RESUMEN
First-in-line benzodiazepine treatment fails to terminate seizures in about 30% of epilepsy patients, highlighting a need for novel anti-seizure strategies. It is emerging that impaired K+/Cl- cotransporter 2 (KCC2) activity leads to deficits in GABAergic inhibition and increased seizure vulnerability in patients. In neurons, the with-no-lysine (WNK) kinase-STE20/SPS1-related proline/alanine-rich (SPAK) kinase signalling pathway inhibits KCC2 activity via T1007 phosphorylation. Here, we exploit the selective WNK kinase inhibitor WNK463 to test the effects of pharmacological WNK inhibition on KCC2 function, GABAergic inhibition, and epileptiform activity. Immunoprecipitation and western blotting analysis revealed that WNK463 reduces KCC2-T1007 phosphorylation in vitro and in vivo. Using patch-clamp recordings in primary rat neurons, we further observed that WNK463 hyperpolarized the Cl- reversal potential, and enhanced KCC2-mediated Cl- extrusion. In the 4-aminopyridine slice model of acute seizures, WNK463 administration reduced the frequency and number of seizure-like events. In vivo, C57BL/6 mice that received intrahippocampal WNK463 experienced delayed onset of kainic acid-induced status epilepticus, less epileptiform EEG activity, and did not develop pharmaco-resistance to diazepam. Our findings demonstrate that acute WNK463 treatment potentiates KCC2 activity in neurons and limits seizure burden in two well-established models of seizures and epilepsy. In summary, our work suggests that agents which act to increase KCC2 activity may be useful adjunct therapeutics to alleviate diazepam-resistant status epilepticus.
Asunto(s)
Epilepsia , Estado Epiléptico , Simportadores , Animales , Diazepam/metabolismo , Diazepam/farmacología , Hipocampo/metabolismo , Humanos , Lisina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratas , Estado Epiléptico/inducido químicamente , Estado Epiléptico/tratamiento farmacológico , Estado Epiléptico/metabolismo , Simportadores/metabolismoRESUMEN
A robust body of evidence supports the concept that phosphodiesterase 10A (PDE10A) activity in the basal ganglia orchestrates the control of coordinated movement in human subjects. Although human mutations in the PDE10A gene manifest in hyperkinetic movement disorders that phenocopy many features of early Huntington's disease, characterization of the maladapted molecular mechanisms and aberrant signaling processes that underpin these conditions remains scarce. Recessive mutations in the GAF-A domain have been shown to impair PDE10A function due to the loss of striatal PDE10A protein levels, but here we show that this paucity is caused by irregular intracellular trafficking and increased PDE10A degradation in the cytosolic compartment. In contrast to GAF-A mutants, dominant mutations in the GAF-B domain of PDE10A induce PDE10A misfolding, a common pathological phenotype in many neurodegenerative diseases. These data demonstrate that the function of striatal PDE10A is compromised in disorders where disease-associated mutations trigger a reduction in the fidelity of PDE compartmentalization.
Asunto(s)
Membrana Celular/metabolismo , Enfermedad de Huntington/genética , Neuronas/enzimología , Hidrolasas Diéster Fosfóricas/genética , Dominios Proteicos/genética , Animales , Autofagia/genética , Cuerpo Estriado/citología , Cuerpo Estriado/patología , AMP Cíclico/metabolismo , Embrión de Mamíferos , Células HEK293 , Humanos , Enfermedad de Huntington/patología , Hidrólisis , Isoenzimas/genética , Isoenzimas/metabolismo , Mutación , Neuronas/citología , Técnicas de Placa-Clamp , Hidrolasas Diéster Fosfóricas/metabolismo , Cultivo Primario de Células , Proteolisis , Ratas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
The type 2 K+/Cl- cotransporter (KCC2) allows neurons to maintain low intracellular levels of Cl-, a prerequisite for efficient synaptic inhibition. Reductions in KCC2 activity are evident in epilepsy; however, whether these deficits directly contribute to the underlying pathophysiology remains controversial. To address this issue, we created knock-in mice in which threonines 906 and 1007 within KCC2 have been mutated to alanines (KCC2-T906A/T1007A), which prevents its phospho-dependent inactivation. The respective mice appeared normal and did not show any overt phenotypes, and basal neuronal excitability was unaffected. KCC2-T906A/T1007A mice exhibited increased basal neuronal Cl- extrusion, without altering total or plasma membrane accumulation of KCC2. Critically, activity-induced deficits in synaptic inhibition were reduced in the mutant mice. Consistent with this, enhanced KCC2 was sufficient to limit chemoconvulsant-induced epileptiform activity. Furthermore, this increase in KCC2 function mitigated induction of aberrant high-frequency activity during seizures, highlighting depolarizing GABA as a key contributor to the pathological neuronal synchronization seen in epilepsy. Thus, our results demonstrate that potentiating KCC2 represents a therapeutic strategy to alleviate seizures.
Asunto(s)
Epilepsia/metabolismo , Neuronas/metabolismo , Convulsiones/metabolismo , Simportadores/metabolismo , Membranas Sinápticas/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Sustitución de Aminoácidos , Animales , Epilepsia/genética , Epilepsia/patología , Técnicas de Sustitución del Gen , Ratones , Mutación Missense , Neuronas/patología , Convulsiones/genética , Convulsiones/patología , Simportadores/genética , Membranas Sinápticas/genética , Membranas Sinápticas/patología , Ácido gamma-Aminobutírico/genética , Cotransportadores de K ClRESUMEN
Estrogen plays a critical role in many physiological processes and exerts profound effects on behavior by regulating neuronal excitability. While estrogen has been established to exert effects on dendritic morphology and excitatory neurotransmission its role in regulating neuronal inhibition is poorly understood. Fast synaptic inhibition in the adult brain is mediated by specialized populations of γ-c aA receptors (GABAARs) that are selectively enriched at synapses, a process dependent upon their interaction with the inhibitory scaffold protein gephyrin. Here we have assessed the role that estradiol (E2) plays in regulating the dynamics of GABAARs and stability of inhibitory synapses. Treatment of cultured cortical neurons with E2 reduced the accumulation of GABAARs and gephyrin at inhibitory synapses. However, E2 exposure did not modify the expression of either the total or the plasma membrane GABAARs or gephyrin. Mechanistically, single-particle tracking revealed that E2 treatment selectively reduced the dwell time and thereby decreased the confinement of GABAARs at inhibitory synapses. Consistent with our cell biology measurements, we observed a significant reduction in amplitude of inhibitory synaptic currents in both cultured neurons and hippocampal slices exposed to E2, while their frequency was unaffected. Collectively, our results suggest that acute exposure of neurons to E2 leads to destabilization of GABAARs and gephyrin at inhibitory synapses, leading to reductions in the efficacy of GABAergic inhibition via a postsynaptic mechanism.
Asunto(s)
Estradiol/farmacología , Inhibición Neural/efectos de los fármacos , Receptores de GABA-A/metabolismo , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Animales , Proteínas Portadoras/farmacología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Células Cultivadas , Femenino , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Proteínas de la Membrana/farmacología , Ratones , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Transmisión Sináptica/efectos de los fármacosRESUMEN
apoE is the primary lipid carrier within the CNS and the strongest genetic risk factor for late onset Alzheimer's disease (AD). apoE is primarily lipidated via ABCA1, and both are under transcriptional regulation by the nuclear liver X receptor (LXR). Considerable evidence from genetic (using ABCA1 overexpression) and pharmacological (using synthetic LXR agonists) studies in AD mouse models suggests that increased levels of lipidated apoE can improve cognitive performance and, in some strains, can reduce amyloid burden. However, direct synthetic LXR ligands have hepatotoxic side effects that limit their clinical use. Here, we describe a set of small molecules, previously annotated as antagonists of the purinergic receptor, P2X7, which enhance ABCA1 expression and activity as well as apoE secretion, and are not direct LXR ligands. Furthermore, P2X7 is not required for these molecules to induce ABCA1 upregulation and apoE secretion, demonstrating that the ABCA1 and apoE effects are mechanistically independent of P2X7 inhibition. Hence, we have identified novel dual activity compounds that upregulate ABCA1 across multiple CNS cell types, including human astrocytes, pericytes, and microglia, through an indirect LXR mechanism and that also independently inhibit P2X7 receptor activity.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/agonistas , Apolipoproteínas E/agonistas , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Transportador 1 de Casete de Unión a ATP/metabolismo , Adamantano/análogos & derivados , Adamantano/química , Adamantano/farmacología , Animales , Apolipoproteínas E/metabolismo , Aziridinas/química , Aziridinas/farmacología , Benzamidas/química , Benzamidas/farmacología , Células Cultivadas , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Naftoquinonas/química , Naftoquinonas/farmacología , Antagonistas del Receptor Purinérgico P2X/química , Receptores Purinérgicos P2X7/deficiencia , Bibliotecas de Moléculas Pequeñas/química , Sulfonamidas/química , Sulfonamidas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
K+/Cl- cotransporter 2 (KCC2) is selectively expressed in the adult nervous system and allows neurons to maintain low intracellular Cl- levels. Thus, KCC2 activity is an essential prerequisite for fast hyperpolarizing synaptic inhibition mediated by type A γ-aminobutyric acid (GABAA) receptors, which are Cl--permeable, ligand-gated ion channels. Consistent with this, deficits in the activity of KCC2 lead to epilepsy and are also implicated in neurodevelopmental disorders, neuropathic pain, and schizophrenia. Accordingly, there is significant interest in developing activators of KCC2 as therapeutic agents. To provide insights into the cellular processes that determine KCC2 activity, we have investigated the mechanism by which N-ethylmaleimide (NEM) enhances transporter activity using a combination of biochemical and electrophysiological approaches. Our results revealed that, within 15 min, NEM increased cell surface levels of KCC2 and modulated the phosphorylation of key regulatory residues within the large cytoplasmic domain of KCC2 in neurons. More specifically, NEM increased the phosphorylation of serine 940 (Ser-940), whereas it decreased phosphorylation of threonine 1007 (Thr-1007). NEM also reduced with no lysine (WNK) kinase phosphorylation of Ste20-related proline/alanine-rich kinase (SPAK), a kinase that directly phosphorylates KCC2 at residue Thr-1007. Mutational analysis revealed that Thr-1007 dephosphorylation mediated the effects of NEM on KCC2 activity. Collectively, our results suggest that compounds that either increase the surface stability of KCC2 or reduce Thr-1007 phosphorylation may be of use as enhancers of KCC2 activity.
Asunto(s)
Etilmaleimida/metabolismo , Simportadores/metabolismo , Animales , Membrana Celular/metabolismo , Embrión de Mamíferos , Humanos , Moduladores del Transporte de Membrana/metabolismo , Neuronas/metabolismo , Fosforilación/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de GABA/metabolismo , Simportadores/fisiología , Cotransportadores de K ClRESUMEN
The behavioral and anatomical deficits seen in fragile X syndrome (FXS) are widely believed to result from imbalances in the relative strengths of excitatory and inhibitory neurotransmission. Although modified neuronal excitability is thought to be of significance, the contribution that alterations in GABAergic inhibition play in the pathophysiology of FXS are ill defined. Slow sustained neuronal inhibition is mediated by γ-aminobutyric acid type B (GABAB) receptors, which are heterodimeric G-protein-coupled receptors constructed from R1a and R2 or R1b and R2 subunits. Via the activation of Gi/o, they limit cAMP accumulation, diminish neurotransmitter release, and induce neuronal hyperpolarization. Here we reveal that selective deficits in R1a subunit expression are seen in Fmr1 knock-out mice (KO) mice, a widely used animal model of FXS, but the levels of the respective mRNAs were unaffected. Similar trends of R1a expression were seen in a subset of FXS patients. GABAB receptors (GABABRs) exert powerful pre- and postsynaptic inhibitory effects on neurotransmission. R1a-containing GABABRs are believed to mediate presynaptic inhibition in principal neurons. In accordance with this result, deficits in the ability of GABABRs to suppress glutamate release were seen in Fmr1-KO mice. In contrast, the ability of GABABRs to suppress GABA release and induce postsynaptic hyperpolarization was unaffected. Significantly, this deficit contributes to the pathophysiology of FXS as the GABABR agonist (R)-baclofen rescued the imbalances between excitatory and inhibitory neurotransmission evident in Fmr1-KO mice. Collectively, our results provided evidence that selective deficits in the activity of presynaptic GABABRs contribute to the pathophysiology of FXS.
Asunto(s)
Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/genética , Receptores de GABA-B/metabolismo , Anciano , Anciano de 80 o más Años , Animales , Conducta Animal , Modelos Animales de Enfermedad , Síndrome del Cromosoma X Frágil/metabolismo , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Fosforilación , ARN Mensajero/metabolismo , Receptores de GABA-B/genética , Serina/química , Transducción de Señal , Transmisión Sináptica , Ácido gamma-Aminobutírico/metabolismoRESUMEN
The K(+)/Cl(-) cotransporter (KCC2) allows adult neurons to maintain low intracellular Cl(-) levels, which are a prerequisite for efficient synaptic inhibition upon activation of γ-aminobutyric acid receptors. Deficits in KCC2 activity are implicated in epileptogenesis, but how increased neuronal activity leads to transporter inactivation is ill defined. In vitro, the activity of KCC2 is potentiated via phosphorylation of serine 940 (S940). Here we have examined the role this putative regulatory process plays in determining KCC2 activity during status epilepticus (SE) using knockin mice in which S940 is mutated to an alanine (S940A). In wild-type mice, SE induced by kainate resulted in dephosphorylation of S940 and KCC2 internalization. S940A homozygotes were viable and exhibited comparable basal levels of KCC2 expression and activity relative to WT mice. However, exposure of S940A mice to kainate induced lethality within 30 min of kainate injection and subsequent entrance into SE. We assessed the effect of the S940A mutation in cultured hippocampal neurons to explore the mechanisms underlying this phenotype. Under basal conditions, the mutation had no effect on neuronal Cl(-) extrusion. However, a selective deficit in KCC2 activity was seen in S940A neurons upon transient exposure to glutamate. Significantly, whereas the effects of glutamate on KCC2 function could be ameliorated in WT neurons with agents that enhance S940 phosphorylation, this positive modulation was lost in S940A neurons. Collectively our results suggest that phosphorylation of S940 plays a critical role in potentiating KCC2 activity to limit the development of SE.
Asunto(s)
Estado Epiléptico/metabolismo , Estado Epiléptico/patología , Simportadores/metabolismo , Animales , Cloruros/metabolismo , Endocitosis , Técnicas de Sustitución del Gen , Glutamatos/farmacología , Ratones , Ratones Mutantes Neurológicos , Proteínas Mutantes/metabolismo , Mutación/genética , Fosforilación , Fosfoserina/metabolismo , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 1/metabolismo , Simportadores/genética , Ácido gamma-Aminobutírico/metabolismo , Cotransportadores de K ClRESUMEN
The accumulation of γ-aminobutyric acid receptors (GABAARs) at the appropriate postsynaptic sites is critical for determining the efficacy of fast inhibitory neurotransmission. Although we know that the majority of synaptic GABAAR subtypes are assembled from α1-3, ß, and γ2 subunits, our understanding of how neurons facilitate their targeting to and stabilization at inhibitory synapses is rudimentary. To address these issues, we have created knock-in mice in which the pH-sensitive green fluorescent protein (GFP) and the Myc epitope were introduced to the extracellular domain of the mature receptor α2 subunit (pHα2). Using immunoaffinity purification and mass spectroscopy, we identified a stable complex of 174 proteins that were associated with pHα2, including other GABAAR subunits, and previously identified receptor-associated proteins such as gephyrin and collybistin. 149 of these proteins were novel GABAAR binding partners and included G-protein-coupled receptors and ion channel subunits, proteins that regulate trafficking and degradation, regulators of protein phosphorylation, GTPases, and a number of proteins that regulate their activity. Notably, members of the postsynaptic density family of proteins that are critical components of excitatory synapses were not associated with GABAARs. Crucially, we demonstrated for a subset of these novel proteins (including cullin1, ephexin, potassium channel tetramerization domain containing protein 12, mitofusin2, metabotropic glutamate receptor 5, p21-activated kinase 7, and Ras-related protein 5A) bind directly to the intracellular domains of GABAARs, validating our proteomic analysis. Thus, our experiments illustrate the complexity of the GABAAR proteome and enhance our understanding of the mechanisms neurons use to construct inhibitory synapses.
Asunto(s)
Proteínas Fluorescentes Verdes/metabolismo , Proteoma/metabolismo , Proteómica/métodos , Receptores de GABA-A/metabolismo , Sinapsis/metabolismo , Animales , Western Blotting , Fenómenos Electrofisiológicos , Proteínas Fluorescentes Verdes/genética , Células HEK293 , Hipocampo/metabolismo , Hipocampo/fisiología , Humanos , Concentración de Iones de Hidrógeno , Potenciales Postsinápticos Inhibidores , Espectrometría de Masas , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/metabolismo , Neuronas/fisiología , Proteoma/genética , Receptores de GABA-A/genética , Sinapsis/fisiologíaRESUMEN
GABA(A) receptors form Cl(-) permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K(+)/Cl(-) cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl(-) levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in E(GABA) values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg(2+) conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factor ex vivo and in vivo.
Asunto(s)
Hipocampo/fisiología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Simportadores/antagonistas & inhibidores , Simportadores/fisiología , Animales , Animales Recién Nacidos , Células Cultivadas , Células HEK293 , Hipocampo/efectos de los fármacos , Humanos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Ratas , Ratas Sprague-Dawley , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Cotransportadores de K ClRESUMEN
The KCC2 cotransporter establishes the low neuronal Cl(-) levels required for GABAA and glycine (Gly) receptor-mediated inhibition, and KCC2 deficiency in model organisms results in network hyperexcitability. However, no mutations in KCC2 have been documented in human disease. Here, we report two non-synonymous functional variants in human KCC2, R952H and R1049C, exhibiting clear statistical association with idiopathic generalized epilepsy (IGE). These variants reside in conserved residues in the KCC2 cytoplasmic C-terminus, exhibit significantly impaired Cl(-)-extrusion capacities resulting in less hyperpolarized Gly equilibrium potentials (EG ly), and impair KCC2 stimulatory phosphorylation at serine 940, a key regulatory site. These data describe a novel KCC2 variant significantly associated with a human disease and suggest genetically encoded impairment of KCC2 functional regulation may be a risk factor for the development of human IGE.
Asunto(s)
Epilepsia Generalizada/genética , Epilepsia Generalizada/metabolismo , Simportadores/genética , Simportadores/metabolismo , Potenciales de Acción , Alelos , Animales , Estudios de Casos y Controles , Línea Celular , Cloruros/metabolismo , Frecuencia de los Genes , Variación Genética , Hipocampo/metabolismo , Humanos , Modelos Moleculares , Mutación , Fosforilación , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Células Piramidales/metabolismo , Quebec , Ratas , Simportadores/química , Cotransportadores de K ClRESUMEN
Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.
Asunto(s)
Proteínas del Citoesqueleto/antagonistas & inhibidores , Potenciales Postsinápticos Excitadores/fisiología , Memoria a Largo Plazo/fisiología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Neuronas/metabolismo , Receptores de GABA-B/fisiología , Conducta Espacial/fisiología , Sinapsis/metabolismo , Animales , Células Cultivadas , Proteínas del Citoesqueleto/biosíntesis , Proteínas del Citoesqueleto/genética , Técnicas de Sustitución del Gen , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Aprendizaje por Laberinto/fisiología , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Técnicas de Cultivo de Órganos , Receptores de GABA-B/genéticaRESUMEN
The 5-HT3A receptor homology model, based on the partial structure of the nicotinic acetylcholine receptor from Torpedo marmorata, reveals an asymmetric ion channel with five portals framed by adjacent helical amphipathic (HA) stretches within the 114-residue loop between the M3 and M4 membrane-spanning domains. The positive charge of Arg-436, located within the HA stretch, is a rate-limiting determinant of single channel conductance (γ). Further analysis reveals that positive charge and volume of residue 436 are determinants of 5-HT3A receptor inward rectification, exposing an additional role for portals. A structurally unresolved stretch of 85 residues constitutes the bulk of the M3-M4 loop, leaving a >45-Šgap in the model between M3 and the HA stretch. There are no additional structural data for this loop, which is vestigial in bacterial pentameric ligand-gated ion channels and was largely removed for crystallization of the Caenorhabditis elegans glutamate-activated pentameric ligand-gated ion channels. We created 5-HT3A subunit loop truncation mutants, in which sequences framing the putative portals were retained, to determine the minimum number of residues required to maintain their functional integrity. Truncation to between 90 and 75 amino acids produced 5-HT3A receptors with unaltered rectification. Truncation to 70 residues abolished rectification and increased γ. These findings reveal a critical M3-M4 loop length required for functions attributable to cytoplasmic portals. Examination of all 44 subunits of the human neurotransmitter-activated Cys-loop receptors reveals that, despite considerable variability in their sequences and lengths, all M3-M4 loops exceed 70 residues, suggesting a fundamental requirement for portal integrity.
Asunto(s)
Conformación Proteica , Estructura Secundaria de Proteína , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/fisiología , Secuencia de Aminoácidos , Animales , Arginina/química , Arginina/genética , Arginina/fisiología , Sitios de Unión/genética , Células HEK293 , Humanos , Cinética , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/genética , Potenciales de la Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Técnicas de Placa-Clamp , Multimerización de Proteína , Receptores de Serotonina 5-HT3/genética , Homología de Secuencia de Aminoácido , Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Torpedo , TransfecciónRESUMEN
LMTK3 is a brain-specific transmembrane serine/threonine protein kinase that acts as a scaffold for protein phosphatase-1 (PP1). Although LMKT3 has been identified as a risk factor for autism and epilepsy, its physiological significance is unknown. Here, we demonstrate that LMTK3 copurifies and binds to KCC2, a neuron-specific K+/Cl- transporter. KCC2 activity is essential for Cl--mediated hyperpolarizing GABAAR receptor currents, the unitary events that underpin fast synaptic inhibition. LMTK3 acts to promote the association of KCC2 with PP1 to promote the dephosphorylation of S940 within its C-terminal cytoplasmic domain, a process the diminishes KCC2 activity. Accordingly, acute inhibition of LMTK3 increases KCC2 activity dependent upon S940 and increases neuronal Cl- extrusion. Consistent with this, LMTK3 inhibition reduced intrinsic neuronal excitability and the severity of seizure-like events in vitro. Thus, LMTK3 may have profound effects on neuronal excitability as an endogenous modulator of KCC2 activity.
RESUMEN
The K(+) -Cl(-) cotransporter type 2 is the major Cl(-) extrusion mechanism in most adult neurons. This process in turn leads to Cl(-) influx upon activation of γ-aminobutyric acid type A (GABAA ) receptors and the canonical hyperpolarising inhibitory postsynaptic potential. Several neurological disorders are treated with drugs that target and enhance GABAA receptor signaling, including the commonly used benzodiazepine diazepam and the anesthetic propofol. Some of these disorders are also associated with deficits in GABAA signaling and become less sensitive to therapeutic drugs that target GABAA receptors. To date, it is unknown if alterations in the neuronal Cl(-) gradient affect the efficacies of diazepam and propofol. We therefore used the in vitro model of glutamate-induced hyperexcitability to test if alterations in the Cl(-) gradient affect the efficacy of GABAA modulators. We exclusively utilised the gramicidin perforated-patch-clamp configuration to preserve the endogenous Cl(-) gradient in rat neurons. Brief exposure to glutamate reduced the inhibitory efficacy of diazepam within 5 min, which was caused by the collapse of the Cl(-) gradient, and not due to reductions in GABAA receptor number. Unlike diazepam, propofol retained its efficacy by shunting the membrane conductance despite the glutamate-induced appearance of depolarising GABAA -mediated currents. Similarly, pharmacological inhibition of K(+) -Cl(-) cotransporter type 2 by furosemide disrupted Cl(-) homeostasis and reduced the efficacy of diazepam but not propofol. Collectively our results suggest that pathological hyperexcitable conditions could cause the rapid accumulation of intracellular Cl(-) and the appearance of depolarising GABAA -mediated currents that would decrease the efficacy of diazepam.
Asunto(s)
Diazepam/farmacología , Moduladores del GABA/farmacología , Inhibición Neural/efectos de los fármacos , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Simportadores/metabolismo , Animales , Células Cultivadas , Cloruros/metabolismo , Furosemida/farmacología , Ácido Glutámico/farmacología , Homeostasis , Muscimol/farmacología , Propofol/farmacología , Ratas , Ratas Sprague-Dawley , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Simportadores/efectos de los fármacos , Cotransportadores de K ClRESUMEN
Hyperpolarizing GABAAR currents, the unitary events that underlie synaptic inhibition, are dependent upon efficient Cl- extrusion, a process that is facilitated by the neuronal specific K+/Cl- co-transporter KCC2. Its activity is also a determinant of the anticonvulsant efficacy of the canonical GABAAR-positive allosteric: benzodiazepines (BDZs). Compromised KCC2 activity is implicated in the pathophysiology of status epilepticus (SE), a medical emergency that rapidly becomes refractory to BDZ (BDZ-RSE). Here, we have identified small molecules that directly bind to and activate KCC2, which leads to reduced neuronal Cl- accumulation and excitability. KCC2 activation does not induce any overt effects on behavior but prevents the development of and terminates ongoing BDZ-RSE. In addition, KCC2 activation reduces neuronal cell death following BDZ-RSE. Collectively, these findings demonstrate that KCC2 activation is a promising strategy to terminate BDZ-resistant seizures and limit the associated neuronal injury.
Asunto(s)
Estado Epiléptico , Simportadores , Ratones , Animales , Benzodiazepinas/farmacología , Benzodiazepinas/uso terapéutico , Estado Epiléptico/tratamiento farmacológico , Convulsiones/metabolismo , Ácido gamma-Aminobutírico/metabolismo , Simportadores/metabolismoRESUMEN
Picrotoxin and t-butylbicyclophosphorothionate (TBPS) are GABA(A) receptor (GABA(A)R) open channel blockers. However, picrotoxin displaceable [(35)S]TBPS binding to α1ß2γ2 GABA(A)Rs occurs in the absence of GABA, suggesting that access to the binding site is independent of activation. Alternatively, spontaneous gating may provide access to the channel. In the absence of episodic GABA application, picrotoxin and TBPS blocked (by 91 ± 3% and 85 ± 5%, respectively) GABA-evoked currents mediated by α1ß2γ2 receptors. We used two approaches to inhibit spontaneous GABA(A)R gating, bicuculline, which inhibits spontaneous current in the absence of exogenous agonist and the α1(K278M) mutant subunit. Whole-cell patch-clamp recordings demonstrated that α1(K278M)ß2γ2 receptors have negligible spontaneous gating. Application of bicuculline to α1ß2γ2 receptors in the absence of exogenous GABA caused a 35% reduction of current blockade by TBPS and reduced [(35)S]TBPS binding by 25%. Consistent with this, in the absence of exogenous GABA, α1(K278M)ß2γ2 receptors exhibited reduced blockade by TBPS current compared to wild-type receptors. These data suggest that a decrease in spontaneous gating reduces accessibility of TBPS to its binding site. GABA application during picrotoxin or TBPS administration enhanced α1ß2γ2 receptor blockade (to 98% in both cases). The GABA-dependent component of TBPS blockade accounts for the stimulation of [(35)S]TBPS binding to α1ß2γ2 receptors seen with GABA (1 µm) application. Moreover, application of GABA at concentrations that cause significant steady-state desensitization reduced [(35)S]TBPS binding. The α1(K278M) subunit slowed desensitization kinetics and increased the rate of deactivation of GABA-evoked currents. Furthermore, there was a marked increase in the GABA EC(50) for desensitization of α1(K278M)ß2γ2 receptors associated with a large increase in the GABA-dependent stimulation of [(35)S]TBPS binding. These data establish a relationship between GABA(A)R function and the three phases of [(35)S]TBPS binding seen in the absence and the presence of GABA.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Antagonistas de Receptores de GABA-A/farmacología , Activación del Canal Iónico/efectos de los fármacos , Receptores de GABA-A/metabolismo , Bicuculina/farmacología , Sitios de Unión , Línea Celular , Glicina/metabolismo , Células HEK293 , Humanos , Mutación , Técnicas de Placa-Clamp/métodos , Picrotoxina/farmacología , Receptores de Serotonina 5-HT3/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Benzodiazepines have been used for decades as first-line treatment for status epilepticus (SE). For reasons that are not fully understood, the efficacy of benzodiazepines decreases with increasing duration of seizure activity. This often forces clinicians to resort to more drastic second- and third-line treatments that are not always successful. The antiseizure properties of benzodiazepines are mediated by γ-aminobutyric acid type A (GABA(A) ) receptors. Decades of research have focused on the failure of GABAergic inhibition after seizure onset as the likely cause of the development benzodiazepine resistance during SE. However, the details of the deficits in GABA(A) signaling are still largely unknown. Therefore, it is necessary to improve our understanding of the mechanisms of benzodiazepine resistance so that more effective strategies can be formulated. In this review we discuss evidence supporting the role of altered GABA(A) receptor function as the major underlying cause of benzodiazepine-resistant SE in both humans and animal models. We specifically address the prevailing hypothesis, which is based on changes in the number and subtypes of GABA(A) receptors, as well as the potential influence of perturbed chloride homeostasis in the mature brain.
Asunto(s)
Anticonvulsivantes/farmacología , Benzodiazepinas/farmacología , Epilepsia/tratamiento farmacológico , Receptores de GABA-A/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Animales , Anticonvulsivantes/uso terapéutico , Benzodiazepinas/uso terapéutico , Cloruros/metabolismo , Resistencia a Medicamentos , Moduladores del GABA/farmacología , Moduladores del GABA/uso terapéutico , Homeostasis , Humanos , Iones/metabolismo , Ratones , Ratas , Receptores de GABA-A/metabolismo , Convulsiones/tratamiento farmacológico , Convulsiones/fisiopatologíaRESUMEN
The cysteine (Cys) residue at position 312 in the third transmembrane domain (M3) is conserved among 5-hydroxytryptamine type 3 (5-HT(3)) receptor subunits and many other subunits of the nicotinic acetylcholine (nACh) related Cys-loop receptor family, including most of the gamma-aminobutyric acid type A (GABA(A)) and glycine receptor subunits. To elucidate a possible role for the Cys-312 in human 5-HT(3)A receptors, we replaced it with alanine and expressed the 5-HT(3)A(C312A) mutant in HEK293 cells. The mutation resulted in an absence of 5-HT-induced whole-cell current without reducing homopentamer formation, surface expression or 5-HT binding. The 5-HT(3)A(C312A) mutant, when co-expressed with the wild-type 5-HT(3)A subunit, did not affect functional expression of receptors, suggesting that the mutant is not dominant negative. Interestingly, co-expression of 5-HT(3)A(C312A) with 5-HT(3)B led to surface expression of heteropentamers that mediated small 5-HT responses. This suggests that the Cys-312 is essential for homomeric but not heteromeric receptor gating. To further investigate the relationship between residue 312 and gating we replaced it with amino acids located at the equivalent position within other Cys-loop subunits that are either capable or incapable of forming functional homopentamers. Replacement of 5-HT(3)A Cys-312 by Gly or Leu (equivalent residues in the nACh receptor delta and gamma subunits) abolished and severely attenuated function, respectively, whereas replacement by Thr or Ser (equivalent residues in nACh receptor alpha7 and GABA(A) subunits) supported robust function. Thus, 5-HT(3)A residue 312 and equivalent polar residues in the M3 of other Cys-loop subunits are essential determinants of homopentameric gating.
Asunto(s)
Secuencia Conservada , Cisteína/química , Cisteína/fisiología , Receptores de Serotonina 5-HT3/química , Receptores de Serotonina 5-HT3/fisiología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Cisteína/genética , Humanos , Datos de Secuencia Molecular , Mutación Missense , Técnicas de Placa-Clamp , Receptores Nicotínicos/fisiología , Receptores de Serotonina 5-HT3/genética , Serotonina/fisiología , Receptor Nicotínico de Acetilcolina alfa 7RESUMEN
Kcc2 plays a critical role in determining the efficacy of synaptic inhibition, however, the cellular mechanisms neurons use to regulate its membrane trafficking, stability and activity are ill-defined. To address these issues, we used affinity purification to isolate stable multi-protein complexes of K-Cl Co-transporter 2 (Kcc2) from the plasma membrane of murine forebrain. We resolved these using blue-native polyacrylamide gel electrophoresis (BN-PAGE) coupled to LC-MS/MS and label-free quantification. Data are available via ProteomeXchange with identifier PXD021368. Purified Kcc2 migrated as distinct molecular species of 300, 600, and 800 kDa following BN-PAGE. In excess of 90% coverage of the soluble N- and C-termini of Kcc2 was obtained. In total we identified 246 proteins significantly associated with Kcc2. The 300 kDa species largely contained Kcc2, which is consistent with a dimeric quaternary structure for this transporter. The 600 and 800 kDa species represented stable multi-protein complexes of Kcc2. We identified a set of novel structural, ion transporting, immune related and signaling protein interactors, that are present at both excitatory and inhibitory synapses, consistent with the proposed localization of Kcc2. These included spectrins, C1qa/b/c and the IP3 receptor. We also identified interactors more directly associated with phosphorylation; Akap5, Akap13, and Lmtk3. Finally, we used LC-MS/MS on the same purified endogenous plasma membrane Kcc2 to detect phosphorylation sites. We detected 11 sites with high confidence, including known and novel sites. Collectively our experiments demonstrate that Kcc2 is associated with components of the neuronal cytoskeleton and signaling molecules that may act to regulate transporter membrane trafficking, stability, and activity.