RESUMEN
Excitatory synaptic inputs from specific brain regions are often targeted to distinct dendritic arbors on hippocampal pyramidal neurons. Recent work has suggested that CA2 pyramidal neurons respond robustly and preferentially to excitatory input into the stratum lacunosum moleculare (SLM), with a relatively modest response to Schaffer collateral excitatory input into stratum radiatum (SR) in acute mouse hippocampal slices, but the extent to which this difference may be explained by morphology is unknown. In an effort to replicate these findings and to better understand the role of dendritic morphology in shaping responses from proximal and distal synaptic sites, we measured excitatory postsynaptic currents and action potentials in CA2 pyramidal cells in response to SR and SLM stimulation and subsequently analyzed confocal images of the filled cells. We found that, in contrast to previous reports, SR stimulation evoked substantial responses in all recorded CA2 pyramidal cells. Strikingly, however, we found that not all neurons responded to SLM stimulation, and in those neurons that did, responses evoked by SLM and SR were comparable in size and effectiveness in inducing action potentials. In a comprehensive morphometric analysis of CA2 pyramidal cell apical dendrites, we found that the neurons that were unresponsive to SLM stimulation were the same ones that lacked substantial apical dendritic arborization in the SLM. Neurons responsive to both SR and SLM stimulation had roughly equal amounts of dendritic branching in each layer. Remarkably, our study in mouse CA2 generally replicates the work characterizing the diversity of CA2 pyramidal cells in the guinea pig hippocampus. We conclude, then, that like in guinea pig, mouse CA2 pyramidal cells have a diverse apical dendrite morphology that is likely to be reflective of both the amount and source of excitatory input into CA2 from the entorhinal cortex and CA3.
Asunto(s)
Región CA2 Hipocampal/fisiología , Dendritas/fisiología , Corteza Entorrinal/fisiología , Potenciales Postsinápticos Excitadores/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Animales , Región CA2 Hipocampal/citología , Corteza Entorrinal/citología , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de ÓrganosRESUMEN
Ca(V)2.2 (N-type) calcium channels control the entry of calcium into neurons to regulate essential functions but most notably presynaptic transmitter release. Ca(V)2.2 channel expression levels are precisely controlled, but we know little of the cellular mechanisms involved. The ubiquitin proteasome system (UPS) is known to regulate expression of many synaptic proteins, including presynaptic elements, to optimize synaptic efficiency. However, we have limited information about ubiquitination of Ca(V)2 channels. Here we show that Ca(V)2.2 proteins are ubiquitinated, and that elements in the proximal C terminus of Ca(V)2.2 encoded by exon 37b of the mouse Cacna1b gene predispose cloned and native channels to downregulation by the UPS. Ca(V)2.2 channels containing e37b are expressed throughout the mammalian nervous system, but in some cells, notably nociceptors, sometimes e37a--not e37b--is selected during alternative splicing of Ca(V)2.2 pre-mRNA. By a combination of biochemical and functional analyses we show e37b promotes a form of ubiquitination that is coupled to reduced Ca(V)2.2 current density and increased sensitivity to the UPS. Cell-specific alternative splicing of e37a in nociceptors reduces Ca(V)2.2 channel ubiquitination and sensitivity to the UPS, suggesting a role in pain processing.
Asunto(s)
Canales de Calcio Tipo N/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación/fisiología , Empalme Alternativo , Animales , Canales de Calcio Tipo N/genética , Ganglios Espinales/metabolismo , Ratones , Neuronas/metabolismo , Complejo de la Endopetidasa Proteasomal/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
Endocytosis of AMPA receptors and other postsynaptic cargo occurs at endocytic zones (EZs), stably positioned sites of clathrin adjacent to the postsynaptic density (PSD). The tight localization of postsynaptic endocytosis is thought to control spine composition and regulate synaptic transmission. However, the mechanisms that situate the EZ near the PSD and the role of spine endocytosis in synaptic transmission are unknown. Here, we report that a physical link between dynamin-3 and the postsynaptic adaptor Homer positions the EZ near the PSD. Disruption of dynamin-3 or its interaction with Homer uncouples the PSD from the EZ, resulting in synapses lacking postsynaptic clathrin. Loss of the EZ leads to a loss of synaptic AMPA receptors and reduced excitatory synaptic transmission that corresponds with impaired synaptic recycling. Thus, a physical link between the PSD and the EZ ensures localized endocytosis and recycling by recapturing and maintaining a proximate pool of cycling AMPA receptors.
Asunto(s)
Proteínas Portadoras/fisiología , Dinamina III/fisiología , Receptores AMPA/fisiología , Vesículas Transportadoras/fisiología , Animales , Proteínas Portadoras/química , Clatrina/fisiología , ADN/genética , Dinamina III/química , Electrofisiología , GTP Fosfohidrolasas/deficiencia , GTP Fosfohidrolasas/genética , Proteínas de Andamiaje Homer , Humanos , Inmunohistoquímica , Metabolismo de los Lípidos/fisiología , Microscopía Confocal , Microscopía Electrónica , Neuronas/fisiología , Neuronas/ultraestructura , Técnicas de Placa-Clamp , Interferencia de ARN/fisiología , Transmisión Sináptica/fisiología , Vesículas Transportadoras/ultraestructuraRESUMEN
Mutations in the PARK2 gene cause hereditary Parkinson disease (PD). The PARK2 gene product, termed parkin, is an E3 ubiquitin ligase that mediates the transfer of ubiquitin onto diverse substrate proteins. Despite progress in defining the molecular properties and substrates of parkin, little is known about its physiological function. Here, we show that parkin regulates the function and stability of excitatory glutamatergic synapses. Postsynaptic expression of parkin dampens excitatory synaptic transmission and causes a marked loss of excitatory synapses onto hippocampal neurons. Conversely, knockdown of endogenous parkin or expression of PD-linked parkin mutants profoundly enhances synaptic efficacy and triggers a proliferation of glutamatergic synapses. This proliferation is associated with increased vulnerability to synaptic excitotoxicity. Thus, parkin negatively regulates the number and strength of excitatory synapses. Increased excitatory drive produced by disruption of parkin may contribute to the pathophysiology of PD.
Asunto(s)
Neuronas/fisiología , Enfermedad de Parkinson/fisiopatología , Sinapsis/enzimología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Células Cultivadas , Corteza Cerebral/citología , Potenciales Postsinápticos Excitadores/fisiología , Ácido Glutámico/metabolismo , Hipocampo/citología , Mutación , Neuronas/patología , Neurotoxinas/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Técnicas de Placa-Clamp , Ratas , Sinapsis/patología , Transmisión Sináptica/fisiologíaRESUMEN
Dendritic spines are micron-sized membrane protrusions receiving most excitatory synaptic inputs in the mammalian brain. Spines form and grow during long-term potentiation (LTP) of synaptic strength. However, the source of membrane for spine formation and enlargement is unknown. Here we report that membrane trafficking from recycling endosomes is required for the growth and maintenance of spines. Using live-cell imaging and serial section electron microscopy, we demonstrate that LTP-inducing stimuli promote the mobilization of recycling endosomes and vesicles into spines. Preventing recycling endosomal transport abolishes LTP-induced spine formation. Using a pH-sensitive recycling cargo, we show that exocytosis from recycling endosomes occurs locally in spines, is triggered by activation of synaptic NMDA receptors, and occurs concurrently with spine enlargement. Thus, recycling endosomes provide membrane for activity-dependent spine growth and remodeling, defining a novel membrane trafficking mechanism for spine morphological plasticity and providing a mechanistic link between structural and functional plasticity during LTP.
Asunto(s)
Espinas Dendríticas/fisiología , Endosomas/fisiología , Exocitosis/fisiología , Plasticidad Neuronal/fisiología , Animales , Transporte Biológico Activo/fisiología , Tamaño de la Célula , ADN/biosíntesis , ADN/genética , Espinas Dendríticas/ultraestructura , Electrofisiología , Endosomas/ultraestructura , Hipocampo/citología , Hipocampo/crecimiento & desarrollo , Procesamiento de Imagen Asistido por Computador , Inmunohistoquímica , Potenciación a Largo Plazo/fisiología , Microscopía Electrónica , Microscopía Electrónica de Transmisión , Ratas , Receptores de N-Metil-D-Aspartato/fisiologíaRESUMEN
N-type calcium channels are critical for pain transduction. Inhibitors of these channels are powerful analgesics, but clinical use of current N-type blockers remains limited by undesirable actions in other regions of the nervous system. We now demonstrate that a unique splice isoform of the N-type channel is restricted exclusively to dorsal root ganglia. By a combination of functional and molecular analyses at the single-cell level, we show that the DRG-specific exon, e37a, is preferentially present in Ca(V)2.2 mRNAs expressed in neurons that contain nociceptive markers, VR1 and Na(V)1.8. Cell-specific inclusion of exon 37a correlates closely with significantly larger N-type currents in nociceptive neurons. This unique splice isoform of the N-type channel could represent a novel target for pain management.
Asunto(s)
Empalme Alternativo , Canales de Calcio Tipo L/genética , Canales de Calcio Tipo L/metabolismo , Ganglios Espinales/metabolismo , Dolor/fisiopatología , Animales , Canales de Calcio Tipo N/metabolismo , Capsaicina/farmacología , Conductividad Eléctrica , Exones , Ganglios Espinales/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nociceptores/efectos de los fármacos , Nociceptores/metabolismo , Dolor/genética , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Phosphatidylinositol-4,5-bisphosphate (PIP2) has been shown to regulate many ion channels, transporters, and other signaling proteins, but it is not known whether it also regulates neurotransmitter-gated channels. The NMDA receptors (NMDARs) are gated by glutamate and serve as a critical control point in synaptic function. Here we demonstrate that PIP2 supports NMDAR activity. In Xenopus oocytes, overexpression of phospholipase Cgamma (PLCgamma) or preincubation with 10 microm wortmannin markedly reduced NMDA currents. Stimulation of the epidermal growth factor receptor (EGFR) promoted the formation of an immunocomplex between PLCgamma and NMDAR subunits. Stimulation of EGFR or the PLCbeta-coupled M1 acetylcholine receptor produced a robust transient inhibition of NMDA currents. Wortmannin application blocked the recovery of NMDA currents from the inhibition. Using mutagenesis, we identified the structural elements on NMDAR intracellular tails that transduce the receptor-mediated inhibition, which pinpoint to the binding site for the cytoskeletal protein alpha-actinin. Mutation of the PIP2-binding residues of alpha-actinin dramatically reduced NMDA currents and occluded the effect of EGF. Interestingly, EGF or wortmannin affected the interaction between NMDAR subunits and alpha-actinin, suggesting that this protein mediates the effect of PIP2 on NMDARs. In mature hippocampal neurons, expression of the mutant alpha-actinin reduced NMDA currents and accelerated inactivation. We propose a model in which alpha-actinin supports NMDAR activity via tethering their intracellular tails to plasma membrane PIP2. Thus, our results extend the influence of PIP2 to the NMDA ionotropic glutamate receptors and introduce a novel mechanism of "indirect" regulation of transmembrane protein activity by PIP2.
Asunto(s)
Actinina/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Receptores de N-Metil-D-Aspartato/metabolismo , Animales , Sitios de Unión/fisiología , Femenino , Mutación , Ratas , Receptores de N-Metil-D-Aspartato/genética , Xenopus laevisRESUMEN
Neuronal L-type calcium channels are essential for regulating activity-dependent gene expression, but they are thought to open too slowly to contribute to action potential-dependent calcium entry. A complication of studying native L-type channels is that they represent a minor fraction of the whole-cell calcium current in most neurons. Dihydropyridine antagonists are therefore widely used to establish the contribution of L-type channels to various neuronal processes and to study their underlying biophysical properties. The effectiveness of these antagonists on L-type channels, however, varies with stimulus and channel subtype. Here, we study recombinant neuronal L-type calcium channels, CaV1.2 and CaV1.3. We show that these channels open with fast kinetics and carry substantial calcium entry in response to individual action potential waveforms, contrary to most studies of native L-type currents. Neuronal CaV1.3 L-type channels were as efficient as CaV2.2 N-type channels at supporting calcium entry during action potential-like stimuli. We conclude that the apparent slow activation of native L-type currents and their lack of contribution to single action potentials reflect the state-dependent nature of the dihydropyridine antagonists used to study them, not the underlying properties of L-type channels.
Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo L/metabolismo , Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Neuronas/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Canales de Calcio/fisiología , Canales de Calcio Tipo L/fisiología , Activación del Canal Iónico/efectos de los fármacos , Cinética , Ratones , Neuronas/efectos de los fármacos , Neuronas/metabolismo , RatasRESUMEN
Ca2+ channel beta subunits are important molecular determinants of the kinetics and voltage dependence of Ca2+ channel gating. Through direct interactions with channel-forming alpha1 subunits, beta subunits enhance expression levels, accelerate activation, and have variable effects on inactivation. Four distinct beta subunit genes each encode five homologous sequence domains (D1-5), three of which (D1, D3, and D5) undergo alternative splicing. We have isolated from human spinal cord a novel alternatively spliced beta4 subunit containing a short form of domain D1 (beta4a) that is highly homologous to N termini of Xenopus and rat beta3 subunits. The purpose of this study was to compare the gating properties of various alpha1 subunit complexes containing beta4a with those of complexes containing a beta4 subunit with a longer form of domain D1, beta4b. Expression in Xenopus oocytes revealed that, relative to alpha1A and alpha1B complexes containing beta4b, the voltage dependence of activation and inactivation of complexes containing beta4a were shifted to more depolarized potentials. Moreover, alpha1A and alpha1B complexes containing beta4a inactivated at a faster rate. Interestingly, beta4 subunit alternative splicing did not influence the gating properties of alpha1C and alpha1E subunits. Experiments with beta4 deletion mutants revealed that both the N and C termini of the beta4 subunit play critical roles in setting voltage-dependent gating parameters and that their effects are alpha1 subunit specific. Our data are best explained by a model in which distinct modes of activation and inactivation result from beta-subunit splice variant-specific interactions with an alpha1 subunit gating structure.
Asunto(s)
Empalme Alternativo , Canales de Calcio/metabolismo , Activación del Canal Iónico/fisiología , Subunidades de Proteína , Adolescente , Adulto , Anciano , Animales , Canales de Calcio/genética , Clonación Molecular , Femenino , Expresión Génica , Humanos , Masculino , Potenciales de la Membrana/fisiología , Persona de Mediana Edad , Modelos Biológicos , Datos de Secuencia Molecular , Oocitos/metabolismo , Técnicas de Placa-Clamp , ARN Complementario/genética , ARN Complementario/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Médula Espinal/metabolismo , Relación Estructura-Actividad , Población Blanca , XenopusRESUMEN
Ca2+ channel beta subunits modify alpha1 subunit gating properties through direct interactions with intracellular linker domains. In a previous report (Helton and Horne, 2002), we showed that alternative splicing of the beta4 subunit had alpha1 subunit subtype-specific effects on Ca2+ channel activation and fast inactivation. We extend these findings in the present report to include effects on slow inactivation and block by the peptide toxin omega-conotoxin (CTx)-MVIIC. N-terminal deletion and site-directed mutagenesis experiments revealed that the effects of alternative splicing on toxin block and all aspects of gating could be attributed to a proline-rich motif found within N-terminal beta4b amino acids 10-20. Interestingly, this motif is conserved within the third postsynaptic density-95 (PSD-95)/Discs large/zona occludens-1 domain of the distantly related membrane-associated guanylate kinase homolog, PSD-95. Sequence identity of approximately 30% made possible the building of beta4a and beta4b three-dimensional structural models using PSD-95 as the target sequence. The models (1) reveal that alternative splicing of the beta4 N terminus results in dramatic differences in surface charge distribution and (2) localize the proline-rich motif of beta4b to an extended arm structure that flanks what would be the equivalent of a highly modified PSD-95 carboxylate binding loop. Northern blot analysis revealed a markedly different pattern of distribution for beta4a versus beta4b in the human CNS. Whereas beta4a is distributed throughout evolutionarily older regions of the CNS, beta4b is concentrated heavily in the forebrain. These results raise interesting questions about the functional role that alternative splicing of the beta4 subunit has played in the evolution of complex neural networks.
Asunto(s)
Empalme Alternativo/fisiología , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Activación del Canal Iónico/fisiología , omega-Conotoxinas/farmacología , Secuencias de Aminoácidos/fisiología , Animales , Encéfalo/metabolismo , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/efectos de los fármacos , Humanos , Activación del Canal Iónico/efectos de los fármacos , Potenciales de la Membrana/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas del Tejido Nervioso/genética , Oocitos/metabolismo , Especificidad de Órganos , Técnicas de Placa-Clamp , Estructura Terciaria de Proteína/fisiología , Subunidades de Proteína/efectos de los fármacos , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Médula Espinal/metabolismo , Relación Estructura-Actividad , Xenopus laevisRESUMEN
L-type calcium channels couple membrane depolarization in neurons to numerous processes including gene expression, synaptic efficacy, and cell survival. To establish the contribution of L-type calcium channels to various signaling cascades, investigators have relied on their unique pharmacological sensitivity to dihydropyridines. The traditional view of dihydropyridine-sensitive L-type calcium channels is that they are high-voltage-activating and have slow activation kinetics. These properties limit the involvement of L-type calcium channels to neuronal functions triggered by strong and sustained depolarizations. This review highlights literature, both long-standing and recent, that points to significant functional diversity among L-type calcium channels expressed in neurons and other excitable cells. Past literature contains several reports of low-voltage-activated neuronal L-type calcium channels that parallel the unique properties of recently cloned CaV1.3 L-type channels. The fast kinetics and low activation thresholds of CaV1.3 channels stand in stark contrast to criteria currently used to describe L-type calcium channels. A more accurate view of neuronal L-type calcium channels encompasses a broad range of activation thresholds and recognizes their potential contribution to signaling cascades triggered by subthreshold depolarizations.