RESUMO
The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.
Assuntos
Cálcio , Receptores Nicotínicos , Animais , Ratos , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Sequência de Aminoácidos , Sítios de Ligação , Cálcio/metabolismo , Simulação de Dinâmica Molecular , Técnicas de Patch-Clamp , Subunidades Proteicas/metabolismo , Subunidades Proteicas/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/química , Xenopus laevisRESUMO
Growth hormone (GH) binding to GH receptor activates janus kinase 2 (JAK2)-signal transducer and activator of transcription 5b (STAT5b) pathway, which stimulates transcription of insulin-like growth factor-1 (IGF1), insulin-like growth factor binding protein 3 (IGFBP3) and insulin-like growth factor acid-labile subunit (IGFALS). Although STAT5B deficiency was established as an autosomal recessive disorder, heterozygous dominant-negative STAT5B variants have been reported in patients with less severe growth deficit and milder immune dysfunction. We developed an in vivo functional assay in zebrafish to characterize the pathogenicity of three human STAT5B variants (p.Ala630Pro, p.Gln474Arg and p.Lys632Asn). Overexpression of human wild-type (WT) STAT5B mRNA and its variants led to a significant reduction of body length together with developmental malformations in zebrafish embryos. Overexpression of p.Ala630Pro, p.Gln474Arg or p.Lys632Asn led to an increased number of embryos with pericardial edema, cyclopia and bent spine compared with WT STAT5B. Although co-injection of WT and p.Gln474Arg and WT and p.Lys632Asn STAT5B mRNA in zebrafish embryos partially or fully rescues the length and the developmental malformations in zebrafish embryos, co-injection of WT and p.Ala630Pro STAT5B mRNA leads to a greater number of embryos with developmental malformations and a reduction in body length of these embryos. These results suggest that these variants could interfere with endogenous stat5.1 signaling through different mechanisms. In situ hybridization of zebrafish embryos overexpressing p.Gln474Arg and p.Lys632Asn STAT5B mRNA shows a reduction in igf1 expression. In conclusion, our study reveals the pathogenicity of the STAT5B variants studied.
Assuntos
Fator de Transcrição STAT5 , Peixe-Zebra , Animais , Humanos , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Fator de Transcrição STAT5/genética , Fator de Transcrição STAT5/metabolismo , Hormônio do Crescimento , Transdução de Sinais/genética , RNA Mensageiro , Fator de Crescimento Insulin-Like I/genéticaRESUMO
Vertebrate hair cell (HC) systems are innervated by efferent fibers that modulate their response to external stimuli. In mammals, the best studied efferent-HC synapse, the cholinergic medial olivocochlear (MOC) efferent system, makes direct synaptic contacts with HCs. The net effect of MOC activity is to hyperpolarize HCs through the activation of α9α10 nicotinic cholinergic receptors (nAChRs) and the subsequent activation of Ca2+-dependent SK2 potassium channels. A serious obstacle in research on many mammalian sensory systems in their native context is that their constituent neurons are difficult to access even in newborn animals, hampering circuit observation, mapping, or controlled manipulation. By contrast, fishes and amphibians have a superficial and accessible mechanosensory system, the lateral line (LL), which circumvents many of these problems. LL responsiveness is modulated by efferent neurons which aid to distinguish between external and self-generated stimuli. One component of the LL efferent system is cholinergic and its activation inhibits LL afferent activity, similar to what has been described for MOC efferents. The zebrafish (Danio rerio) has emerged as a powerful model system for studying human hearing and balance disorders, since LL HC are structurally and functionally analogous to cochlear HCs, but are optically and pharmacologically accessible within an intact specimen. Complementing mammalian studies, zebrafish have been used to gain significant insights into many facets of HC biology, including mechanotransduction and synaptic physiology as well as mechanisms of both hereditary and acquired HC dysfunction. With the rise of the zebrafish LL as a model in which to study auditory system function and disease, there has been an increased interest in studying its efferent system and evaluate the similarity between mammalian and piscine efferent synapses. Advances derived from studies in zebrafish include understanding the effect of the LL efferent system on HC and afferent activity, and revealing that an α9-containing nAChR, functionally coupled to SK channels, operates at the LL efferent synapse. In this review, we discuss the tools and findings of these recent investigations into zebrafish efferent-HC synapse, their commonalities with the mammalian counterpart and discuss several emerging areas for future studies.
RESUMO
The α9α10 nicotinic acetylcholine receptor (nAChR) plays a fundamental role in inner ear physiology. It mediates synaptic transmission between efferent olivocochlear fibers that descend from the brainstem and hair cells of the auditory sensory epithelium. The α9 and α10 subunits have undergone a distinct evolutionary history within the family of nAChRs. Predominantly in mammalian vertebrates, the α9α10 receptor has accumulated changes at the protein level that may ultimately relate to the evolutionary history of the mammalian hearing organ. In the present work, we investigated the responses of α9α10 nAChRs to choline, the metabolite of acetylcholine degradation at the synaptic cleft. Whereas choline is a full agonist of chicken α9α10 receptors it is a partial agonist of the rat receptor. Making use of the expression of α9α10 heterologous receptors, encompassing wild-type, heteromeric, homomeric, mutant, chimeric, and hybrid receptors, and in silico molecular docking, we establish that the mammalian (rat) α10 nAChR subunit underscores the reduced efficacy of choline. Moreover, we show that whereas the complementary face of the α10 subunit does not play an important role in the activation of the receptor by ACh, it is strictly required for choline responses. Thus, we propose that the evolutionary changes acquired in the mammalian α9α10 nAChR resulted in the loss of choline acting as a full agonist at the efferent synapse, without affecting the triggering of ACh responses. This may have accompanied the fine-tuning of hair cell post-synaptic responses to the high-frequency activity of efferent medial olivocochlear fibers that modulate the cochlear amplifier.
RESUMO
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 expansion and pruning of ion channel families has played a crucial role in the evolution of nervous systems. Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels with distinct roles in synaptic transmission at the neuromuscular junction, the central and peripheral nervous system, and the inner ear. Remarkably, the complement of nAChR subunits has been highly conserved along vertebrate phylogeny. To ask whether the different subtypes of receptors underwent different evolutionary trajectories, we performed a comprehensive analysis of vertebrate nAChRs coding sequences, mouse single-cell expression patterns, and comparative functional properties of receptors from three representative tetrapod species. We found significant differences between hair cell and neuronal receptors that were most likely shaped by the differences in coexpression patterns and coassembly rules of component subunits. Thus, neuronal nAChRs showed high degree of coding sequence conservation, coupled to greater coexpression variance and conservation of functional properties across tetrapod clades. In contrast, hair cell α9α10 nAChRs exhibited greater sequence divergence, narrow coexpression pattern, and great variability of functional properties across species. These results point to differential substrates for random change within the family of gene paralogs that relate to the segregated roles of nAChRs in synaptic transmission.
Assuntos
Evolução Molecular , Receptores Nicotínicos/genética , Vertebrados/genética , Animais , Neurônios Colinérgicos/metabolismo , Células Ciliadas Auditivas/metabolismo , Camundongos , Ratos , Receptores Nicotínicos/metabolismoRESUMO
We characterized, by electrophysiological methods, two biophysical properties of murine recombinant alpha4beta2 nicotinic acetylcholine receptors (nAChR) bearing a mutation (alpha4:+L264alpha4:beta2 or alpha4:S252Falpha4:beta2) linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Sensitivity to acetylcholine (ACh) was increased by the S252F substitution expressed in heterozygosis (alpha4:S252Falpha4:beta2) but was markedly reduced when this mutation was expressed in homozygosis (S252Falpha4:beta2). ACh sensitivity was not altered by the +L264 insertion. Moreover, receptor desensitization was significantly increased by both mutations expressed in heterozygosis. These results are in general agreement to those of rat and human recombinant receptors bearing the same mutations, thus contributing to validate the use of knock-in mice harboring ADNFLE mutations as models to study this pathology.
Assuntos
Epilepsias Parciais/genética , Epilepsias Parciais/fisiopatologia , Ativação do Canal Iônico/fisiologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/fisiologia , Acetilcolina/farmacologia , Animais , Colinérgicos/farmacologia , Genes Dominantes , Heterozigoto , Homozigoto , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Modelos Químicos , Mutagênese , Oócitos/fisiologia , Técnicas de Patch-Clamp , Receptores Nicotínicos/química , XenopusRESUMO
In this study we report the effects of neramexane, a novel amino-alkyl-cyclohexane derivative that is a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, on recombinant rat alpha9alpha10 nicotinic acetylcholine receptors expressed in Xenopus laevis oocytes. We compared its effects with those of memantine, a well-studied pore blocker of NMDA receptors, currently used in therapeutics for the treatment of Alzheimer's disease. Our results indicate that both compounds block acetylcholine-evoked responses at micromolar concentrations with a rank order of potency of neramexane>memantine, P<0.05. Block by neramexane of acetylcholine responses was not overcome at high concentrations of the agonist, indicative of a non-competitive inhibition. The lack of interaction of neramexane with the ligand binding domain was confirmed by radioligand binding experiments in transfected tsA201 cells. Moreover, block did not involve an increase in desensitization kinetics, it was independent of the resting potential of the membrane at low concentrations of neramexane and slightly voltage-dependent at concentrations higher than 1 microM. Finally, clinically-relevant concentrations of neramexane blocked native alpha9alpha10-containing nicotinic acetylcholine receptors of rat inner hair cells, thus demonstrating a possible in vivo relevance in potentially unexplored therapeutic areas.
Assuntos
Ciclopentanos/farmacologia , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Antagonistas Nicotínicos/farmacologia , Subunidades Proteicas/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacologia , Animais , Linhagem Celular , Células Ciliadas Auditivas Internas/fisiologia , Humanos , Memantina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/fisiologia , Subunidades Proteicas/genética , Ratos , Ratos Sprague-Dawley , Receptores Nicotínicos/genética , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , Xenopus laevisRESUMO
The alpha9 and alpha10 nicotinic cholinergic subunits assemble to form the receptor that mediates synaptic transmission between efferent olivocochlear fibers and hair cells of the cochlea. They are the latest vertebrate nicotinic cholinergic receptor (nAChR) subunits that have been cloned, and their identification has established a distant early divergent branch within the nAChR gene family. The alpha10 subunit serves as a "structural" component leading to heteromeric alpha9alpha10 nAChRs with distinct properties. We now have probed the stoichiometry of recombinant alpha9alpha10 nAChRs expressed in Xenopus oocytes. We have made use of the analysis of the population of receptors assembled from a wild-type subunit and its partner alpha9 or alpha10 subunit bearing a reporter mutation of a valine to threonine at position 13' of the second transmembrane domain (TM2). Because the mutation increased the sensitivity of the receptor for acetylcholine (ACh) but mutations at different subunits were not equivalent, the number of alpha9 and alpha10 subunits could be inferred from the number of components in compound concentration-response curves to ACh. The results were confirmed via the analysis of the effects of a mutation to threonine at position 17' of TM2. Because at this position the mutations at different subunits were equivalent, the stoichiometry was inferred directly from the shifts in the ACh EC50 values. We conclude that the recombinant alpha9alpha10 receptor is a pentamer with a (alpha9)2(alpha10)3 stoichiometry.
Assuntos
Subunidades Proteicas/química , Receptores Nicotínicos/química , Acetilcolina/farmacologia , Sequência de Aminoácidos , Animais , Sequência Conservada , Feminino , Matemática , Oócitos , Concentração Osmolar , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina , Valina , Xenopus laevisRESUMO
In this study, we report the effects of the quinoline derivatives quinine, its optical isomer quinidine, and chloroquine on alpha9alpha10-containing nicotinic acetylcholine receptors (nAChRs). The compounds blocked acetylcholine (ACh)-evoked responses in alpha9alpha10-injected Xenopus laevis oocytes in a concentration-dependent manner, with a rank order of potency of chloroquine (IC50 = 0.39 microM) > quinine (IC50 = 0.97 microM) approximately quinidine (IC50= 1.37 microM). Moreover, chloroquine blocked ACh-evoked responses on rat cochlear inner hair cells with an IC50 value of 0.13 microM, which is within the same range as that observed for recombinant receptors. Block by chloroquine was purely competitive, whereas quinine inhibited ACh currents in a mixed competitive and noncompetitive manner. The competitive nature of the blockage produced by the three compounds was confirmed by equilibrium binding experiments using [3H]methyllycaconitine. Binding affinities (Ki values) were 2.3, 5.5, and 13.0 microM for chloroquine, quinine, and quinidine, respectively. Block by quinine was found to be only slightly voltage-dependent, thus precluding open-channel block as the main mechanism of interaction of quinine with alpha9alpha10 nAChRs. The present results add to the pharmacological characterization of alpha9alpha10-containing nicotinic receptors and indicate that the efferent olivocochlear system that innervates the cochlear hair cells is a target of these ototoxic antimalarial compounds.
Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Quinidina/farmacologia , Quinina/farmacologia , Receptores Nicotínicos/efeitos dos fármacos , Animais , Antimaláricos/toxicidade , Cloroquina/toxicidade , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Quinidina/toxicidade , Quinina/toxicidade , Ensaio Radioligante , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/efeitos dos fármacos , Xenopus laevisRESUMO
We have performed a systematic mutagenesis of three hydrophobic rings (17', 13' and 9') within transmembrane region (TM) 2 of the alpha9alpha10 nicotinic cholinergic receptor (nAChR) to a hydrophilic (threonine) residue and compared the properties of mutant receptors reconstituted in Xenopus laevis oocytes. Phenotypic changes in alpha9alpha10 mutant receptors were evidenced by a decrease in the desensitization rate, an increase in both the EC(50) for ACh as well as the efficacy of partial agonists and the reduction of the allosteric modulation by extracellular Ca(2+). Mutated receptors exhibited spontaneous openings and, at the single-channel level, an increased apparent mean open time with no major changes in channel conductance, thus suggesting an increase in gating of the channel as the underlying mechanism. Overall, the degrees of the phenotypes of mutant receptors were more overt in the case of the centrally located V13'T mutant. Based on the atomic model of the pore of the electric organ of the Torpedo ray, we can propose that the interactions of side chains at positions 13' and 9' are key ones in creating an energetic barrier to ion permeation. In spite of the fact that the roles of the TM2 residues are mostly conserved in the distant alpha9alpha10 member of the nAChR family, their mechanistic contributions to channel gating show significant differences when compared to other nAChRs. These differences might be originated from slight differential intramolecular rearrangements during gating for the different receptors and might lead each nAChR to be in tune with their physiological roles.
Assuntos
Ativação do Canal Iônico/genética , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Acetilcolina/farmacologia , Regulação Alostérica , Sequência de Aminoácidos , Animais , Cálcio/farmacologia , Colinérgicos/farmacologia , Relação Dose-Resposta a Droga , Potenciais da Membrana/efeitos dos fármacos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Técnicas de Patch-Clamp , Fenótipo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Transfecção , Xenopus laevisRESUMO
We describe the functional properties of a nicotinic alpha9/serotonin subtype 3A (5HT3A) chimeric receptor expressed in Xenopus laevis oocytes. The chimera preserved ligand-binding properties of alpha9 and channel properties of 5HT3A. Thus, it responded to acetylcholine in a concentration-dependent manner with an EC50 of 70 microM but not to serotonin. It was blocked by methyllycaconitine, strychnine, atropine and nicotine, with the same rank order of potency as alpha9 receptors. The current-voltage relationship of currents through the alpha9/5HT3A chimera was similar to that of the 5HT3A receptors. These results are an evidence of functional coupling between the ligand-binding and the channel domains of the chimeric receptor.
Assuntos
Aconitina/análogos & derivados , Receptores Nicotínicos/biossíntese , Receptores 5-HT3 de Serotonina/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Aconitina/farmacologia , Animais , Atropina/farmacologia , DNA Complementar/biossíntese , DNA Complementar/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Antagonistas Muscarínicos/farmacologia , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ratos , Receptores Nicotínicos/efeitos dos fármacos , Receptores 5-HT3 de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Estricnina/farmacologia , Xenopus laevisRESUMO
In the present work, we characterized the effects of serotonin type 3 receptor ligands on recombinant and native alpha 9 alpha 10-containing nicotinic acetylcholine receptors (nAChRs). Our results indicate that the recombinant alpha 9 alpha 10 nAChR shares striking pharmacological properties with 5-HT(3) ligand-gated ion channels. Thus, 5-HT(3) receptor antagonists block ACh-evoked currents in alpha 9 alpha 10-injected Xenopus laevis oocytes with a rank order of potency of tropisetron (IC(50), 70.1 +/- 0.9 nM) > ondansetron (IC(50), 0.6 +/- 0.1 microM) = MDL 72222 (IC(50), 0.7 +/- 0.1 microM). Although serotonin does not elicit responses in alpha 9 alpha 10-injected oocytes, it blocks recombinant alpha 9 alpha 10 receptors in a noncompetitive and voltage-dependent manner (IC(50), 5.4 +/- 0.6 microM). On the other hand, we demonstrate an in vivo correlate of these properties of the recombinant receptor, with those of the alpha 9 alpha 10-containing nAChR of frog saccular hair cells. The possibility that the biogenic amine serotonin might act as a neuromodulator of the cholinergic efferent transmission in the vestibular apparatus and in the organ of Corti is discussed.