RESUMO
OBJECTIVE: To elucidate the biophysical basis underlying the distinct and severe clinical presentation in patients with the recurrent missense SCN1A variant, p.Thr226Met. Patients with this variant show a well-defined genotype-phenotype correlation and present with developmental and early infantile epileptic encephalopathy that is far more severe than typical SCN1A Dravet syndrome. METHODS: Whole cell patch clamp and dynamic action potential clamp were used to study T226M Nav 1.1 channels expressed in mammalian cells. Computational modeling was used to explore the neuronal scale mechanisms that account for altered action potential firing. RESULTS: T226M channels exhibited hyperpolarizing shifts of the activation and inactivation curves and enhanced fast inactivation. Dynamic action potential clamp hybrid simulation showed that model neurons containing T226M conductance displayed a left shift in rheobase relative to control. At current stimulation levels that produced repetitive action potential firing in control model neurons, depolarization block and cessation of action potential firing occurred in T226M model neurons. Fully computationally simulated neuron models recapitulated the findings from dynamic action potential clamp and showed that heterozygous T226M models were also more susceptible to depolarization block. INTERPRETATION: From a biophysical perspective, the T226M mutation produces gain of function. Somewhat paradoxically, our data suggest that this gain of function would cause interneurons to more readily develop depolarization block. This "functional dominant negative" interaction would produce a more profound disinhibition than seen with haploinsufficiency that is typical of Dravet syndrome and could readily explain the more severe phenotype of patients with T226M mutation. Ann Neurol 2019;85:514-525.
Assuntos
Epilepsias Mioclônicas/genética , Mutação com Ganho de Função/genética , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Espasmos Infantis/genética , Animais , Células CHO , Cricetulus , Bases de Dados Genéticas , Epilepsias Mioclônicas/diagnóstico , Humanos , Espasmos Infantis/diagnósticoRESUMO
Ionotropic glutamate receptors are the most important excitatory receptors in the central nervous system, and their impairment can lead to multiple neuronal diseases. Here, we show that glutamate-induced currents in oocytes expressing GluA1 are increased by coexpression of the schizophrenia-associated phosphoinositide kinase PIP5K2A. This effect was due to enhanced membrane abundance and was blunted by a point mutation (N251S) in PIP5K2A. An increase in GluA1 currents was also observed upon acute injection of PI(4,5)P2, the main product of PIP5K2A. By expression of wild-type and mutant PIP5K2A in human embryonic kidney cells, we were able to provide evidence of impaired kinase activity of the mutant PIP5K2A. We defined the region K813-K823 of GluA1 as critical for the PI(4,5)P2 effect by performing an alanine scan that suggested PI(4,5)P2 binding to this area. A PIP strip assay revealed PI(4,5)P2 binding to the C-terminal GluA1 peptide. The present observations disclose a novel mechanism in the regulation of GluA1.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/química , Receptores de AMPA/química , Alanina/química , Alanina/genética , Alanina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células HEK293 , Humanos , Dados de Sequência Molecular , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , XenopusRESUMO
Xenopus laevis oocytes are an outstanding heterologous expression system for the investigation of ion channels. However, oocytes express an amazing variety of endogenous ion channels that can severely interfere with electrophysiological measurements. It is therefore necessary to be aware of the channels present in the oocyte and to be able to exclude artifacts they might cause during the analysis of heterologously expressed ion channels. Research on Xenopus endogenous ion channels has started over 30 years ago, and many channels have been described since then. This does not only include voltage-gated channels conducting Na(+), K(+), Ca(2+), and Cl(-), but also ion channels activated by ligand binding such as ionotropic neurotransmitter receptors. Furthermore, there are other channels such as those triggered by changes in osmolarity or mechanical stress, as well as conductances caused by yet uncharacterized molecules. Here, we present an overview of ion channels endogenous to the oocyte described in the literature so far, and provide procedures and methods to abolish or minimize their impact on electrophysiological recordings of exogenous channels.
Assuntos
Canais Iônicos/metabolismo , Oócitos/citologia , Xenopus laevis/metabolismo , Animais , Canais de Cálcio/metabolismo , Canais de Cloreto/metabolismo , Eletrofisiologia/métodos , Canais Iônicos/química , Íons , Ligantes , Modelos Biológicos , Neurotransmissores , Oócitos/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Sódio/metabolismoRESUMO
The involvement of neurotransmission in neuronal development is a generally accepted concept. Nevertheless, the precise regulation of neurotransmitter receptor expression is still unclear. To investigate the expression profiles of the most important ionotropic neurotransmitter receptors, namely GABA(A) receptors (GABA(A)Rs), NMDA receptors (NMDARs), and AMPA receptors (AMPARs), quantitative RT-PCR, immunoblot analysis and patch clamp studies were performed in in vitro-generated neural stem cells (NSCs). This clearly defined cell line is closely related to radial glia cells, the stem cells in the neonate brain. We found functional GABA(A)Rs of the subunit composition alpha2, beta3, and gamma1 to be expressed. Unexpectedly, functional ionotropic glutamate receptors were absent. However, NSCs expressed the NMDAR subunits NR2A and NR3A, and the AMPAR subunit GluR4 at the protein level, and GluR3 at the mRNA level. The overexpression of functional NMDARs in NSCs led to an increased mRNA level of AMPAR subunits, indicating a role in synaptogenesis. Early neuronal markers remained unchanged. These data extend our knowledge about ionotropic neurotransmitter receptor expression during neuronal development and will aid further investigations on activity-dependent neurogenesis.
Assuntos
Células-Tronco Embrionárias/fisiologia , Neuroglia/metabolismo , Neurônios/metabolismo , Receptores de GABA-A/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Bicuculina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Embrião de Mamíferos , Células-Tronco Embrionárias/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Antagonistas GABAérgicos/farmacologia , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , N-Metilaspartato/farmacologia , Neurogênese/fisiologia , Neuroglia/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp/métodos , Bloqueadores dos Canais de Potássio/farmacologia , Subunidades Proteicas/genética , RNA Mensageiro/metabolismo , Receptores de GABA-A/genética , Receptores de N-Metil-D-Aspartato/genética , Fatores de Transcrição SOXB1/metabolismo , Simportadores de Cloreto de Sódio-Potássio/genética , Simportadores de Cloreto de Sódio-Potássio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Tetraetilamônio/farmacologia , Transfecção/métodos , Ácido gama-Aminobutírico/farmacologiaRESUMO
Alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptors are essential players in fast synaptic transmission in the vertebrate central nervous system. Their synaptic delivery and localization as well as their electrophysiological properties are regulated by transmembrane AMPA receptor regulatory proteins (TARPs). However, the exact mechanisms of how the four originally designated TARPs (gamma2, gamma3, gamma4, and gamma8) modulate AMPA receptor function remain largely unknown. Previous studies suggested the C-terminal domain (CTD) of gamma2 to mediate increased trafficking and reduced desensitization of AMPA receptors. As it remained unclear whether these findings extend to other TARPs, we set out to investigate and compare the role of the CTDs of the four original TARPs in AMPA receptor modulation. To address this issue, we replaced the TARP CTDs with the CTD of the homologous subunit gamma1, a voltage-dependent calcium channel subunit expressed in skeletal muscle that lacks TARP properties. We analyzed the impact of the resulting chimeras on GluR1 functional properties in Xenopus oocytes and HEK293 cells. Interestingly, the CTDs of all TARPs not only modulate the extent and kinetics of desensitization but also modulate agonist potencies of AMPA receptors. Furthermore, the CTDs are required for TARP-induced modulation of AMPA receptor gating, including conversion of antagonists to partial agonists and constitutive channel openings. Strikingly, we found a special role of the cytoplasmic tail of gamma4, suggesting that the underlying mechanisms of modulation of AMPA receptor function are different among the TARPs. We propose that the intracellularly located CTD is the origin of TARP-specific functional modulation and not merely a facilitator of trafficking.
Assuntos
Regulação da Expressão Gênica , Receptores de AMPA/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Citoplasma/metabolismo , Humanos , Cinética , Modelos Biológicos , Oócitos/metabolismo , Estrutura Terciária de Proteína , Ratos , Receptores de AMPA/química , Transmissão Sináptica , Xenopus laevisRESUMO
N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, learning and memory, and are implicated in various neuronal disorders. We synthesized a diffusible photochromic glutamate analogue, azobenzene-triazole-glutamate (ATG), which is specific for NMDARs and functions as a photoswitchable agonist. ATG is inactive in its dark-adapted trans-isoform, but can be converted into its active cis-isoform using one-photon (near UV) or two-photon (740 nm) excitation. Irradiation with violet light photo-inactivates ATG within milliseconds, allowing agonist removal on the timescale of NMDAR deactivation. ATG is compatible with Ca(2+) imaging and can be used to optically mimic synaptic coincidence detection protocols. Thus, ATG can be used like traditional caged glutamate compounds, but with the added advantages of NMDAR specificity, low antagonism of GABAR-mediated currents, and precise temporal control of agonist delivery.
Assuntos
Região CA1 Hipocampal/metabolismo , Córtex Cerebral/metabolismo , Ácido Glutâmico/análogos & derivados , Luz , Células Piramidais/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Animais , Hipocampo/metabolismo , Camundongos , Oócitos , Técnicas de Patch-Clamp , Isoformas de Proteínas , Ratos , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Raios Ultravioleta , Xenopus laevisRESUMO
Ionotropic glutamate receptors (iGluRs) do not only mediate the majority of excitatory neurotransmission in the vertebrate CNS, but also modulate pre- and postnatal neurogenesis. Most of the studies on the developmental role of iGluRs are performed on neural progenitors and neural stem cells (NSCs). We took a step back in our study by examining the role of iGluRs in the earliest possible cell type, embryonic stem cells (ESCs), by looking at the mRNA expression of the major iGluR subfamilies in undifferentiated mouse ESCs. For that, we used two distinct murine ES cell lines, 46C ESCs and J1 ESCs. Regarding 46C ESCs, we found transcripts of kainate receptors (KARs) (GluK2 to GluK5), AMPA receptors (AMPARs) (GluA1, GluA3, and GluA4), and NMDA receptors (NMDARs) (GluN1, and GluN2A to GluN2D). Analysis of 46C-derived cells of later developmental stages, namely neuroepithelial precursor cells (NEPs) and NSCs, revealed that the mRNA expression of KARs is significantly upregulated in NEPs and, subsequently, downregulated in NSCs. However, we could not detect any protein expression of any of the KAR subunits present on the mRNA level either in ESCs, NEPs, or NSCs. Regarding AMPARs and NMDARs, GluN2A is weakly expressed at the protein level only in NSCs. Matching our findings for iGluRs, all three cell types were found to weakly express pre- and postsynaptic markers of glutamatergic synapses only at the mRNA level. Finally, we performed patch-clamp recordings of 46C ESCs and could not detect any current upon iGluR agonist application. Similar to 46C ESCs, J1 ESCs express KARs (GluK2 to GluK5), AMPARs (GluA3), and NMDARs (GluN1, and GluN2A to GluN2D) at the mRNA level, but these transcripts are not translated into receptor proteins either. Thus, we conclude that ESCs do not contain functional iGluRs, although they do express an almost complete set of iGluR subunit mRNAs.
RESUMO
Ionotropic glutamate receptors are major players in synaptic transmission and are critically involved in many cognitive events. Although receptors of different subfamilies serve different functions, they all show a conserved domain topology. For most of these domains, structure-function relationships have been established and are well understood. However, up to date the role of the transmembrane domain C in receptor function has been investigated only poorly. We have constructed a series of receptor chimeras and point mutants designed to shed light on the structural and/or functional importance of this domain. We here present evidence that the role of transmembrane domain C exceeds that of a mere scaffolding domain and that several amino acid residues located within the domain are crucial for receptor gating and desensitization. Furthermore, our data suggest that the domain may be involved in receptor interaction with transmembrane AMPA receptor regulatory proteins.