RESUMEN
Proline-rich transmembrane protein 2 (PRRT2) is the single causative gene for pleiotropic paroxysmal syndromes, including epilepsy, kinesigenic dyskinesia, episodic ataxia, and migraine. PRRT2 is a neuron-specific type-2 membrane protein with a COOH-terminal intramembrane domain and a long proline-rich NH2-terminal cytoplasmic region. A large array of experimental data indicates that PRRT2 is a neuron stability gene that negatively controls intrinsic excitability by regulating surface membrane localization and biophysical properties of voltage-dependent Na+ channels Nav1.2 and Nav1.6, but not Nav1.1. To further investigate the regulatory role of PRRT2, we studied the structural features of this membrane protein with molecular dynamics simulations, and its structure-function relationships with Nav1.2 channels by biochemical and electrophysiological techniques. We found that the intramembrane COOH-terminal region maintains a stable conformation over time, with the first transmembrane domain forming a helix-loop-helix motif within the bilayer. The unstructured NH2-terminal cytoplasmic region bound to the Nav1.2 better than the isolated COOH-terminal intramembrane domain, mimicking full-length PRRT2, while the COOH-terminal intramembrane domain was able to modulate Na+ current and channel biophysical properties, still maintaining the striking specificity for Nav1.2 versus Nav1.1. channels. The results identify PRRT2 as a dual-domain protein in which the NH2-terminal cytoplasmic region acts as a binding antenna for Na+ channels, while the COOH-terminal membrane domain regulates channel exposure on the membrane and its biophysical properties.
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Proteínas de la Membrana , Modelos Moleculares , Proteínas del Tejido Nervioso , Canales de Sodio , Humanos , Biofisica , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Simulación de Dinámica Molecular , Canales de Sodio/química , Canales de Sodio/metabolismo , Mutación , Células HEK293 , Estructura Terciaria de Proteína , Unión ProteicaRESUMEN
Dietary restriction, such as low glycemic index diet (LGID), have been successfully used to treat drug-resistant epilepsy. However, if such diet could also counteract antiepileptogenesis is still unclear. Here, we investigated whether the administration of LGID during the latent pre-epileptic period, prevents or delays the appearance of the overt epileptic phenotype. To this aim, we used the Synapsin II knockout (SynIIKO) mouse, a model of temporal lobe epilepsy in which seizures manifest 2-3 months after birth, offering a temporal window in which LGID may affect epileptogenesis. Pregnant SynIIKO mice were fed with either LGID or standard diet during gestation and lactation. Both diets were maintained in weaned mice up to 5 months of age. LGID delayed the seizure onset and induced a reduction of seizures severity only in female SynIIKO mice. In parallel with the epileptic phenotype, high-density multielectrode array recordings revealed a reduction of frequency, amplitude, duration, velocity of propagation and spread of interictal events by LGID in the hippocampus of SynIIKO females, but not mutant males, confirming the gender-specific effect. ELISA-based analysis revealed that LGID increased cortico-hippocampal allopregnanolone (ALLO) levels only in females, while it was unable to affect ALLO plasma concentrations in either sex. The results indicate that the gender-specific interference of LGID with the epileptogenic process can be ascribed to a gender-specific increase in cortical ALLO, a neurosteroid known to strengthen GABAergic transmission. The study highlights the possibility of developing a personalized gender-based therapy for temporal lobe epilepsy.
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Epilepsia del Lóbulo Temporal , Epilepsia , Masculino , Embarazo , Femenino , Ratones , Animales , Epilepsia del Lóbulo Temporal/genética , Epilepsia del Lóbulo Temporal/inducido químicamente , Índice Glucémico , Convulsiones , Hipocampo , Epilepsia/genética , DietaRESUMEN
Neuron-restrictive silencer factor/repressor element 1 (RE1)-silencing transcription factor (NRSF/REST) is a transcriptional repressor of a large cluster of neural genes containing RE1 motifs in their promoter region. NRSF/REST is ubiquitously expressed in non-neuronal cells, including astrocytes, while it is down-regulated during neuronal differentiation. While neuronal NRSF/REST homeostatically regulates intrinsic excitability and synaptic transmission, the role of the high NRSF/REST expression levels in the homeostatic functions of astrocytes is poorly understood. Here, we investigated the functional consequences of NRSF/REST deletion in primary cortical astrocytes derived from NRSF/REST conditional knockout mice (KO). We found that NRSF/REST KO astrocyte displayed a markedly reduced activity of inward rectifying K+ channels subtype 4.1 (Kir4.1) underlying spatial K+ buffering that was associated with a decreased expression and activity of the glutamate transporter-1 (GLT-1) responsible for glutamate uptake by astrocytes. The effects of the impaired astrocyte homeostatic functions on neuronal activity were investigated by co-culturing wild-type hippocampal neurons with NRSF/REST KO astrocytes. Interestingly, neurons experienced increased neuronal excitability at high firing rates associated with decrease after hyperpolarization and increased amplitude of excitatory postsynaptic currents. The data indicate that astrocytic NRSF/REST directly participates in neural circuit homeostasis by regulating intrinsic excitability and excitatory transmission and that dysfunctions of NRSF/REST expression in astrocytes may contribute to the pathogenesis of neurological disorders.
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Astrocitos , Factores de Transcripción , Ratones , Animales , Factores de Transcripción/genética , Astrocitos/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Regulación de la Expresión GénicaRESUMEN
PRRT2 is a neuronal protein that controls neuronal excitability and network stability by modulating voltage-gated Na+ channel (Nav). PRRT2 pathogenic variants cause pleiotropic syndromes including epilepsy, paroxysmal kinesigenic dyskinesia and episodic ataxia attributable to loss-of-function pathogenetic mechanism. Based on the evidence that the transmembrane domain of PRRT2 interacts with Nav1.2/1.6, we focused on eight missense mutations located within the domain that show expression and membrane localization similar to the wild-type protein. Molecular dynamics simulations showed that the mutants do not alter the structural stability of the PRRT2 membrane domain and preserve its conformation. Using affinity assays, we found that the A320V and V286M mutants displayed respectively decreased and increased binding to Nav1.2. Accordingly, surface biotinylation showed an increased Nav1.2 surface exposure induced by the A320V mutant. Electrophysiological analysis confirmed the lack of modulation of Nav1.2 biophysical properties by the A320V mutant with a loss-of-function phenotype, while the V286M mutant displayed a gain-of-function with respect to wild-type PRRT2 with a more pronounced left-shift of the inactivation kinetics and delayed recovery from inactivation. The data confirm the key role played by the PRRT2-Nav interaction in the pathogenesis of the PRRT2-linked disorders and suggest an involvement of the A320 and V286 residues in the interaction site. Given the similar clinical phenotype caused by the two mutations, we speculate that circuit instability and paroxysmal manifestations may arise when PRRT2 function is outside the physiological range.
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Mutación Missense , Canal de Sodio Activado por Voltaje NAV1.2 , Canal de Sodio Activado por Voltaje NAV1.2/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Mutación/genéticaRESUMEN
Neuroinflammation is involved in the pathogenesis of several neurologic disorders, including epilepsy. Both changes in the input/output functions of synaptic circuits and cell Ca2+ dysregulation participate in neuroinflammation, but their impact on neuron function in epilepsy is still poorly understood. Lipopolysaccharide (LPS), a toxic byproduct of bacterial lysis, has been extensively used to stimulate inflammatory responses both in vivo and in vitro LPS stimulates Toll-like receptor 4, an important mediator of the brain innate immune response that contributes to neuroinflammation processes. Although we report that Toll-like receptor 4 is expressed in both excitatory and inhibitory mouse hippocampal neurons (both sexes), its chronic stimulation by LPS induces a selective increase in the excitatory synaptic strength, characterized by enhanced synchronous and asynchronous glutamate release mechanisms. This effect is accompanied by a change in short-term plasticity with decreased facilitation, decreased post-tetanic potentiation, and increased depression. Quantal analysis demonstrated that the effects of LPS on excitatory transmission are attributable to an increase in the probability of release associated with an overall increased expression of L-type voltage-gated Ca2+ channels that, at presynaptic terminals, abnormally contributes to evoked glutamate release. Overall, these changes contribute to the excitatory/inhibitory imbalance that scales up neuronal network activity under inflammatory conditions. These results provide new molecular clues for treating hyperexcitability of hippocampal circuits associated with neuroinflammation in epilepsy and other neurologic disorders.SIGNIFICANCE STATEMENT Neuroinflammation is thought to have a pathogenetic role in epilepsy, a disorder characterized by an imbalance between excitation/inhibition. Fine adjustment of network excitability and regulation of synaptic strength are both implicated in the homeostatic maintenance of physiological levels of neuronal activity. Here, we focused on the effects of chronic neuroinflammation induced by lipopolysaccharides on hippocampal glutamatergic and GABAergic synaptic transmission. Our results show that, on chronic stimulation with lipopolysaccharides, glutamatergic, but not GABAergic, neurons exhibit an enhanced synaptic strength and changes in short-term plasticity because of an increased glutamate release that results from an anomalous contribution of L-type Ca2+ channels to neurotransmitter release.
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Canales de Calcio Tipo L/metabolismo , Epilepsia/metabolismo , Potenciales Postsinápticos Excitadores , Ácido Glutámico/metabolismo , Hipocampo/metabolismo , Animales , Células Cultivadas , Hipocampo/citología , Hipocampo/fisiología , Lipopolisacáridos/farmacología , Ratones , Ratones Endogámicos C57BL , Plasticidad Neuronal , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Receptor Toll-Like 4/metabolismoRESUMEN
PRoline-Rich Transmembrane protein-2 (PRRT2) is a recently described neuron-specific type-2 integral membrane protein with a large cytosolic N-terminal domain that distributes in presynaptic and axonal domains where it interacts with several presynaptic proteins and voltage-gated Na+ channels. Several PRRT2 mutations are the main cause of a wide and heterogeneous spectrum of paroxysmal disorders with a loss-of-function pathomechanism. The highest expression levels of PRRT2 in brain occurs in cerebellar granule cells (GCs) and cerebellar dysfunctions participate in the dyskinetic phenotype of PRRT2 knockout (KO) mice. We have investigated the effects of PRRT2 deficiency on the intrinsic excitability of GCs and the input-output relationships at the mossy fiber-GC synapses. We show that PRRT2 KO primary GCs display increased expression of Na+ channels, increased amplitude of Na+ currents and increased length of the axon initial segment, leading to an overall enhancement of intrinsic excitability. In acute PRRT2 KO cerebellar slices, GCs were more prone to action potential discharge in response to mossy fiber activation and exhibited an enhancement of transient and persistent Na+ currents, in the absence of changes at the mossy fiber-GC synapses. The results support a key role of PRRT2 expressed in GCs in the physiological regulation of the excitatory input to the cerebellum and are consistent with a major role of a cerebellar dysfunction in the pathogenesis of the PRRT2-linked paroxysmal pathologies.
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Cerebelo/fisiopatología , Distonía/fisiopatología , Proteínas de la Membrana/metabolismo , Neuronas/patología , Neuronas/fisiología , Animales , Cerebelo/metabolismo , Modelos Animales de Enfermedad , Distonía/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Mutations in PRoline-Rich Transmembrane protein 2 (PRRT2) underlie a group of paroxysmal disorders including epilepsy, kinesigenic dyskinesia and migraine. Most of the mutations lead to impaired PRRT2 expression and/or function, emphasizing the pathogenic role of the PRRT2 deficiency. In this work, we investigated the phenotype of primary hippocampal neurons obtained from mouse embryos in which the PRRT2 gene was constitutively inactivated. Although PRRT2 is expressed by both excitatory and inhibitory neurons, its deletion decreases the number of excitatory synapses without significantly affecting the number of inhibitory synapses or the nerve terminal ultrastructure. Analysis of synaptic function in primary PRRT2 knockout excitatory neurons by live imaging and electrophysiology showed slowdown of the kinetics of exocytosis, weakened spontaneous and evoked synaptic transmission and markedly increased facilitation. Inhibitory neurons showed strengthening of basal synaptic transmission, accompanied by faster depression. At the network level these complex synaptic effects resulted in a state of heightened spontaneous and evoked activity that was associated with increased excitability of excitatory neurons in both PRRT2 knockout primary cultures and acute hippocampal slices. The data indicate the existence of network instability/hyperexcitability as the possible basis of the paroxysmal phenotypes associated with PRRT2 mutations.
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Hipocampo/fisiología , Proteínas de la Membrana/fisiología , Plasticidad Neuronal , Neuronas/fisiología , Transmisión Sináptica , Animales , Células Cultivadas , Exocitosis , Masculino , Potenciales de la Membrana , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Vías Nerviosas/fisiología , Sinapsis/fisiología , Sinapsis/ultraestructuraRESUMEN
Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain scaffolding protein with kinase and GTPase activities involved in synaptic vesicle (SV) dynamics. While its role in Parkinson's disease has been largely investigated, little is known about LRRK2 physiological role and until now few proteins have been described as substrates. We have previously demonstrated that LRRK2 through its WD40 domain interacts with synapsin I, an important SV-associated phosphoprotein involved in neuronal development and in the regulation of neurotransmitter release. To test whether synapsin I is substrate for LRRK2 and characterize the properties of its phosphorylation, we used in vitro kinase and binding assays as well as cellular model and site-direct mutagenesis. Using synaptosomes in superfusion, patch-clamp recordings in autaptic WT and synapsin I KO cortical neurons and SypHy assay on primary cortical culture from wild-type and BAC human LRRK2 G2019S mice we characterized the role of LRRK2 kinase activity on glutamate release and SV trafficking. Here we reported that synapsin I is phosphorylated by LRRK2 and demonstrated that the interaction between LRRK2 WD40 domain and synapsin I is crucial for this phosphorylation. Moreover, we showed that LRRK2 phosphorylation of synapsin I at threonine 337 and 339 significantly reduces synapsin I-SV/actin interactions. Using complementary experimental approaches, we demonstrated that LRRK2 controls glutamate release and SV dynamics in a kinase activity and synapsin I-dependent manner. Our findings show that synapsin I is a LRRK2 substrate and describe a novel mechanisms of regulation of glutamate release by LRRK2 kinase activity.
Asunto(s)
Ácido Glutámico/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Sinapsinas/metabolismo , Transmisión Sináptica/fisiología , Animales , Encéfalo/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/metabolismo , Fosforilación , Vesículas Sinápticas/metabolismoRESUMEN
See Lerche (doi:10.1093/brain/awy073) for a scientific commentary on this article.Proline-rich transmembrane protein 2 (PRRT2) is the causative gene for a heterogeneous group of familial paroxysmal neurological disorders that include seizures with onset in the first year of life (benign familial infantile seizures), paroxysmal kinesigenic dyskinesia or a combination of both. Most of the PRRT2 mutations are loss-of-function leading to haploinsufficiency and 80% of the patients carry the same frameshift mutation (c.649dupC; p.Arg217Profs*8), which leads to a premature stop codon. To model the disease and dissect the physiological role of PRRT2, we studied the phenotype of neurons differentiated from induced pluripotent stem cells from previously described heterozygous and homozygous siblings carrying the c.649dupC mutation. Single-cell patch-clamp experiments on induced pluripotent stem cell-derived neurons from homozygous patients showed increased Na+ currents that were fully rescued by expression of wild-type PRRT2. Closely similar electrophysiological features were observed in primary neurons obtained from the recently characterized PRRT2 knockout mouse. This phenotype was associated with an increased length of the axon initial segment and with markedly augmented spontaneous and evoked firing and bursting activities evaluated, at the network level, by multi-electrode array electrophysiology. Using HEK-293 cells stably expressing Nav channel subtypes, we demonstrated that the expression of PRRT2 decreases the membrane exposure and Na+ current of Nav1.2/Nav1.6, but not Nav1.1, channels. Moreover, PRRT2 directly interacted with Nav1.2/Nav1.6 channels and induced a negative shift in the voltage-dependence of inactivation and a slow-down in the recovery from inactivation. In addition, by co-immunoprecipitation assays, we showed that the PRRT2-Nav interaction also occurs in brain tissue. The study demonstrates that the lack of PRRT2 leads to a hyperactivity of voltage-dependent Na+ channels in homozygous PRRT2 knockout human and mouse neurons and that, in addition to the reported synaptic functions, PRRT2 is an important negative modulator of Nav1.2 and Nav1.6 channels. Given the predominant paroxysmal character of PRRT2-linked diseases, the disturbance in cellular excitability by lack of negative modulation of Na+ channels appears as the key pathogenetic mechanism.
Asunto(s)
Regulación de la Expresión Génica/genética , Proteínas de la Membrana/metabolismo , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Canal de Sodio Activado por Voltaje NAV1.6/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/fisiología , Animales , Segmento Inicial del Axón/fisiología , Diferenciación Celular , Corteza Cerebral/citología , Consanguinidad , Fibroblastos/patología , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas , Potenciales de la Membrana/genética , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.6/genética , Proteína Homeótica Nanog/genética , Proteína Homeótica Nanog/metabolismo , Proteínas del Tejido Nervioso/genética , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/patología , Neuronas/citología , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , HermanosRESUMEN
L1 (also known as L1CAM) is a trans-membrane glycoprotein mediating neuron-neuron adhesion through homophilic and heterophilic interactions. Although experimental evidence has implicated L1 in axonal outgrowth, fasciculation and pathfinding, its contribution to voltage-gated Na(+) channel function and membrane excitability has remained unknown. Here, we show that firing rate, single cell spiking frequency and Na(+) current density are all reduced in hippocampal excitatory neurons from L1-deficient mice both in culture and in slices owing to an overall reduced membrane expression of Na(+) channels. Remarkably, normal firing activity was restored when L1 was reintroduced into L1-deficient excitatory neurons, indicating that abnormal firing patterns are not related to developmental abnormalities, but are a direct consequence of L1 deletion. Moreover, L1 deficiency leads to impairment of action potential initiation, most likely due to the loss of the interaction of L1 with ankyrin G that produces the delocalization of Na(+) channels at the axonal initial segment. We conclude that L1 contributes to functional expression and localization of Na(+) channels to the neuronal plasma membrane, ensuring correct initiation of action potential and normal firing activity.
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Membrana Celular/metabolismo , Regulación de la Expresión Génica/fisiología , Hipocampo/metabolismo , Molécula L1 de Adhesión de Célula Nerviosa/metabolismo , Neuronas/metabolismo , Canales de Sodio Activados por Voltaje/biosíntesis , Animales , Membrana Celular/genética , Hipocampo/citología , Ratones , Ratones Noqueados , Molécula L1 de Adhesión de Célula Nerviosa/genética , Neuronas/citología , Canales de Sodio Activados por Voltaje/genéticaRESUMEN
The expression and function of TRPV1 are influenced by its interaction with cellular proteins. Here, we identify Whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the WhirlinTRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Nociceptores/metabolismo , Canales Catiónicos TRPV/biosíntesis , Animales , Células Cultivadas , Proteínas de la Membrana/genética , Ratones , Complejos Multiproteicos/genética , Nociceptores/citología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño , Ratas , Ratas Wistar , Canales Catiónicos TRPV/genéticaRESUMEN
The functional consequence of γ-aminobutyric acid (GABA) release at mossy fiber terminals is still a debated topic. Here, we provide multiple evidence of GABA release in cultured autaptic hippocampal granule cells. In â¼50% of the excitatory autaptic neurons, GABA, VGAT, or GAD67 colocalized with vesicular glutamate transporter 1-positive puncta, where both GABAB and GABAA receptors (Rs) were present. Patch-clamp recordings showed a clear enhancement of autaptic excitatory postsynaptic currents in response to the application of the GABABR antagonist CGP58845 only in neurons positive to the selective granule cell marker Prox1, and expressing low levels of GAD67. Indeed, GCP non-responsive excitatory autaptic neurons were both Prox1- and GAD67-negative. Although the amount of released GABA was not sufficient to activate functional postsynaptic GABAARs, it effectively activated presynaptic GABABRs that maintain a tonic "brake" on the probability of release and on the size of the readily releasable pool and contributed to resting potential hyperpolarization possibly through extrasynaptic GABAAR activation. The autocrine inhibition exerted by GABABRs on glutamate release enhanced both paired-pulse facilitation and post-tetanic potentiation. Such GABABR-mediated changes in short-term plasticity confer to immature granule cells the capability to modulate their filtering properties in an activity-dependent fashion, with remarkable consequences on the dynamic behavior of neural circuits.
Asunto(s)
Hipocampo/fisiología , Plasticidad Neuronal/fisiología , Neuronas/fisiología , Ácido gamma-Aminobutírico/metabolismo , Animales , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Glutamato Descarboxilasa/metabolismo , Ratones Endogámicos C57BL , Ratones Transgénicos , Técnicas de Placa-Clamp , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Transient receptor potential vanilloid subtype I (TRPV1) is a thermosensory ion channel that is also gated by chemical substances such as vanilloids. Adjacent to the channel gate, this polymodal thermoTRP channel displays a TRP domain, referred to as AD1, that plays a role in subunit association and channel gating. Previous studies have shown that swapping the AD1 in TRPV1 with the cognate from the TRPV2 channel (AD2) reduces protein expression and produces a nonfunctional chimeric channel (TRPV1-AD2). Here, we used a stepwise, sequential, cumulative site-directed mutagenesis approach, based on rebuilding the AD1 domain in the TRPV1-AD2 chimera, to unveil the minimum number of amino acids needed to restore protein expression and polymodal channel activity. Unexpectedly, we found that virtually full restitution of the AD1 sequence is required to reinstate channel expression and responses to capsaicin, temperature, and voltage. This strategy identified E692, R701, and T704 in the TRP domain as important for TRPV1 activity. Even conservative mutagenesis at these sites (E692D/R701K/T704S) impaired channel expression and abolished TRPV1 activity. However, the sole mutation of these positions in the TRPV1-AD2 chimera (D692E/K701R/S704T) was not sufficient to rescue channel gating, implying that other residues in the TRP domain are necessary to endow activity to TRPV1-AD2. A biophysical analysis of a functional chimera suggested that mutations in the TRP domain raised the energetics of channel gating by altering the coupling of stimuli sensing and pore opening. These findings indicate that inter- and/or intrasubunit interactions in the TRP domain are essential for correct TRPV1 gating.
Asunto(s)
Activación del Canal Iónico , Canales Catiónicos TRPV/química , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Células HEK293 , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Ratas , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismoRESUMEN
The unique properties of few-layered graphene (FLG) make it interesting for a variety of applications, including biomedical applications, such as tissue engineering and drug delivery. Although different studies focus on applications in the central nervous system, its interaction with the peripheral nervous system has been so far overlooked. Here, we investigated the effects of exposure to colloidal dispersions of FLG on the sensory neurons of the rat dorsal root ganglia (DRG). We found that the FLG flakes were actively internalized by sensory neurons, accumulated in large intracellular vesicles, and possibly degraded over time, without major toxicological concerns, as neuronal viability, morphology, protein content, and basic electrical properties of DRG neurons were preserved. Interestingly, in our electrophysiological investigation under noxious stimuli, we observed an increased functional response upon FLG treatment of the nociceptive subpopulation of DRG neurons in response to irritants specific for chemoreceptors TRPV1 and TRPA1. The observed effects of FLG on DRG neurons may open-up novel opportunities for applications of these materials in specific disease models.
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Grafito , Nociceptores , Ratas , Animales , Nociceptores/metabolismo , Irritantes/metabolismo , Irritantes/farmacología , Grafito/farmacología , Grafito/metabolismo , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/farmacología , Ganglios Espinales/metabolismoRESUMEN
A large amount of experimental evidence has highlighted the rapid changes in synaptic efficacy induced by high-frequency stimulation and BDNF at central excitatory synapses. We clarified the quantal mechanisms and the involvement of Synapsin I (SynI) phosphorylation in the expression of post-tetanic potentiation (PTP) and in its modulation by BDNF in mouse glutamatergic autapses. We found that PTP is associated with an elevation in the probability of release and a concomitant increase in the size of the readily releasable pool (RRP). The latter component was virtually absent in SynI knock-out (KO) neurons, which indeed displayed impaired PTP. PTP was fully rescued by the expression of wild-type SynI, but not of its dephosphomimetic mutants in the phosphorylation sites for cAMP-dependent protein kinase and Ca²âº/calmodulin-dependent protein kinases I/II. BDNF potently enhanced PTP through a further increase in the RRP size, which was missing in SynI KO neurons. In these neurons, the BDNF-induced PTP enhancement was rescued by the expression of wild-type SynI, but not of its dephosphomimetic mutant at the mitogen-dependent protein kinase sites. The results indicate that the increase in RRP size necessary for the full expression of PTP, and its sensitivity to BDNF, involve phosphorylation of SynI at distinct sites, thus implicating SynI as an essential downstream effector for the expression of PTP and for its enhancement by BDNF.
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Fenómenos Biofísicos/genética , Factor Neurotrófico Derivado del Encéfalo/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Neuronas/efectos de los fármacos , Mutación Puntual/fisiología , Sinapsinas/deficiencia , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Análisis de Varianza , Animales , Fenómenos Biofísicos/efectos de los fármacos , Calcio/metabolismo , Carbazoles/farmacología , Células Cultivadas , Estimulación Eléctrica , Embrión de Mamíferos , Inhibidores Enzimáticos/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/genética , Potenciales Postsinápticos Excitadores/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Alcaloides Indólicos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Modelos Biológicos , Mutagénesis Sitio-Dirigida/métodos , Neuronas/fisiología , Técnicas de Placa-Clamp , Fosforilación/fisiología , Sinapsinas/genética , TransfecciónRESUMEN
Proline-rich transmembrane protein 2 (PRRT2) is a neuron-specific protein implicated in the control of neurotransmitter release and neural network stability. Accordingly, PRRT2 loss-of-function mutations associate with pleiotropic paroxysmal neurological disorders, including paroxysmal kinesigenic dyskinesia, episodic ataxia, benign familial infantile seizures, and hemiplegic migraine. PRRT2 is a negative modulator of the membrane exposure and biophysical properties of Na+ channels NaV1.2/NaV1.6 predominantly expressed in brain glutamatergic neurons. NaV channels form complexes with ß-subunits that facilitate the membrane targeting and the activation of the α-subunits. The opposite effects of PRRT2 and ß-subunits on NaV channels raises the question of whether PRRT2 and ß-subunits interact or compete for common binding sites on the α-subunit, generating Na+ channel complexes with distinct functional properties. Using a heterologous expression system, we have observed that ß-subunits and PRRT2 do not interact with each other and act as independent non-competitive modulators of NaV1.2 channel trafficking and biophysical properties. PRRT2 antagonizes the ß4-induced increase in expression and functional activation of the transient and persistent NaV1.2 currents, without affecting resurgent current. The data indicate that ß4-subunit and PRRT2 form a push-pull system that finely tunes the membrane expression and function of NaV channels and the intrinsic neuronal excitability.
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Proteínas de la Membrana , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso , Neuronas , Humanos , Ataxia , Encéfalo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Enfermedades del Sistema Nervioso , Canal de Sodio Activado por Voltaje NAV1.2/química , Canal de Sodio Activado por Voltaje NAV1.2/metabolismo , Neuronas/química , Neuronas/citologíaRESUMEN
The transient receptor potential vanilloid 1 (TRPV1) channel is a thermosensory receptor implicated in diverse physiological and pathological processes. The TRP domain, a highly conserved region in the C terminus adjacent to the internal channel gate, is critical for subunit tetramerization and channel gating. Here, we show that cell-penetrating, membrane-anchored peptides patterned after this protein domain are moderate and selective TRPV1 antagonists both in vitro and in vivo, blocking receptor activity in intact rat primary sensory neurons and their peripheral axons with mean decline time of 30 min. The most potent lipopeptide, TRP-p5, blocked all modes of TRPV1 gating with micromolar efficacy (IC(50)<10 µM), without significantly affecting other thermoTRP channels. In contrast, its retrosequence or the corresponding sequences of other TRPV channels did not alter TRPV1 channel activity (IC(50)>100 µM). TRP-p5 did not affect the capsaicin sensitivity of the vanilloid receptor. Our data suggest that TRP-p5 interferes with protein-protein interactions at the level of the TRP domain that are essential for the "conformational" change that leads to gate opening. Therefore, these palmitoylated peptides, which we termed TRPducins, are noncompetitive, voltage-independent, sequence-specific TRPV1 blockers. Our findings indicate that TRPducin-like peptides may embody a novel molecular strategy that can be exploited to generate a selective pharmacological arsenal for the TRP superfamily of ion channels.
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Péptidos/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Canales Catiónicos TRPV/metabolismo , Animales , Animales Recién Nacidos , Capsaicina/farmacología , Línea Celular , Células Cultivadas , Electrofisiología , Células HEK293 , Humanos , Inmunohistoquímica , Péptidos/química , Ratas , Canales Catiónicos TRPV/químicaRESUMEN
The capsaicin receptor TRPV1 (Transient Receptor Potential, Vanilloid family member 1), the founding member of the heat-sensitive TRP ("thermo-TRP") channel family, plays a pivotal role in pain transduction. There is mounting evidence that TRPV1 regulation is complex and is manifest at many levels, from gene expression through post-translational modification and formation of receptor heteromers to subcellular compartmentalization and association with regulatory proteins. These mechanisms are believed to be involved both in disease-related changes in TRPV1 expression, and the long-lasting refractory state, referred to as "desensitization", that follows TRPV1 agonist treatment. The signaling cascades that regulate TRPV1 and related thermo-TRP channels are only beginning to be understood. Here we review our current knowledge in this rapidly changing field. We propose that the complex regulation of TRPV1 may be exploited for therapeutic purposes, with the ultimate goal being the development of novel, innovative agents that target TRPV1 in diseased, but not healthy, tissues. Such compounds are expected to be devoid of the side-effects (e.g. hyperthermia and impaired noxious heat sensation) that plague the clinical use of existing TRPV1 antagonists.
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Dolor/fisiopatología , Canales Catiónicos TRPV/fisiología , Animales , Humanos , Transducción de SeñalRESUMEN
The repressor-element 1-silencing transcription/neuron-restrictive silencer factor (REST/NRSF) controls hundreds of neuron-specific genes. We showed that REST/NRSF downregulates glutamatergic transmission in response to hyperactivity, thus contributing to neuronal homeostasis. However, whether GABAergic transmission is also implicated in the homeostatic action of REST/NRSF is unknown. Here, we show that hyperactivity-induced REST/NRSF activation, triggers a homeostatic rearrangement of GABAergic inhibition, with increased frequency of miniature inhibitory postsynaptic currents (IPSCs) and amplitude of evoked IPSCs in mouse cultured hippocampal neurons. Notably, this effect is limited to inhibitory-onto-excitatory neuron synapses, whose density increases at somatic level and decreases in dendritic regions, demonstrating a complex target- and area-selectivity. The upscaling of perisomatic inhibition was occluded by TrkB receptor inhibition and resulted from a coordinated and sequential activation of the Npas4 and Bdnf gene programs. On the opposite, the downscaling of dendritic inhibition was REST-dependent, but BDNF-independent. The findings highlight the central role of REST/NRSF in the complex transcriptional responses aimed at rescuing physiological levels of network activity in front of the ever-changing environment.
Asunto(s)
Potenciales Postsinápticos Inhibidores/fisiología , Neuronas/metabolismo , Proteínas Represoras/metabolismo , Animales , Células Cultivadas , GABAérgicos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Hipocampo/citología , Homeostasis , Ratones Endogámicos C57BL , Neuronas/fisiología , Receptor trkB/metabolismo , Sinapsis/metabolismo , Factores de TranscripciónRESUMEN
Loss-of-function mutations in proline-rich transmembrane protein-2 (PRRT2) cause paroxysmal disorders associated with defective Ca2+ dependence of glutamatergic transmission. We find that either acute or constitutive PRRT2 deletion induces a significant decrease in the amplitude of evoked excitatory postsynaptic currents (eEPSCs) that is insensitive to extracellular Ca2+ and associated with a reduced contribution of P/Q-type Ca2+ channels to the EPSC amplitude. This synaptic phenotype parallels a decrease in somatic P/Q-type Ca2+ currents due to a decreased membrane targeting of the channel with unchanged total expression levels. Co-immunoprecipitation, pull-down assays, and proteomics reveal a specific and direct interaction of PRRT2 with P/Q-type Ca2+ channels. At presynaptic terminals lacking PRRT2, P/Q-type Ca2+ channels reduce their clustering at the active zone, with a corresponding decrease in the P/Q-dependent presynaptic Ca2+ signal. The data highlight the central role of PRRT2 in ensuring the physiological Ca2+ sensitivity of the release machinery at glutamatergic synapses.