced the channel sensitivity to DDT. Our results illustrate the molecular basis of resistance mediates by L982W mutation, which will be helpful to understand the interacions of pyrethroids or DDT with sodium channels and develop molecular markers for monitoring pest resistance to pyrethroids and DDT.
Assuntos
Aedes , Inseticidas , Piretrinas , Animais , DDT/farmacologia , Leucina , Piretrinas/farmacologia , Inseticidas/farmacologia , Canais de Sódio/genética , Mutação , Resistência a Inseticidas/genética , Aedes/genética , Mosquitos Vetores/genéticaRESUMO
BACKGROUND: In insects, airborne chemical signals are mainly detected by two receptor families, odorant receptors (ORs) and ionotropic receptors (IRs). Functions of ORs have been intensively investigated in Diptera and Lepidoptera, while the functions and evolution of the more ancient IR family remain largely unexplored beyond Diptera. RESULTS: Here, we identified a repertoire of 26 IRs from transcriptomes of female and male antennae, and ovipositors in the moth Agrotis segetum. We observed that a large clade formed by IR75p and IR75q expansions is closely related to the acid-sensing IRs identified in Diptera. We functionally assayed each of the five AsegIRs from this clade using Xenopus oocytes and found that two receptors responded to the tested ligands. AsegIR75p.1 responded to several compounds but hexanoic acid was revealed to be the primary ligand, and AsegIR75q.1 responded primarily to octanoic acid, and less so to nonanoic acid. It has been reported that the C6-C10 medium-chain fatty acids repel various insects including many drosophilids and mosquitos. We show that the C6-C10 medium-chain fatty acids elicited antennal responses of both sexes of A. segetum, while only octanoic acid had repellent effect to the moths in a behavioral assay. In addition, using fluorescence in situ hybridization, we demonstrated that the five IRs and their co-receptor AsegIR8a are not located in coeloconic sensilla as found in Drosophila, but in basiconic or trichoid sensilla. CONCLUSIONS: Our results significantly expand the current knowledge of the insect IR family. Based on the functional data in combination with phylogenetic analysis, we propose that subfunctionalization after gene duplication plays an important role in the evolution of ligand specificities of the acid-sensing IRs in Lepidoptera.
Assuntos
Brassica napus , Dípteros , Mariposas , Receptores Odorantes , Animais , Antenas de Artrópodes , Caprilatos , Dípteros/genética , Feminino , Hibridização in Situ Fluorescente , Proteínas de Insetos/genética , Ligantes , Masculino , Mariposas/genética , Filogenia , Receptores Odorantes/genéticaRESUMO
Epilepsy is a neurological disorder characterized by abnormal neuronal excitability, with glutamate playing a key role as the predominant excitatory neurotransmitter involved in seizures. Animal models of epilepsy are crucial in advancing epilepsy research by faithfully replicating the diverse symptoms of this disorder. In particular, the GASH/Sal (genetically audiogenic seizure-prone hamster from Salamanca) model exhibits seizures resembling human generalized tonic-clonic convulsions. A single nucleotide polymorphism (SNP; C9586732T, p.His289Tyr) in the Grik1 gene (which encodes the kainate receptor GluK1) has been previously identified in this strain. The H289Y mutation affects the amino-terminal domain of GluK1, which is related to the subunit assembly and trafficking. We used confocal microscopy in Xenopus oocytes to investigate how the H289Y mutation, compared to the wild type (WT), affects the expression and cell-surface trafficking of GluK1 receptors. Additionally, we employed the two-electrode voltage-clamp technique to examine the functional effects of the H289Y mutation. Our results indicate that this mutation increases the expression and incorporation of GluK1 receptors into an oocyte's membrane, enhancing kainate-evoked currents, without affecting their functional properties. Although further research is needed to fully understand the molecular mechanisms responsible for this epilepsy, the H289Y mutation in GluK1 may be part of the molecular basis underlying the seizure-prone circuitry in the GASH/Sal model.
Assuntos
Epilepsia Reflexa , Cricetinae , Animais , Humanos , Xenopus laevis/metabolismo , Epilepsia Reflexa/genética , Convulsões/metabolismo , Receptores de Ácido Caínico/metabolismo , Oócitos/metabolismoRESUMO
The lateral line (LL) is a sensory system that allows fish and amphibians to detect water currents. LL responsiveness is modulated by efferent neurons that aid in distinguishing between external and self-generated stimuli, maintaining sensitivity to relevant cues. One component of the efferent system is cholinergic, the activation of which inhibits afferent activity. LL hair cells (HCs) share structural, functional, and molecular similarities with those of the cochlea, making them a popular model for studying human hearing and balance disorders. Because of these commonalities, one could propose that the receptor at the LL efferent synapse is a α9α10 nicotinic acetylcholine receptor (nAChR). However, the identities of the molecular players underlying ACh-mediated inhibition in the LL remain unknown. Surprisingly, through the analysis of single-cell expression studies and in situ hybridization, we describe that α9, but not the α10, subunits are enriched in zebrafish HCs. Moreover, the heterologous expression of zebrafish α9 subunits indicates that homomeric receptors are functional and exhibit robust ACh-gated currents blocked by α-bungarotoxin and strychnine. In addition, in vivo Ca2+ imaging on mechanically stimulated zebrafish LL HCs show that ACh elicits a decrease in evoked Ca2+ signals, regardless of HC polarity. This effect is blocked by both α-bungarotoxin and apamin, indicating coupling of ACh-mediated effects to small-conductance Ca2+-activated potassium (SKs) channels. Our results indicate that an α9-containing (α9*) nAChR operates at the zebrafish LL efferent synapse. Moreover, the activation of α9* nAChRs most likely leads to LL HC hyperpolarization served by SK channels.SIGNIFICANCE STATEMENT The fish lateral line (LL) mechanosensory system shares structural, functional, and molecular similarities with those of the mammalian cochlea. Thus, it has become an accessible model for studying human hearing and balance disorders. However, the molecular players serving efferent control of LL hair cell (HC) activity have not been identified. Here we demonstrate that, different from the hearing organ of vertebrate species, a nicotinic acetylcholine receptor composed only of α9 subunits operates at the LL efferent synapse. Activation of α9-containing receptors leads to LL HC hyperpolarization because of the opening of small-conductance Ca2+-activated potassium channels. These results will further aid in the interpretation of data obtained from LL HCs as a model for cochlear HCs.
Assuntos
Vias Eferentes/fisiologia , Sistema da Linha Lateral/fisiologia , Sistema Nervoso Parassimpático/fisiologia , Sinapses/fisiologia , Animais , Bungarotoxinas/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Regulação da Expressão Gênica , Células Ciliadas Auditivas/fisiologia , Antagonistas Nicotínicos/farmacologia , Oócitos , Estimulação Física , Receptores Nicotínicos/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Baixa/efeitos dos fármacos , Estricnina/farmacologia , Xenopus , Peixe-ZebraRESUMO
The dopamine D2 receptor (D2 R) functions as an autoreceptor on dopaminergic cell bodies and terminals and as a postsynaptic receptor on a variety of neurons in the central nervous system. As a result of alternative splicing, the D2 R is expressed as two isoforms: long (D2L R) and short (D2S R) differing by a stretch of 29 residues in the third intracellular loop, with D2S R being the predominant presynaptic isoform. Recent reports described a Ca2+ sensitivity of the desensitization time course of potassium currents elicited via D2S R, but not via D2L R, when either isoform was selectively expressed in dopaminergic neurons. Here, we aimed to study the mechanism behind this subtype-specific Ca2+ sensitivity. Thus, we measured the desensitization of potassium channel responses evoked by D2L R and D2S R using two-electrode voltage clamp in Xenopus oocytes in the absence and presence of different amounts of ß-arrestin2 and G protein-coupled receptor kinase-2 (GRK2), both of which are known to play important roles in D2 R desensitization in native cells. We found that co-expression of both GRK2 and ß-arrestin2 was necessary for reconstitution of the Ca2+ sensitivity of D2S R desensitization, while D2L R did not display Ca2+ sensitivity under these conditions. The effect of Ca2+ chelation by BAPTA-AM to slow the rate of D2S R desensitization was mimicked by the GRK2 inhibitor, Cmpd101, and by the kinase-inactivating GRK2 mutation, K220R, but not by the PKC inhibitor, Gö6976, nor by the calmodulin antagonist, KN-93. Thus, Ca2+ -sensitive desensitization of D2S R appears to be mediated via a GRK2 phosphorylation-dependent mechanism.
Assuntos
Cálcio/metabolismo , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Oócitos/metabolismo , Receptores de Dopamina D2/metabolismo , beta-Arrestina 2/metabolismo , Animais , Células Cultivadas , Feminino , Oócitos/citologia , Xenopus laevisRESUMO
The P2X5 receptor, an ATP-gated cation channel, is believed to be involved in tumor development, inflammatory bone loss and inflammasome activation after bacterial infection. Therefore, it is a worthwhile pharmacological target to treat the corresponding diseases, especially in minority populations that have a gene variant coding for functional homotrimeric P2X5 channels. Here, we investigated the effects of dihydropyridines on the human full-length P2X5 receptor (hP2X5FL) heterologously expressed in Xenopus oocytes using the two-microelectrode voltage clamp method. Agonist dependency, kinetics and permeation behavior, including Cl- permeability, were similar to hP2X5FL expressed in HEK293 or 1321N1 cells. Additionally, 1,4-dihydropyridines have been shown to interact with various other purinergic receptors, and we have examined them as potential hP2X5 modulators. Of seven commercially available and four newly synthesized dihydropyridines tested at hP2X5FL, only amlodipine exerted an inhibitory effect, but only at a high concentration of 300 µM. Isradipine and-even more-nimodipine stimulated ATP-induced currents in the low micromolar range. We conclude that common dihydropyridines or four new derivatives of amlodipine are not suitable as hP2X5 antagonists, but amlodipine might serve as a lead for future synthesis to increase its affinity. Furthermore, a side effect of nimodipine therapy could be a stimulatory effect on inflammatory processes.
Assuntos
Di-Hidropiridinas , Trifosfato de Adenosina/farmacologia , Di-Hidropiridinas/farmacologia , Células HEK293 , Humanos , Técnicas de Patch-Clamp , Receptores PurinérgicosRESUMO
We report the case of a patient with severe progressive epilepsy and peripheral neuropathy and a novel de novo inactivating variant (p.E79X) in Protein Kinase D1 (PKD1). Using CRISPR/Cas9, we engineered the homologous variant in mice and showed that in the homozygote mouse, it recapitulated the patient peripheral nerve hypermyelination pathology. The lethality of the homozygote mouse prevented us from performing an assessment of locomotor behavior. The mutant heterozygote mouse; however, exhibited a significant increase in kainate-induced seizure activity over wild-type mice, supporting the hypothesis that the PKD1 variant is a candidate for the cause of the patient epilepsy. Because PKD1 was previously identified in a kinomic screen as an interacting partner of the K-Cl cotransporter 3 (KCC3), and since KCC3 is involved in peripheral nerve disease and brain hyperexcitability, one possible mechanism of action of PKD1 in disease is through KCC3. We show that catalytically inactive PKD1 stimulates KCC3 activity, consistent with tonic relief of inhibitory phosphorylation. Our findings implicate a novel role for PKD1 in the human nervous system, and uncover a mechanism that could serve as a potential target to promote nervous system myelination.
Assuntos
Epilepsia/genética , Bainha de Mielina , Doenças do Sistema Nervoso Periférico/etiologia , Proteína Quinase C/genética , Animais , Criança , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Bainha de Mielina/metabolismo , Bainha de Mielina/patologia , Oócitos/metabolismo , Doenças do Sistema Nervoso Periférico/genética , Potássio/metabolismo , Proteína Quinase C/metabolismo , Teste de Desempenho do Rota-Rod , Convulsões/induzido quimicamente , Convulsões/genética , Simportadores/genética , Simportadores/metabolismo , Xenopus laevisRESUMO
The water channel aquaporin 2 (AQP2) is responsible for water reabsorption by kidney collecting duct cells. A substitution of amino acid leucine 137 to proline in AQP2 (AQP2-L137P) causes Nephrogenic Diabetes Insipidus (NDI). This study aimed to determine the cell biological consequences of this mutation on AQP2 function. Studies were performed in HEK293 and MDCK type I cells, transfected with wildtype (WT) AQP2 or an AQP2-L137P mutant. AQP2-L137P was predominantly detected as a high-mannose form of AQP2, whereas AQP2-WT was observed in both non-glycosylated and complex glycosylated forms. In contrast to AQP2-WT, the AQP2-L137P mutant did not accumulate on the apical plasma membrane following stimulation with forskolin. Ubiquitylation of AQP2-L137P was different from AQP2-WT, with predominance of non-distinct protein bands at various molecular weights. The AQP2-L137P mutant displayed reduced half-life compared to AQP2-WT. Treatment of cells with chloroquine increased abundance of AQP2-WT, but not AQP2-L137P. In contrast, treatment with MG132 increased abundance of AQP2-L137P but not AQP2-WT. Xenopus oocytes injected with AQP2-WT had increased osmotic water permeability when compared to AQP2-L137P, which correlated with lack of the mutant form in the plasma membrane. From the localization of the mutation and nature of the substitution it is likely that AQP2-L137P causes protein misfolding, which may be responsible for the observed functional defects. The data suggest that the L137P mutation results in altered AQP2 protein maturation, increased AQP2 degradation via the proteasomal pathway and limited plasma membrane expression. These combined mechanisms are likely responsible for the phenotype observed in this class of NDI patients.
Assuntos
Aquaporina 2/genética , Permeabilidade da Membrana Celular/genética , Diabetes Insípido Nefrogênico/genética , Diabetes Insípido Nefrogênico/patologia , Túbulos Renais Coletores/patologia , Animais , Linhagem Celular , Cloroquina/farmacologia , Cães , Células HEK293 , Humanos , Leupeptinas/farmacologia , Células Madin Darby de Rim Canino , Oócitos/fisiologia , Dobramento de Proteína , Deficiências na Proteostase/genética , Ubiquitinação/genética , Xenopus laevisRESUMO
The forward (kon) and reverse (koff) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon estimates correlated well with EC50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D2R agonist candidate drugs.
Assuntos
Agonistas de Dopamina/metabolismo , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Serina/metabolismo , 8-Hidroxi-2-(di-n-propilamino)tetralina/farmacologia , Animais , Sequência Conservada , Dopamina/metabolismo , Agonistas de Dopamina/química , Humanos , Cinética , Proteínas Mutantes/metabolismo , Mutação/genética , Fenetilaminas/farmacologia , Ligação Proteica , Relação Estrutura-Atividade , Tiramina/metabolismo , Xenopus laevisRESUMO
Fritillaria bulbs are used in Traditional Chinese Medicine to treat several illnesses. Peimine (Pm), an anti-inflammatory compound from Fritillaria, is known to inhibit some voltage-dependent ion channels and muscarinic receptors, but its interaction with ligand-gated ion channels remains unexplored. We have studied if Pm affects nicotinic acetylcholine receptors (nAChRs), since they play broad functional roles, both in the nervous system and non-neuronal tissues. Muscle-type nAChRs were incorporated to Xenopus oocytes and the action of Pm on the membrane currents elicited by ACh (IAChs) was assessed. Functional studies were combined with virtual docking and molecular dynamics assays. Co-application of ACh and Pm reversibly blocked IACh, with an IC50 in the low micromolar range. Pm inhibited nAChR by: (i) open-channel blockade, evidenced by the voltage-dependent inhibition of IAch, (ii) enhancement of nAChR desensitization, revealed by both an accelerated IACh decay and a decelerated IACh deactivation, and (iii) resting-nAChR blockade, deduced from the IACh inhibition elicited by Pm when applied before ACh superfusion. In good concordance, virtual docking and molecular dynamics assays demonstrated that Pm binds to different sites at the nAChR, mostly at the transmembrane domain. Thus, Pm from Fritillaria bulbs, considered therapeutic herbs, targets nAChRs with high affinity, which might account for its anti-inflammatory actions.
Assuntos
Anti-Inflamatórios/farmacologia , Cevanas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Músculos/efeitos dos fármacos , Oócitos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Medicamentos de Ervas Chinesas/farmacologia , Músculos/metabolismo , Oócitos/metabolismo , Receptores Nicotínicos/genética , Xenopus laevisRESUMO
Many retinal diseases are associated with pathological cell swelling, but the underlying etiology remains to be established. A key component of the volume-sensitive machinery, the transient receptor potential vanilloid 4 (TRPV4) ion channel, may represent a sensor and transducer of cell swelling, but the molecular link between the swelling and TRPV4 activation is unresolved. Here, our results from experiments using electrophysiology, cell volumetric measurements, and fluorescence imaging conducted in murine retinal cells and Xenopus oocytes indicated that cell swelling in the physiological range activated TRPV4 in Müller glia and Xenopus oocytes, but required phospholipase A2 (PLA2) activity exclusively in Müller cells. Volume-dependent TRPV4 gating was independent of cytoskeletal rearrangements and phosphorylation. Our findings also revealed that TRPV4-mediated transduction of volume changes is dependent by its N terminus, more specifically by its distal-most part. We conclude that the volume sensitivity and function of TRPV4 in situ depend critically on its functional and cell type-specific interactions.
Assuntos
Células Ependimogliais/metabolismo , Ativação do Canal Iônico/fisiologia , Neuroglia/metabolismo , Oócitos/metabolismo , Canais de Cátion TRPV/metabolismo , Animais , Tamanho Celular , Células Ependimogliais/citologia , Feminino , Ativação do Canal Iônico/genética , Camundongos , Neuroglia/citologia , Neurônios/citologia , Neurônios/metabolismo , Oócitos/citologia , Técnicas de Patch-Clamp , Fosfolipases A2/metabolismo , Fosforilação , Ratos , Canais de Cátion TRPV/genética , Xenopus laevisRESUMO
Mutations in the KCNK18 gene that encodes the TRESK K2P potassium channel have previously been linked with typical familial migraine with aura. Recently, an atypical clinical case has been reported in which a male individual carrying the p.Trp101Arg (W101R) missense mutation in the KCNK18 gene was diagnosed with intellectual disability and migraine with brainstem aura. Here we report the functional characterization of this new missense variant. This mutation is located in a highly conserved residue close to the selectivity filter, and our results show although these mutant channels retain their K+ selectivity and calcineurin-dependent regulation, the variant causes an overall dramatic loss of TRESK channel function as well as an initial dominant-negative effect when co-expressed with wild-type channels in Xenopus laevis oocytes. The dramatic functional consequences of this mutation thereby support a potentially pathogenic role for this variant and provide further insight into the relationship between the structure and function of this ion channel.
Assuntos
Deficiência Intelectual/genética , Transtornos de Enxaqueca/genética , Mutação de Sentido Incorreto/genética , Canais de Potássio/genética , Animais , Calcineurina/genética , Humanos , Deficiência Intelectual/metabolismo , Potenciais da Membrana/genética , Transtornos de Enxaqueca/metabolismo , Oócitos/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismoRESUMO
HKT1;5 loci/alleles are important determinants of crop salinity tolerance. HKT1;5s encode plasmalemma-localized Na+ transporters, which move xylem Na+ into xylem parenchyma cells, reducing shoot Na+ accumulation. Allelic variation in rice OsHKT1;5 sequence in specific landraces (Nona Bokra OsHKT1;5-NB/Nipponbare OsHKT1;5-Ni) correlates with variation in salt tolerance. Oryza coarctata, a halophytic wild rice, grows in fluctuating salinity at the seawater-estuarine interface in Indian and Bangladeshi coastal regions. The distinct transport characteristics of the shoots and roots expressing the O. coarctata OcHKT1;5 transporter are reported vis-à-vis OsHKT1;5-Ni. Yeast sodium extrusion-deficient cells expressing OcHKT1;5 are sensitive to increasing Na+ (10-100 mM). Electrophysiological measurements in Xenopus oocytes expressing O. coarctata or rice HKT1;5 transporters indicate that OcHKT1;5, like OsHKT1;5-Ni, is a Na+-selective transporter, but displays 16-fold lower affinity for Na+ and 3.5-fold higher maximal conductance than OsHKT1;5-Ni. For Na+ concentrations >10 mM, OcHKT1;5 conductance is higher than that of OsHKT1;5-Ni, indicating the potential of OcHKT1;5 for increasing domesticated rice salt tolerance. Homology modeling/simulation suggests that four key amino-acid changes in OcHKT1;5 (in loops on the extracellular side; E239K, G207R, G214R, L363V) account for its lower affinity and higher Na+ conductance vis-à-vis OsHKT1;5-Ni. Of these, E239K in OcHKT1;5 confers lower affinity for Na+ transport, as evidenced by Na+ transport assays of reciprocal site-directed mutants for both transporters (OcHKT1;5-K239E, OsHKT1;5-Ni-E270K) in Xenopus oocytes. Both transporters have likely analogous roles in xylem sap desalinization, and differences in xylem sap Na+ concentrations in both species are attributed to differences in Na+ transport affinity/conductance between the transporters.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Aminoácidos , Animais , Proteínas de Transporte de Cátions/genética , Membrana Celular/metabolismo , Oócitos/metabolismo , Organismos Geneticamente Modificados , Oryza/genética , Proteínas de Plantas/genética , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Homologia de Sequência de Aminoácidos , Sódio/metabolismo , Xenopus , Xilema/metabolismoRESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease affecting motor neurons that leads to progressive paralysis of skeletal muscle. Studies of ALS have revealed defects in expression of acetylcholine receptors (AChRs) in skeletal muscle that occur even in the absence of motor neuron anomalies. The endocannabinoid palmitoylethanolamide (PEA) modified the clinical conditions in one ALS patient, improving muscle force and respiratory efficacy. By microtransplanting muscle membranes from selected ALS patients into Xenopus oocytes, we show that PEA reduces the desensitization of acetylcholine-evoked currents after repetitive neurotransmitter application (i.e., rundown). The same effect was observed using muscle samples from denervated (non-ALS) control patients. The expression of human recombinant α1ß1γδ (γ-AChRs) and α1ß1εδ AChRs (ε-AChRs) in Xenopus oocytes revealed that PEA selectively affected the rundown of ACh currents in ε-AChRs. A clear up-regulation of the α1 subunit in muscle from ALS patients compared with that from non-ALS patients was found by quantitative PCR, but no differential expression was found for other subunits. Clinically, ALS patients treated with PEA showed a lower decrease in their forced vital capacity (FVC) over time as compared with untreated ALS patients, suggesting that PEA can enhance pulmonary function in ALS. In the present work, data were collected from a cohort of 76 ALS patients and 17 denervated patients. Our results strengthen the evidence for the role of skeletal muscle in ALS pathogenesis and pave the way for the development of new drugs to hamper the clinical effects of the disease.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Etanolaminas/uso terapêutico , Terapia de Alvo Molecular , Músculo Esquelético/efeitos dos fármacos , Ácidos Palmíticos/uso terapêutico , Receptores Nicotínicos/efeitos dos fármacos , Potenciais de Ação/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Amidas , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/fisiopatologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/transplante , Etanolaminas/farmacologia , Feminino , Humanos , Masculino , Microinjeções , Pessoa de Meia-Idade , Denervação Muscular , Músculo Esquelético/ultraestrutura , Junção Neuromuscular/fisiopatologia , Oócitos , Ácidos Palmíticos/farmacologia , Receptores Nicotínicos/fisiologia , Proteínas Recombinantes de Fusão/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Método Simples-Cego , Xenopus laevisRESUMO
We previously published a series of 8-methoxypirazolo[1,5-a]quinazolines (PQs) and their 4,5-dihydro derivatives (4,5(H)PQ) bearing the (hetero)arylalkylester group at position 3 as ligands at the γ-aminobutyric type A (GABAA) subtype receptor. Continuing the study in this field, we report here the design and synthesis of 3-(hetero)arylpyrazolo[1,5-a]quinazoline and 3-(hetero)aroylpyrazolo[1,5-a]quinazoline 8-methoxy substituted as interesting analogs of the above (hetero)arylalkylester, in which the shortening or the removal of the linker between the 3-(hetero)aryl ring and the PQ was performed. Only compounds that are able to inhibit radioligand binding by more than 80% at 10 µM have been selected for electrophysiological studies on recombinant α1ß2γ2L GABAA receptors. Some compounds show a promising profile. For example, compounds 6a and 6b are able to modulate the GABAAR in an opposite manner, since 6b enhances and 6a reduces the variation of the chlorine current, suggesting that they act as a partial agonist and an inverse partial agonist, respectively. The most potent derivative was 3-(4-methoxyphenylcarbonyl)-8-methoxy-4,5-dihydropyrazolo[1,5-a] quinazoline 11d, which reaches a maximal activity at 1 µM (+54%), and it enhances the chlorine current at ≥0.01 µM. Finally, compound 6g, acting as a null modulator at α1ß2γ2L, shows the ability to antagonize the full agonist diazepam and the potentiation of CGS 9895 on the new α+/ß- 'non-traditional' benzodiazepine site.
Assuntos
Agonistas de Receptores de GABA-A/síntese química , Antagonistas de Receptores de GABA-A/síntese química , Pirazóis/química , Quinazolinas/química , Receptores de GABA-A/química , Animais , Sítios de Ligação , Células Cultivadas , Técnicas de Química Sintética , Agonistas de Receptores de GABA-A/química , Agonistas de Receptores de GABA-A/farmacologia , Antagonistas de Receptores de GABA-A/química , Antagonistas de Receptores de GABA-A/farmacologia , Ligantes , Estrutura Molecular , Ligação Proteica , Pirazóis/farmacologia , Quinazolinas/farmacologiaRESUMO
Epilepsy is one of the most common chronic neurological diseases, and its pharmacological treatment holds great importance for both physicians and national authorities, especially considering the high proportion of drug-resistant patients (about 30%). Lacosamide (LCM) is an effective and well-tolerated new-generation antiepileptic drug (AED), currently licensed as add-on therapy for partial-onset seizures. However, LCM mechanism of action is still a matter of debate, although its effect on the voltage sensitive sodium channels is by far the most recognized. This study aimed to retrospectively analyze a cohort of 157 drug-resistant patients treated with LCM to describe the most common and effective therapeutic combinations and to investigate if the LCM can affect also GABAA-mediated neurotransmission as previously shown for levetiracetam (LEV). In our cohort, LEV resulted the compound most frequently associated with LCM in the responder subgroup. We therefore translated this clinical observation into the laboratory bench by taking advantage of the technique of "membrane micro-transplantation" in Xenopus oocytes and electrophysiological approaches to study human GABAA-evoked currents. In cortical brain tissues from refractory epileptic patients, we found that LCM reduces the use-dependent GABA impairment (i.e., "rundown") that it is considered one of the specific hallmarks of drug-resistant epilepsies. Notably, in line with our clinical observations, we found that the co-treatment with subthreshold concentrations of LCM and LEV, which had no effect on GABAA currents on their own, reduced GABA impairment in drug-resistant epileptic patients, and this effect was blocked by PKC inhibitors. Our findings demonstrate, for the first time, that LCM targets GABAA receptors and that it can act synergistically with LEV, improving the GABAergic function. This novel mechanism might contribute to explain the clinical efficacy of LCM-LEV combination in several refractory epileptic patients.