RESUMO
This study was undertaken to identify and characterize the first ligands capable of selectively identifying nicotinic acetylcholine receptors containing α7 and ß2 subunits (α7ß2-nAChR subtype). Basal forebrain cholinergic neurons express α7ß2-nAChR. Here, they appear to mediate neuronal dysfunction induced by the elevated levels of oligomeric amyloid-ß associated with early Alzheimer's disease. Additional work indicates that α7ß2-nAChR are expressed across several further critically important cholinergic and GABAergic neuronal circuits within the central nervous system. Further studies, however, are significantly hindered by the inability of currently available ligands to distinguish heteromeric α7ß2-nAChR from the closely related and more widespread homomeric α7-only-nAChR subtype. Functional screening using two-electrode voltage-clamp electrophysiology identified a family of α7ß2-nAChR-selective analogs of α-conotoxin PnIC (α-CtxPnIC). A combined electrophysiology, functional kinetics, site-directed mutagenesis, and molecular dynamics approach was used to further characterize the α7ß2-nAChR selectivity and site of action of these α-CtxPnIC analogs. We determined that α7ß2-nAChR selectivity of α-CtxPnIC analogs arises from interactions at a site distinct from the orthosteric agonist-binding site shared between α7ß2- and α7-only-nAChR. As numerous previously identified α-Ctx ligands are competitive antagonists of orthosteric agonist-binding sites, this study profoundly expands the scope of use of α-Ctx ligands (which have already provided important nAChR research and translational breakthroughs). More immediately, analogs of α-CtxPnIC promise to enable, for the first time, both comprehensive mapping of the distribution of α7ß2-nAChR and detailed investigations of their physiological roles.
Assuntos
Receptores Nicotínicos , Receptor Nicotínico de Acetilcolina alfa7 , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Colinérgicos , Sítios de Ligação , Neurônios GABAérgicos/metabolismo , Antagonistas Nicotínicos/farmacologiaRESUMO
BACKGROUND: Some dendrobatid poison frogs sequester the toxin epibatidine as a defense against predators. We previously identified an amino acid substitution (S108C) at a highly conserved site in a nicotinic acetylcholine receptor ß2 subunit of dendrobatid frogs that decreases sensitivity to epibatidine in the brain-expressing α4ß2 receptor. Introduction of S108C to the orthologous high-sensitivity human receptor similarly decreased sensitivity to epibatidine but also decreased sensitivity to acetylcholine, a potential cost if this were to occur in dendrobatids. This decrease in the acetylcholine sensitivity manifested as a biphasic acetylcholine concentration-response curve consistent with the addition of low-sensitivity receptors. Surprisingly, the addition of the ß2 S108C into the α4ß2 receptor of the dendrobatid Epipedobates anthonyi did not change acetylcholine sensitivity, appearing cost-free. We proposed that toxin-bearing dendrobatids may have additional amino acid substitutions protecting their receptors from alterations in acetylcholine sensitivity. To test this, in the current study, we compared the dendrobatid receptor to its homologs from two non-dendrobatid frogs. RESULTS: The introduction of S108C into the α4ß2 receptors of two non-dendrobatid frogs also does not affect acetylcholine sensitivity suggesting no additional dendrobatid-specific substitutions. However, S108C decreased the magnitude of neurotransmitter-induced currents in Epipedobates and the non-dendrobatid frogs. We confirmed that decreased current resulted from fewer receptors in the plasma membrane in Epipedobates using radiolabeled antibodies against the receptors. To test whether S108C alteration of acetylcholine sensitivity in the human receptor was due to (1) adding low-sensitivity binding sites by changing stoichiometry or (2) converting existing high- to low-sensitivity binding sites with no stoichiometric alteration, we made concatenated α4ß2 receptors in stoichiometry with only high-sensitivity sites. S108C substitutions decreased maximal current and number of immunolabeled receptors but no longer altered acetylcholine sensitivity. CONCLUSIONS: The most parsimonious explanation of our current and previous work is that the S108C substitution renders the ß2 subunit less efficient in assembling/trafficking, thereby decreasing the number of receptors in the plasma membrane. Thus, while ß2 S108C protects dendrobatids against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 receptor function.
Assuntos
Acetilcolina , Venenos , Humanos , Acetilcolina/farmacologia , Piridinas/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologiaRESUMO
Initiated by findings that Alzheimer's disease is associated with a profound loss of cholinergic markers in human brain, decades of studies have examined the interactions between specific subtypes of nicotinic acetylcholine receptors and amyloid-ß [derived from the amyloid precursor protein (APP), which is cleaved to yield variable isoforms of amyloid-ß]. We review the evolving understanding of amyloid-ß's roles in Alzheimer's disease and pioneering studies that highlighted a role of nicotinic acetylcholine receptors in mediating important aspects of amyloid-ß's effects. This review also surveys the current state of research into amyloid-ß / nicotinic acetylcholine receptor interactions. The field has reached an exciting point in which common themes are emerging from the wide range of prior research and a range of accessible, relevant model systems are available to drive further progress. We highlight exciting new areas of inquiry and persistent challenges that need to be considered while conducting this research. Studies of amyloid-ß and the nicotinic acetylcholine receptor populations that it interacts with provide opportunities for innovative basic and translational scientific breakthroughs related to nicotinic receptor biology, Alzheimer's disease, and cholinergic contributions to cognition more broadly.
Assuntos
Doença de Alzheimer , Receptores Nicotínicos , Animais , Humanos , Receptores Nicotínicos/metabolismo , Doença de Alzheimer/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Peptídeos beta-Amiloides/metabolismo , Colinérgicos , Modelos Animais de DoençasRESUMO
Neuronal and network-level hyperexcitability is commonly associated with increased levels of amyloid-ß (Aß) and contribute to cognitive deficits associated with Alzheimer's disease (AD). However, the mechanistic complexity underlying the selective loss of basal forebrain cholinergic neurons (BFCNs), a well-recognized characteristic of AD, remains poorly understood. In this study, we tested the hypothesis that the oligomeric form of amyloid-ß (oAß42), interacting with α7-containing nicotinic acetylcholine receptor (nAChR) subtypes, leads to subnucleus-specific alterations in BFCN excitability and impaired cognition. We used single-channel electrophysiology to show that oAß42 activates both homomeric α7- and heteromeric α7ß2-nAChR subtypes while preferentially enhancing α7ß2-nAChR open-dwell times. Organotypic slice cultures were prepared from male and female ChAT-EGFP mice, and current-clamp recordings obtained from BFCNs chronically exposed to pathophysiologically relevant level of oAß42 showed enhanced neuronal intrinsic excitability and action potential firing rates. These resulted from a reduction in action potential afterhyperpolarization and alterations in the maximal rates of voltage change during spike depolarization and repolarization. These effects were observed in BFCNs from the medial septum diagonal band and horizontal diagonal band, but not the nucleus basalis. Last, aged male and female APP/PS1 transgenic mice, genetically null for the ß2 nAChR subunit gene, showed improved spatial reference memory compared with APP/PS1 aged-matched littermates. Combined, these data provide a molecular mechanism supporting a role for α7ß2-nAChR in mediating the effects of oAß42 on excitability of specific populations of cholinergic neurons and provide a framework for understanding the role of α7ß2-nAChR in oAß42-induced cognitive decline.
Assuntos
Peptídeos beta-Amiloides/genética , Prosencéfalo Basal/fisiopatologia , Disfunção Cognitiva/genética , Disfunção Cognitiva/fisiopatologia , Sistema Nervoso Parassimpático/fisiopatologia , Fragmentos de Peptídeos/genética , Transdução de Sinais/genética , Receptor Nicotínico de Acetilcolina alfa7/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Linhagem Celular , Fenômenos Eletrofisiológicos , Feminino , Genótipo , Humanos , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Transgênicos , Neurônios/patologiaRESUMO
This study investigates-for the first time to our knowledge-the existence and mechanisms of functional interactions between the endogenous mammalian prototoxin, lynx1, and α3- and ß4-subunit-containing human nicotinic acetylcholine receptors (α3ß4*-nAChRs). Concatenated gene constructs were used to express precisely defined α3ß4*-nAChR isoforms (α3ß4)2ß4-, (α3ß4)2α3-, (α3ß4)2α5(398D)-, and (α3ß4)2α5(398N)-nAChR in Xenopus oocytes. In the presence or absence of lynx1, α3ß4*-nAChR agonist responses were recorded by using 2-electrode voltage clamp and single-channel electrophysiology, whereas radioimmunolabeling measured cell-surface expression. Lynx1 reduced (α3ß4)2ß4-nAChR function principally by lowering cell-surface expression, whereas single-channel effects were primarily responsible for reducing (α3ß4)2α3-nAChR function [decreased unitary conductance (≥50%), altered burst proportions (3-fold reduction in the proportion of long bursts), and enhanced closed dwell times (3- to 6-fold increase)]. Alterations in both cell-surface expression and single-channel properties accounted for the reduction in (α3ß4)2α5-nAChR function that was mediated by lynx1. No effects were observed when α3ß4*-nAChRs were coexpressed with mutated lynx1 (control). Lynx1 is expressed in the habenulopeduncular tract, where α3ß4*-α5*-nAChR subtypes are critical contributors to the balance between nicotine aversion and reward. This gives our findings a high likelihood of physiologic significance. The exquisite isoform selectivity of lynx1 interactions provides new insights into the mechanisms and allosteric sites [α(-)-interface containing] by which prototoxins can modulate nAChR function.-George, A. A., Bloy, A., Miwa, J. M., Lindstrom, J. M., Lukas, R. J., Whiteaker, P. Isoform-specific mechanisms of α3ß4*-nicotinic acetylcholine receptor modulation by the prototoxin lynx1.
Assuntos
Proteínas Ligadas por GPI/metabolismo , Receptores Nicotínicos/metabolismo , Potenciais de Ação , Proteínas Adaptadoras de Transdução de Sinal , Animais , Membrana Celular/metabolismo , Membrana Celular/fisiologia , Proteínas Ligadas por GPI/genética , Humanos , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , XenopusRESUMO
Prototoxins are a diverse family of membrane-tethered molecules expressed in the nervous system that modulate nicotinic cholinergic signaling, but their functions and specificity have yet to be completely explored. We tested the selectivity and efficacy of leukocyte antigen, PLAUR (plasminogen activator, urokinase receptor) domain-containing (LYPD)-6B on α3ß4-, α3α5ß4-, and α7-containing nicotinic acetylcholine receptors (nAChRs). To constrain stoichiometry, fusion proteins encoding concatemers of human α3, ß4, and α5 (D and N variants) subunits were expressed in Xenopus laevis oocytes and tested with or without LYPD6B. We used the 2-electrode voltage-clamp method to quantify responses to acetylcholine (ACh): agonist sensitivity (EC50), maximal agonist-induced current (Imax), and time constant (τ) of desensitization. For ß4-α3-α3-ß4-α3 and ß4-α3-ß4-α3-α3, LYPD6B decreased EC50 from 631 to 79 µM, reduced Imax by at least 59%, and decreased τ. For ß4-α3-α5D-ß4-α3 and ß4-α3-ß4-α-α5D, LYPD6B decreased Imax by 63 and 32%, respectively. Thus, LYPD6B acted only on (α3)3(ß4)2 and (α3)2(α5D)(ß4)2 and did not affect the properties of (α3)2(ß4)3, α7, or (α3)2(α5N)(ß4)2 nAChRs. Therefore, LYPD6B acts as a mixed modulator that enhances the sensitivity of (α3)3(ß4)2 nAChRs to ACh while reducing ACh-induced whole-cell currents. LYPD6B also negatively modulates α3ß4 nAChRs that include the α5D common human variant, but not the N variant associated with nicotine dependence.
Assuntos
Receptores Nicotínicos/metabolismo , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Acetilcolina/farmacologia , Animais , Humanos , Nicotina/farmacologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Subunidades Proteicas/metabolismo , Xenopus laevis/metabolismoRESUMO
We examined α7ß2-nicotinic acetylcholine receptor (α7ß2-nAChR) expression in mammalian brain and compared pharmacological profiles of homomeric α7-nAChRs and α7ß2-nAChRs. α-Bungarotoxin affinity purification or immunoprecipitation with anti-α7 subunit antibodies (Abs) was used to isolate nAChRs containing α7 subunits from mouse or human brain samples. α7ß2-nAChRs were detected in forebrain, but not other tested regions, from both species, based on Western blot analysis of isolates using ß2 subunit-specific Abs. Ab specificity was confirmed in control studies using subunit-null mutant mice or cell lines heterologously expressing specific human nAChR subtypes and subunits. Functional expression in Xenopus oocytes of concatenated pentameric (α7)5-, (α7)4(ß2)1-, and (α7)3(ß2)2-nAChRs was confirmed using two-electrode voltage clamp recording of responses to nicotinic ligands. Importantly, pharmacological profiles were indistinguishable for concatenated (α7)5-nAChRs or for homomeric α7-nAChRs constituted from unlinked α7 subunits. Pharmacological profiles were similar for (α7)5-, (α7)4(ß2)1-, and (α7)3(ß2)2-nAChRs except for diminished efficacy of nicotine (normalized to acetylcholine efficacy) at α7ß2- versus α7-nAChRs. This study represents the first direct confirmation of α7ß2-nAChR expression in human and mouse forebrain, supporting previous mouse studies that suggested relevance of α7ß2-nAChRs in Alzheimer disease etiopathogenesis. These data also indicate that α7ß2-nAChR subunit isoforms with different α7/ß2 subunit ratios have similar pharmacological profiles to each other and to α7 homopentameric nAChRs. This supports the hypothesis that α7ß2-nAChR agonist activation predominantly or entirely reflects binding to α7/α7 subunit interface sites.
Assuntos
Prosencéfalo/metabolismo , Receptores Nicotínicos/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Animais , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bungarotoxinas/farmacologia , Cerebelo/metabolismo , Feminino , Hipocampo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Agonistas Nicotínicos/farmacologia , Antagonistas Nicotínicos/farmacologia , Oócitos/metabolismo , Multimerização Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Piridinas/farmacologia , Ensaio Radioligante , Receptores Nicotínicos/genética , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
BACKGROUND AND PURPOSE: Oligomeric amyloid ß 1-42 (oAß1-42) exhibits agonist-like action at human α7- and α7ß2-containing nicotinic receptors. The N-terminal amyloid ß1-15 fragment (N-Aß fragment) modulates presynaptic calcium and enhances hippocampal-based synaptic plasticity via α7-containing nicotinic receptors. Further, the N-Aß fragment and its core sequence, the N-amyloid-beta core hexapeptide (N-Aßcore), protect against oAß1-42-associated synapto- and neurotoxicity. Here, we investigated how oAß1-42, the N-Aß fragment, and the N-Aßcore regulate the single-channel properties of α7- and α7ß2-nicotinic receptors. EXPERIMENTAL APPROACH: Single-channel recordings measured the impact of acetylcholine, oAß1-42, the N-Aß fragment, and the N-Aßcore on the unitary properties of human α7- and α7ß2-containing nicotinic receptors expressed in nicotinic-null SH-EP1 cells. Molecular dynamics simulations identified potential sites of interaction between the N-Aß fragment and orthosteric α7+/α7- and α7+/ß2- nicotinic receptor binding interfaces. KEY RESULTS: The N-Aß fragment and N-Aßcore induced α7- and α7ß2-nicotinic receptor single-channel openings. Relative to acetylcholine, oAß1-42 preferentially enhanced α7ß2-nicotinic receptor single-channel open probability and open-dwell times. Co-application with the N-Aßcore neutralized these effects. Further, administration of the N-Aß fragment alone, or in combination with acetylcholine or oAß1-42, selectively enhanced α7-nicotinic receptor open probability and open-dwell times (compared to acetylcholine or oAß1-42). CONCLUSIONS AND IMPLICATIONS: Amyloid-beta peptides demonstrate functional diversity in regulating α7- and α7ß2-nicotinic receptor function, with implications for a wide range of nicotinic receptor-mediated functions in Alzheimer's disease. The effects of these peptides on α7- and/or α7ß2-nicotinic receptors revealed complex interactions with these subtypes, providing novel insights into the neuroprotective actions of amyloid ß-derived fragments against the toxic effects of oAß1-42.
Assuntos
Peptídeos beta-Amiloides , Fragmentos de Peptídeos , Receptor Nicotínico de Acetilcolina alfa7 , Humanos , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/farmacologia , Peptídeos beta-Amiloides/toxicidade , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/agonistas , Fragmentos de Peptídeos/farmacologia , Fármacos Neuroprotetores/farmacologia , Simulação de Dinâmica Molecular , Acetilcolina/farmacologia , Receptores Nicotínicos/metabolismoRESUMO
Genome-wide studies have strongly associated a non-synonymous polymorphism (rs16969968) that changes the 398th amino acid in the nAChR α5 subunit from aspartic acid to asparagine (D398N), with greater risk for increased nicotine consumption. We have used a pentameric concatemer approach to express defined and consistent populations of α3ß4α5 nAChR in Xenopus oocytes. α5(Asn-398; risk) variant incorporation reduces ACh-evoked function compared with inclusion of the common α5(Asp-398) variant without altering agonist or antagonist potencies. Unlinked α3, ß4, and α5 subunits assemble to form a uniform nAChR population with pharmacological properties matching those of concatemeric α3ß4* nAChRs. α5 subunit incorporation reduces α3ß4* nAChR function after coinjection with unlinked α3 and ß4 subunits but increases that of α3ß4α5 versus α3ß4-only concatemers. α5 subunit incorporation into α3ß4* nAChR also alters the relative efficacies of competitive agonists and changes the potency of the non-competitive antagonist mecamylamine. Additional observations indicated that in the absence of α5 subunits, free α3 and ß4 subunits form at least two further subtypes. The pharmacological profiles of these free subunit α3ß4-only subtypes are dissimilar both to each other and to those of α3ß4α5 nAChR. The α5 variant-induced change in α3ß4α5 nAChR function may underlie some of the phenotypic changes associated with this polymorphism.
Assuntos
Substituição de Aminoácidos , Complexos Multiproteicos/metabolismo , Mutação de Sentido Incorreto , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/metabolismo , Transdução de Sinais/fisiologia , Animais , Estudo de Associação Genômica Ampla , Humanos , Complexos Multiproteicos/genética , Oócitos , Polimorfismo Genético , Subunidades Proteicas/genética , Receptores Nicotínicos/genética , Xenopus laevisRESUMO
Background: Some poison arrow frogs sequester the toxin epibatidine as a defense against predators. We previously identified a single amino acid substitution (S108C) at a highly conserved site in a neuronal nicotinic acetylcholine receptor (nAChR) ß2 subunit that prevents epibatidine from binding to this receptor. When placed in a homologous mammalian nAChR this substitution minimized epibatidine binding but also perturbed acetylcholine binding, a clear cost. However, in the nAChRs of poison arrow frogs, this substitution appeared to have no detrimental effect on acetylcholine binding and, thus, appeared cost-free. Results: The introduction of S108C into the α4ß2 nAChRs of non-dendrobatid frogs also does not affect ACh sensitivity, when these receptors are expressed in Xenopus laevis oocytes. However, α4ß2 nAChRs with C108 had a decreased magnitude of neurotransmitter-induced currents in all species tested ( Epipedobates anthonyi , non-dendrobatid frogs, as well as human), compared with α4ß2 nAChRs with the conserved S108. Immunolabeling of frog or human α4ß2 nAChRs in the plasma membrane using radiolabeled antibody against the ß2 nAChR subunit shows that C108 significantly decreased the number of cell-surface α4ß2 nAChRs, compared with S108. Conclusions: While S108C protects these species against sequestered epibatidine, it incurs a potential physiological cost of disrupted α4ß2 nAChR function. These results may explain the high conservation of a serine at this site in vertebrates, as well as provide an example of a tradeoff between beneficial and deleterious effects of an evolutionary change. They also provide important clues for future work on assembly and trafficking of this important neurotransmitter receptor.
RESUMO
Temporal filtering is a fundamental operation of nervous systems. In peripheral sensory systems, the temporal pattern of spiking activity can encode various stimulus qualities, and temporal filtering allows postsynaptic neurons to detect behaviorally relevant stimulus features from these spike trains. Intrinsic excitability, short-term synaptic plasticity, and voltage-dependent dendritic conductances have all been identified as mechanisms that can establish temporal filtering behavior in single neurons. Here we show that synaptic integration of temporally summating excitation and inhibition can establish diverse temporal filters of presynaptic input. Mormyrid electric fish communicate by varying the intervals between electric organ discharges. The timing of each discharge is coded by peripheral receptors into precisely timed spikes. Within the midbrain posterior exterolateral nucleus, temporal filtering by individual neurons results in selective responses to a particular range of presynaptic interspike intervals. These neurons are diverse in their temporal filtering properties, reflecting the wide range of intervals that must be detected during natural communication behavior. By manipulating presynaptic spike timing with high temporal resolution, we demonstrate that tuning to behaviorally relevant patterns of presynaptic input is similar in vivo and in vitro. We reveal that GABAergic inhibition plays a critical role in establishing different temporal filtering properties. Further, our results demonstrate that temporal summation of excitation and inhibition establishes selective responses to high and low rates of synaptic input, respectively. Simple models of synaptic integration reveal that variation in these two competing influences provides a basic mechanism for generating diverse temporal filters of synaptic input.
Assuntos
Potenciais de Ação/fisiologia , Órgão Elétrico/citologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Potenciais Sinápticos/fisiologia , Potenciais de Ação/efeitos dos fármacos , Análise de Variância , Animais , Axônios/metabolismo , Biofísica , Dendritos/metabolismo , Peixe Elétrico , Órgão Elétrico/fisiologia , Estimulação Elétrica , Feminino , Antagonistas GABAérgicos/farmacologia , Técnicas In Vitro , Masculino , Modelos Neurológicos , Movimento/efeitos dos fármacos , Movimento/fisiologia , Inibição Neural/efeitos dos fármacos , Vias Neurais/fisiologia , Neurônios/citologia , Técnicas de Patch-Clamp/métodos , Picrotoxina/farmacologia , Piridazinas/farmacologia , Potenciais Sinápticos/efeitos dos fármacos , Ácido gama-Aminobutírico/metabolismoRESUMO
We investigated assembly and function of nicotinic acetylcholine receptors (nAChRs) composed of α7 and ß2 subunits. We measured optical and electrophysiological properties of wild-type and mutant subunits expressed in cell lines and Xenopus laevis oocytes. Laser scanning confocal microscopy indicated that fluorescently tagged α7 and ß2 subunits colocalize. Förster resonance energy transfer between fluorescently tagged subunits strongly suggested that α7 and ß2 subunits coassemble. Total internal reflection fluorescence microscopy revealed that assemblies localized to filopodia-like processes of SH-EP1 cells. Gain-of-function α7 and ß2 subunits confirmed that these subunits coassemble within functional receptors. Moreover, α7ß2 nAChRs composed of wild-type subunits or fluorescently tagged subunits had pharmacological properties similar to those of α7 nAChRs, although amplitudes of α7ß2 nAChR-mediated, agonist-evoked currents were generally ~2-fold lower than those for α7 nAChRs. It is noteworthy that α7ß2 nAChRs displayed sensitivity to low concentrations of the antagonist dihydro-ß-erythroidine that was not observed for α7 nAChRs at comparable concentrations. In addition, cysteine mutants revealed that the α7-ß2 subunit interface does not bind ligand in a functionally productive manner, partly explaining lower α7ß2 nAChR current amplitudes and challenges in identifying the function of native α7ß2 nAChRs. On the basis of our findings, we have constructed a model predicting receptor function that is based on stoichiometry and position of ß2 subunits within the α7ß2 nAChRs.
Assuntos
Fenômenos Eletrofisiológicos , Multimerização Proteica , Receptores Nicotínicos/fisiologia , Proteínas de Xenopus/fisiologia , Animais , Linhagem Celular , Eletrofisiologia , Oócitos , Ligação Proteica , Estrutura Quaternária de Proteína , Subunidades Proteicas , Receptores Nicotínicos/química , Receptores Nicotínicos/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Specific types of neurons show stable, predictable excitability properties, while other neurons show transient adaptive plasticity of their excitability. However, little attention has been paid to how the cellular pathways underlying adaptive plasticity interact with those that maintain neuronal stability. We addressed this question in the pacemaker neurons from a weakly electric fish because these neurons show a highly stable spontaneous firing rate as well as an N-methyl-D-aspartate (NMDA) receptor-dependent form of plasticity. We found that basal firing rates were regulated by a serial interaction of conventional and atypical PKC isoforms and that this interaction establishes individual differences within the species. We observed that NMDA receptor-dependent plasticity is achieved by further activation of these kinases. Importantly, the PKC pathway is maintained in an unsaturated baseline state to allow further Ca(2+)-dependent activation during plasticity. On the other hand, the Ca(2+)/calmodulin-dependent phosphatase calcineurin does not regulate baseline firing but is recruited to control the duration of the NMDA receptor-dependent plasticity and return the pacemaker firing rate back to baseline. This work illustrates how neuronal plasticity can be realized by biasing ongoing mechanisms of stability (e.g., PKC) and terminated by recruiting alternative mechanisms (e.g., calcineurin) that constrain excitability. We propose this as a general model for regulating activity-dependent change in neuronal excitability.
Assuntos
Cálcio/metabolismo , Bulbo/fisiologia , Plasticidade Neuronal/fisiologia , Animais , Calcineurina/metabolismo , Calcineurina/fisiologia , Cálcio/fisiologia , Peixe Elétrico/metabolismo , Peixe Elétrico/fisiologia , Feminino , Isoenzimas/metabolismo , Isoenzimas/fisiologia , Masculino , Bulbo/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação , Proteína Quinase C/metabolismo , Proteína Quinase C/fisiologia , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de N-Metil-D-Aspartato/fisiologia , Fatores SexuaisRESUMO
Sleep-related hypermotor epilepsy (SHE) is a group of seizure disorders prominently associated with mutations in nicotinic acetylcholine receptors (nAChR). The most prevalent central nervous system nAChR subtype contains α4 and ß2 subunits, in two ratios. (α4ß2)2ß2-nAChR have high agonist sensitivity (HS-isoform), whereas (α4ß2)2α4-nAChR agonist responses exhibit a small high-sensitivity, and a predominant low-sensitivity, phase of function (LS-isoform). Multiple non-synonymous mutations in the second and third transmembrane domains of α4 and ß2 subunits are associated with SHE. We recently demonstrated that two additional, SHE-associated, missense mutations in the major cytoplasmic loops of these subunits [α4(R336H) and ß2(V337G)] cause increased macroscopic function-per receptor. Here, we use single-channel patch-clamp electrophysiology to show that these mutations influence single-channel amplitudes and open- and closed-state kinetics. Pure populations of HS- or LS-isoform α4ß2-nAChR were expressed by injecting either 1:10 or 30:1 α4:ß2 cRNA ratios, respectively, into Xenopus laevis oocytes. Functional properties of the resulting mutant α4ß2-nAChR isoforms were compared to their wildtype counterparts. α4(R336H) subunit incorporation minimally affected single-channel amplitudes, whereas ß2(V337G) subunit incorporation reduced them significantly in both isoforms. However, for both mutant subunits, increased function-per-receptor was predominantly caused by altered single channel kinetics. The α4(R336H) mutation primarily destabilizes desensitized states between openings. By contrast, the ß2(V337G) mutation principally stabilizes receptor open states. The use of naturally-occurring and physiologically-impactful mutations has allowed us to define valuable new insights regarding the functional roles of nAChR intracellular domains. Further mechanistic context is provided by intracellular-domain structures recently published for other members of the Cys-loop receptor superfamily (α3ß4-nAChR and 5-HT3AR).
Assuntos
Epilepsia do Lobo Frontal/genética , Epilepsia do Lobo Frontal/fisiopatologia , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Sono , Substituição de Aminoácidos , Animais , Microscopia Crioeletrônica , Humanos , Cinética , Mutação , Agonistas Nicotínicos/farmacologia , Oócitos , Técnicas de Patch-Clamp , Domínios Proteicos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Xenopus laevisRESUMO
The mechanisms behind the induction of cellular correlates of memory by sensory input and their contribution to meaningful behavioral changes are largely unknown. We previously reported a graded memory in the form of sensorimotor adaptation in the electromotor output of electric fish. Here we show that the mechanism for this adaptation is a synaptically induced long-lasting shift in intrinsic neuronal excitability. This mechanism rapidly integrates hundreds of spikes in a second, or gradually integrates the same number of spikes delivered over tens of minutes. Thus, this mechanism appears immune to frequency-dependent fluctuations in input and operates as a simple pulse counter over a wide range of time scales, enabling it to transduce graded sensory information into a graded memory and a corresponding change in the behavioral output. This adaptation is based on an NMDA receptor-mediated change in intrinsic excitability of the postsynaptic neurons involving the Ca2+-dependent activation of TRP channels.
Assuntos
Aclimatação , Relógios Biológicos/fisiologia , Órgão Elétrico/fisiologia , Memória/fisiologia , Neurônios/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Sequência de Aminoácidos , Animais , Comportamento Animal , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Peixe Elétrico , Órgão Elétrico/efeitos dos fármacos , Órgão Elétrico/efeitos da radiação , Agonistas de Aminoácidos Excitatórios/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Ácido Flufenâmico/farmacologia , Técnicas In Vitro , Bulbo/citologia , Modelos Neurológicos , N-Metilaspartato/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Estimulação Física/métodos , Piperazinas/farmacologia , Canais de Cátion TRPM/antagonistas & inibidores , Canais de Cátion TRPM/metabolismo , ômega-Conotoxina GVIA/farmacologiaRESUMO
Central nervous system nicotinic acetylcholine receptors (nAChR) are predominantly of the α4ß2 subtype. Two isoforms exist, with high or low agonist sensitivity (HS-(α4ß2)2ß2- and LS-(α4ß2)2α4-nAChR). Both isoforms exhibit similar macroscopic potency and efficacy values at low acetylcholine (ACh) concentrations, mediated by a common pair of high-affinity α4(+)/(-)ß2 subunit binding interfaces. However LS-(α4ß2)2α4-nAChR also respond to higher concentrations of ACh, acting at a third α4(+)/(-)α4 subunit interface. To probe isoform functional differences further, HS- and LS-α4ß2-nAChR were expressed in Xenopus laevis oocytes and single-channel responses were assessed using cell-attached patch-clamp. In the presence of a low ACh concentration, both isoforms produce low-bursting function. HS-(α4ß2)2ß2-nAChR exhibit a single conductance state, whereas LS-(α4ß2)2α4-nAChR display two distinctive conductance states. A higher ACh concentration did not preferentially recruit either conductance state, but did result in increased LS-(α4ß2)2α4-nAChR bursting and reduced closed times. Introduction of an α4(+)/(-)α4-interface loss-of-function α4W182A mutation abolished these changes, confirming this site's role in mediating LS-(α4ß2)2α4-nAChR responses. Small or large amplitude openings are highly-correlated within individual LS-(α4ß2)2α4-nAChR bursts, suggesting that they arise from distinct intermediate states, each of which is stabilized by α4(+)/(-)α4 site ACh binding. These findings are consistent with α4(+)/(-)α4 subunit interface occupation resulting in allosteric potentiation of agonist actions at α4(+)/(-)ß2 subunit interfaces, rather than independent induction of high conductance channel openings.
Assuntos
Receptores Nicotínicos/metabolismo , Acetilcolina/química , Acetilcolina/metabolismo , Acetilcolina/farmacologia , Animais , Sítios de Ligação , Humanos , Potenciais da Membrana/efeitos dos fármacos , Mutagênese Sítio-Dirigida , Oócitos/metabolismo , Ligação Proteica , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/agonistas , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Receptores Nicotínicos/química , Receptores Nicotínicos/genética , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/metabolismoRESUMO
In this paper, we present the results of cellular and molecular studies on the neuroendocrine correlates of male sexual polymorphism in a population of the blenniid fish Salaria pavo (Risso). Bigger and older males defend nests, whereas smaller and younger males mimic female nuptial coloration and behavior to gain access to nests and sneak fertilizations. In this population, sex-role reversal in courtship also occurs (i.e., females are the courting sex). Immunocytochemistry and in situ hybridization were used to examine the production of arginine vasotocin (AVT) peptide and messenger RNA, respectively. The expression of AVT mRNA on a per-cell basis was correlated with mating behavior, rather than with sex morphotype, which suggests that the greater level of AVT mRNA expression in females and sneakers is correlated with the production of courtship behavior. On the other hand, the number and size of AVT peptide-producing cells in both male types is higher or larger, respectively, than in females, which suggests that it is correlated with sex morphotype, rather than with mating behavior.
Assuntos
Peixes/fisiologia , Identidade de Gênero , Prosencéfalo/metabolismo , Comportamento Sexual Animal/fisiologia , Vasotocina/metabolismo , Animais , Feminino , Imuno-Histoquímica , Hibridização In Situ , Masculino , RNA Mensageiro/metabolismo , Vasotocina/genéticaRESUMO
Human nicotinic acetylcholine receptor (nAChR) alpha7 subunits were stably and heterologously expressed in native nAChR-null SH-EP1 human epithelial cells. Immunofluorescence staining shows alpha7 subunit protein expression in virtually every transfected cell. Microautoradiographic analysis identifies 125I-labeled alpha-bungarotoxin (I-Bgt) binding sites corresponding to human alpha7 (halpha7)-nAChRs on the surface of most cells. I-Bgt binds to halpha7-nAChRs in membrane fractions with a typical apparent K(D) value of approximately 5 nM and B(max) value of approximately 1 pmol/mg membrane protein, and 62% of these sites are expressed on the cell surface. Function of heterologously expressed halpha7-nAChRs is evident as rapid, transient inward current responses to (-)-nicotine. Nicotine treatment of transfected cells produces dose- and time-dependent increases (up to approximately 100%) in numbers of I-Bgt binding sites. Epibatidine is a useful ligand for studies of nAChRs containing alpha3 or alpha4 subunits (K(D) values of about 100 or 10 pM, respectively). halpha7-nAChRs expressed in transfected SH-EP1 cells also exhibit picomolar affinity binding of 3H-labeled epibatidine (K(D) value of approximately 0.6 nM). Studies of several forms of native or heterologously expressed rat or human alpha7-nAChRs confirm high-affinity and mutually exclusive interaction with both epibatidine and alpha-bungarotoxin. Rank order potencies for drugs acting to compete for binding of either radioligand are similar (methyllycaconitine > dimethylphenyl-piperazinium > nicotine approximately cytisine > carbamylcholine approximately D-tubocurarine). These results demonstrate that transfected SH-EP1 cells are excellent models for studies of heterologously expressed, human alpha7-nAChRs that exhibit ligand binding and functional properties like native alpha7-nAChRs and that epibatdine is useful as a probe for human alpha7-nAChRs.
Assuntos
Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Agonistas Nicotínicos/metabolismo , Piridinas/metabolismo , Receptores Nicotínicos/metabolismo , Autorradiografia , Northern Blotting , Western Blotting , Bungarotoxinas/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Complementar/biossíntese , DNA Complementar/genética , Células Epiteliais/metabolismo , Humanos , Imuno-Histoquímica , Imunoprecipitação , Potenciais da Membrana/efeitos dos fármacos , Nicotina/farmacologia , Técnicas de Patch-Clamp , Plasmídeos/genética , RNA/biossíntese , RNA/isolamento & purificação , Receptores Nicotínicos/genética , Transfecção , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Dopamine (DA) neurons located in the mammalian midbrain have been generally implicated in reward and drug reinforcement and more specifically in nicotine dependence. However, roles played by nicotinic acetylcholine receptors, including those composed of alpha7-subunits [alpha7-nicotinic acetylcholine receptors (nAChRs)], in modulation of DA signaling and in nicotine dependence are not clearly understood. Although midbrain slice recording has been used previously to identify functional alpha7-nAChRs, these preparations are not optimally designed for extremely rapid and reproducible drug application, and rapidly desensitized, alpha7-nAChR-mediated currents may have been underestimated or not detected. Here, we use patch-clamp, whole-cell current recordings from single neurons acutely dissociated from midbrain nuclei and having features of DA neurons to characterize acetylcholine-induced, inward currents that rapidly activate and desensitize, are mimicked by the alpha7-nAChR-selective agonist, choline, blocked by the alpha7-nAChR-selective antagonists, methyllycaconitine and alpha-bungarotoxin, and are similar to those of heterologously expressed, human alpha7-nAChRs. We also use reverse transcriptase-polymerase chain reaction, in situ hybridization, and immunocytochemical staining to demonstrate nAChR alpha7 subunit gene expression as message and protein in the rat substantia nigra pars compacta and ventral tegmental area. Expression of alpha7 subunit message and of alpha7-nAChR-mediated responses is developmentally regulated, with both being absent in samples taken from rats at postnatal day 7, but later becoming present and increasing over the next 2 weeks. Collectively, this electrophysiological, pharmacological, and molecular evidence indicates that nAChR alpha7 subunits and functional alpha7-nAChRs are expressed somatodendritically by midbrain DA neurons, where they may play important physiological roles and contribute to nicotine reinforcement and dependence.
Assuntos
Mesencéfalo/citologia , Neurônios/metabolismo , Receptores Nicotínicos/isolamento & purificação , Animais , Células Cultivadas , Dopamina/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Expressão Gênica , Cinética , Mesencéfalo/crescimento & desenvolvimento , Ratos , Ratos Wistar , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores Nicotínicos/fisiologia , Transfecção , Receptor Nicotínico de Acetilcolina alfa7RESUMO
Amyloid-beta (Abeta) accumulation and aggregation are thought to contribute to the pathogenesis of Alzheimer's disease (AD). In AD, there is a selective decrease in the numbers of radioligand binding sites corresponding to the most abundant nicotinic acetylcholine receptor (nAChR) subtype, which contains human alpha4 and beta2 subunits (halpha4beta2-nAChR). However, the relationships between these phenomena are uncertain, and effects of Abeta on halpha4beta2-nAChR function have not been investigated in detail. We first confirmed expression of halpha4 and hbeta2 subunits as messenger RNA in transfected, human SHEP1 cells by reverse transcription-polymerase chain reaction and mRNA fluorescence in situ hybridization analyses. Immunoprecipitation Western analyses confirmed alpha4 and beta2 subunit protein expression and co-assembly. Whole cell current recording demonstrated heterologous expression in SH-EP1-halpha4beta2 cells of functional halpha4beta2-nAChRs with characteristic responses to nicotinic agonists or antagonists. Nicotine-induced whole cell currents were suppressed by Abeta(1-42) in a dose-dependent manner. Functional inhibition was selective for Abeta(1-42) compared with the functionally inactive, control peptide Abeta(40-1).Abeta(1-42)-mediated inhibition of halpha4beta2-nAChR function was non-competitive, voltage-independent, and use-independent. Pre-loading of cells with guanyl-5'-yl thiophosphate failed to prevent Abeta(1-42)-induced inhibition, suggesting that down-regulation of halpha4beta2-nAChR function by Abeta(1-42) is not mediated by nAChR internalization. Sensitivity to Abeta(1-42) antagonism at 1 nm was evident for halpha4beta2-nAChRs, but not for heterologously expressed human alpha7-nAChRs, although both nAChR subtypes were functionally inhibited by 100 nm Abeta(1-42), with the magnitude of functional block being higher for 100 nm Abeta(1-42) acting on halpha7-nAChRs. These findings suggest that halpha4beta2-nAChRs are sensitive and perhaps pathophysiologically relevant targets for Abeta neurotoxicity in AD.