RESUMEN
Strengthening of synaptic connections by NMDA (N-methyl-d-aspartate) receptor-dependent long-term potentiation (LTP) shapes neural circuits and mediates learning and memory. During the induction of NMDA-receptor-dependent LTP, Ca2+ influx stimulates recruitment of synaptic AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, thereby strengthening synapses. How Ca2+ induces the recruitment of AMPA receptors remains unclear. Here we show that, in the pyramidal neurons of the hippocampal CA1 region in mice, blocking postsynaptic expression of both synaptotagmin-1 (Syt1) and synaptotagmin-7 (Syt7), but not of either alone, abolished LTP. LTP was restored by expression of wild-type Syt7 but not of a Ca2+-binding-deficient mutant Syt7. Blocking postsynaptic expression of Syt1 and Syt7 did not impair basal synaptic transmission, reduce levels of synaptic or extrasynaptic AMPA receptors, or alter other AMPA receptor trafficking events. Moreover, expression of dominant-negative mutant Syt1 which inhibits Ca2+-dependent presynaptic vesicle exocytosis, also blocked Ca2+-dependent postsynaptic AMPA receptor exocytosis, thereby abolishing LTP. Our results suggest that postsynaptic Syt1 and Syt7 act as redundant Ca2+-sensors for Ca2+-dependent exocytosis of AMPA receptors during LTP, and thereby delineate a simple mechanism for the recruitment of AMPA receptors that mediates LTP.
Asunto(s)
Exocitosis , Potenciación a Largo Plazo/fisiología , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Sinaptotagminas/metabolismo , Animales , Región CA1 Hipocampal/citología , Calcio/metabolismo , Femenino , Masculino , Ratones , Mutación , Transporte de Proteínas , Células Piramidales/metabolismo , Transmisión Sináptica , Sinaptotagminas/genéticaRESUMEN
Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca(2+)- and Mg(2+)-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca(2+)-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca(2+)-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca(2+)-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca(2+) triggering, moves en bloc as Ca(2+) influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.
Asunto(s)
Exocitosis , Neuronas/metabolismo , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Sinaptotagminas/química , Sinaptotagminas/metabolismo , Animales , Sitios de Unión/genética , Calcio/química , Calcio/metabolismo , Membrana Celular/metabolismo , Cristalografía por Rayos X , Electrones , Hipocampo/citología , Rayos Láser , Magnesio/química , Magnesio/metabolismo , Fusión de Membrana , Ratones , Modelos Biológicos , Modelos Moleculares , Mutación/genética , Neuronas/química , Neuronas/citología , Proteínas SNARE/genética , Transmisión Sináptica , Vesículas Sinápticas/química , Vesículas Sinápticas/metabolismo , Sinaptotagminas/genéticaRESUMEN
In forebrain neurons, Ca(2+) triggers exocytosis of readily releasable vesicles by binding to synaptotagmin-1 and -7, thereby inducing fast and slow vesicle exocytosis, respectively. Loss-of-function of synaptotagmin-1 or -7 selectively impairs the fast and slow phase of release, respectively, but does not change the size of the readily-releasable pool (RRP) of vesicles as measured by stimulation of release with hypertonic sucrose, or alter the rate of vesicle priming into the RRP. Here we show, however, that simultaneous loss-of-function of both synaptotagmin-1 and -7 dramatically decreased the capacity of the RRP, again without altering the rate of vesicle priming into the RRP. Either synaptotagmin-1 or -7 was sufficient to rescue the RRP size in neurons lacking both synaptotagmin-1 and -7. Although maintenance of RRP size was Ca(2+)-independent, mutations in Ca(2+)-binding sequences of synaptotagmin-1 or synaptotagmin-7--which are contained in flexible top-loop sequences of their C2 domains--blocked the ability of these synaptotagmins to maintain the RRP size. Both synaptotagmins bound to SNARE complexes; SNARE complex binding was reduced by the top-loop mutations that impaired RRP maintenance. Thus, synaptotagmin-1 and -7 perform redundant functions in maintaining the capacity of the RRP in addition to nonredundant functions in the Ca(2+) triggering of different phases of release.
Asunto(s)
Señalización del Calcio , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagmina I/metabolismo , Sinaptotagminas/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión , Células Cultivadas , Potenciales Postsinápticos Excitadores , Células HEK293 , Hipocampo/citología , Hipocampo/ultraestructura , Humanos , Potenciales Postsinápticos Inhibidores , Ratones Noqueados , Mutación , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Neuronas/ultraestructura , Interferencia de ARN , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas SNARE/metabolismo , Vesículas Sinápticas/ultraestructura , Sinaptotagmina I/química , Sinaptotagmina I/genética , Sinaptotagminas/antagonistas & inhibidores , Sinaptotagminas/química , Sinaptotagminas/genéticaRESUMEN
Glucose stimulates insulin secretion from ß-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca(2+)-triggered exocytosis by direct phosphorylation of a synaptotagmin.
Asunto(s)
Péptido 1 Similar al Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Sinaptotagminas/metabolismo , Secuencia de Aminoácidos , Animales , Colforsina/farmacología , Secuencia Conservada , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Evolución Molecular , Exenatida , Exocitosis/efectos de los fármacos , Receptor del Péptido 1 Similar al Glucagón , Glucosa/farmacología , Células HEK293 , Humanos , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Ratones Noqueados , Datos de Secuencia Molecular , Mutación/genética , Péptidos/farmacología , Fosforilación/efectos de los fármacos , Fosfoserina/metabolismo , Ratas , Receptores de Glucagón/metabolismo , Sinaptotagminas/química , Ponzoñas/farmacologíaRESUMEN
Among SNARE proteins mediating synaptic vesicle fusion, syntaxin-1 uniquely includes an N-terminal peptide ('N-peptide') that binds to Munc18-1, and a large, conserved H(abc)-domain that also binds to Munc18-1. Previous in vitro studies suggested that the syntaxin-1 N-peptide is functionally important, whereas the syntaxin-1 H(abc)-domain is not, but limited information is available about the in vivo functions of these syntaxin-1 domains. Using rescue experiments in cultured syntaxin-deficient neurons, we now show that the N-peptide and the H(abc)-domain of syntaxin-1 perform distinct and independent roles in synaptic vesicle fusion. Specifically, we found that the N-peptide is essential for vesicle fusion as such, whereas the H(abc)-domain regulates this fusion, in part by forming the closed syntaxin-1 conformation. Moreover, we observed that deletion of the H(abc)-domain but not deletion of the N-peptide caused a loss of Munc18-1 which results in a decrease in the readily releasable pool of vesicles at a synapse, suggesting that Munc18 binding to the H(abc)-domain stabilizes Munc18-1. Thus, the N-terminal syntaxin-1 domains mediate different functions in synaptic vesicle fusion, probably via formation of distinct Munc18/SNARE-protein complexes.
Asunto(s)
Proteínas Munc18/metabolismo , Neuronas/metabolismo , Péptidos/metabolismo , Sinapsis/metabolismo , Sintaxina 1/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Células Cultivadas , Técnicas de Silenciamiento del Gen , Fusión de Membrana , Ratones , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Unión Proteica , Conformación Proteica , Mapeo de Interacción de Proteínas , Estructura Terciaria de Proteína , Sinapsis/genética , Transmisión Sináptica , Vesículas Sinápticas/genética , Vesículas Sinápticas/metabolismo , Sintaxina 1/química , Sintaxina 1/genéticaRESUMEN
Complexins are synaptic SNARE complex-binding proteins that cooperate with synaptotagmins in activating Ca(2+)-stimulated, synaptotagmin-dependent synaptic vesicle exocytosis and in clamping spontaneous, synaptotagmin-independent synaptic vesicle exocytosis. Here, we show that complexin sequences are conserved in some non-metazoan unicellular organisms and in all metazoans, suggesting that complexins are a universal feature of metazoans that predate metazoan evolution. We show that complexin from Nematostella vectensis, a cnidarian sea anemone far separated from mammals in metazoan evolution, functionally replaces mouse complexins in activating Ca(2+)-triggered exocytosis, but is unable to clamp spontaneous exocytosis. Thus, the activating function of complexins is likely conserved throughout metazoan evolution.
Asunto(s)
Proteínas Adaptadoras del Transporte Vesicular/química , Proteínas Adaptadoras del Transporte Vesicular/metabolismo , Evolución Biológica , Coanoflagelados/genética , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Unión Proteica/genética , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Coanoflagelados/química , Secuencia Conservada , Exocitosis , Ratones , Filogenia , Estructura Secundaria de Proteína , Proteínas SNARE , Transmisión Sináptica , Sinaptotagminas/genética , Sinaptotagminas/fisiologíaRESUMEN
Most synapses release neurotransmitters in two phases: (1) a fast synchronous phase lasting a few milliseconds; and (2) a delayed "asynchronous" phase lasting hundreds of milliseconds. Ca(2+) triggers fast synchronous neurotransmitter release by binding to synaptotagmin-1, synaptotagmin-2, or synaptotagmin-9, but how Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Recent results suggested that consistent with the Ca(2+)-sensor function of synaptotagmin-7 in neuroendocrine exocytosis, synaptotagmin-7 also functions as a Ca(2+) sensor for synaptic vesicle exocytosis but operates during delayed asynchronous release. Puzzlingly, a subsequent study postulated that synaptotagmin-7 is not a Ca(2+) sensor for release but mediates Ca(2+)-dependent vesicle repriming after intense stimulation. To address these issues, we here analyzed synaptic transmission at rod bipolar neuron-AII amacrine cell synapses in acute mouse retina slices as a model system. Using paired recordings, we show that knock-out of synaptotagmin-7 selectively impairs delayed asynchronous release but not fast synchronous release. Delayed asynchronous release was blocked in wild-type synapses by intracellular addition of high concentrations of the slow Ca(2+)-chelator EGTA, but EGTA had no effect in synaptotagmin-7 knock-out neurons because delayed asynchronous release was already impaired. Moreover, direct measurements of vesicle repriming failed to uncover an effect of the synaptotagmin-7 knock-out on vesicle repriming. Our data demonstrate that synaptotagmin-7 is selectively essential for Ca(2+)-dependent delayed asynchronous release in retinal rod bipolar cell synapses, that its function can be blocked by simply introducing a slow Ca(2+) buffer into the cells, and that synaptotagmin-7 is not required for normal vesicle repriming. SIGNIFICANCE STATEMENT: How Ca(2+) triggers delayed asynchronous release has long remained enigmatic. Synaptotagmin-7 has been implicated recently as Ca(2+) sensor in mediating delayed asynchronous release, or vesicle repriming, in cultured neurons. To test the precise function of synaptotagmin-7 in a physiologically important synapse in situ, we have used pair recordings to study the synaptic transmission between retinal rod bipolar cells and AII amacrine cells. Our data demonstrate that the knock-out of synaptotagmin-7 selectively impaired delayed asynchronous release but not synchronous release. In contrast, the readily releasable vesicles after depletion recover normally in knock-out mice. Therefore, our findings extend our knowledge of synaptotagmins as Ca(2+) sensors in vesicle fusion and support the idea that synapses are governed universally by different synaptotagmin Ca(2+) sensors mediating distinct release.
Asunto(s)
Células Amacrinas/metabolismo , Calcio/metabolismo , Células Bipolares de la Retina/metabolismo , Sinapsis/metabolismo , Vesículas Sinápticas/metabolismo , Sinaptotagminas/metabolismo , Células Amacrinas/citología , Animales , Ratones , Ratones Noqueados , Retina/citología , Retina/metabolismo , Células Bipolares de la Retina/citología , Sinapsis/genética , Transmisión Sináptica/fisiología , Sinaptotagminas/genéticaRESUMEN
Postsynaptic AMPA-type glutamate receptors (AMPARs) are among the major determinants of synaptic strength and can be trafficked into and out of synapses. Neuronal activity regulates AMPAR trafficking during synaptic plasticity to induce long-term changes in synaptic strength, including long-term potentiation (LTP) and long-term depression (LTD). Rab family GTPases regulate most membrane trafficking in eukaryotic cells; particularly, Rab11 and its effectors are implicated in mediating postsynaptic AMPAR insertion during LTP. To explore the synaptic function of Rab11Fip5, a neuronal Rab11 effector and a candidate autism-spectrum disorder gene, we performed shRNA-mediated knock-down and genetic knock-out (KO) studies. Surprisingly, we observed robust shRNA-induced synaptic phenotypes that were rescued by a Rab11Fip5 cDNA but that were nevertheless not observed in conditional KO neurons. Both in cultured neurons and acute slices, KO of Rab11Fip5 had no significant effect on basic parameters of synaptic transmission, indicating that Rab11Fip5 is not required for fundamental synaptic operations, such as neurotransmitter release or postsynaptic AMPAR insertion. KO of Rab11Fip5 did, however, abolish hippocampal LTD as measured both in acute slices or using a chemical LTD protocol in cultured neurons but did not affect hippocampal LTP. The Rab11Fip5 KO mice performed normally in several behavioral tasks, including fear conditioning, but showed enhanced contextual fear extinction. These are the first findings to suggest a requirement for Rab11Fip5, and presumably Rab11, during LTD.
Asunto(s)
Proteínas Portadoras/metabolismo , Hipocampo/citología , Depresión Sináptica a Largo Plazo/fisiología , Proteínas Mitocondriales/metabolismo , Sinapsis/genética , Animales , Animales Recién Nacidos , Proteínas Portadoras/genética , Condicionamiento Psicológico/fisiología , Conducta Exploratoria/fisiología , Miedo/fisiología , Antagonistas del GABA/farmacología , Técnicas In Vitro , Depresión Sináptica a Largo Plazo/genética , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales/genética , Mutación/genética , Picrotoxina/farmacología , Receptores AMPA/metabolismo , Conducta Social , Sinapsis/fisiología , Proteínas de Unión al GTP rabRESUMEN
Inositol hexakisphosphate (InsP(6)) levels rise and fall with neuronal excitation and silence, respectively, in the hippocampus, suggesting potential signaling functions of this inositol polyphosphate in hippocampal neurons. We now demonstrate that intracellular application of InsP(6) caused a concentration-dependent inhibition of autaptic excitatory postsynaptic currents (EPSCs) in cultured hippocampal neurons. The treatment did not alter the size and replenishment rate of the readily releasable pool in autaptic neurons. Intracellular exposure to InsP(6) did not affect spontaneous EPSCs or excitatory amino acid-activated currents in neurons lacking autapses. The InsP(6)-induced inhibition of autaptic EPSCs was effectively abolished by coapplication of an antibody to synaptotagmin-1 C2B domain. Importantly, preabsorption of the antibody with a GST-WT synaptotagmin-1 C2B domain fragment but not with a GST-mutant synaptotagmin-1 C2B domain fragment that poorly reacted with the antibody impaired the activity of the antibody on the InsP(6)-induced inhibition of autaptic EPSCs. Furthermore, K(+) depolarization significantly elevated endogenous levels of InsP(6) and occluded the inhibition of autaptic EPSCs by exogenous InsP(6). These data reveal that InsP(6) suppresses excitatory neurotransmission via inhibition of the presynaptic synaptotagmin-1 C2B domain-mediated fusion via an interaction with the synaptotagmin Ca(2+)-binding sites rather than via interference with presynaptic Ca(2+) levels, synaptic vesicle trafficking, or inactivation of postsynaptic ionotropic glutamate receptors. Therefore, elevated InsP(6) in activated neurons serves as a unique negative feedback signal to control hippocampal excitatory neurotransmission.
Asunto(s)
Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Ácido Fítico/metabolismo , Células Piramidales/fisiología , Transmisión Sináptica/fisiología , Sinaptotagmina I/metabolismo , Análisis de Varianza , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Retroalimentación Fisiológica/fisiología , Femenino , Técnicas de Placa-Clamp , Ácido Fítico/farmacología , Embarazo , Estructura Terciaria de Proteína/fisiología , Ratas , Ratas Sprague-Dawley , Conteo por Cintilación , TritioRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0158295.].
RESUMEN
Sensory neuron cilia are evolutionarily conserved dendritic appendages that convert environmental stimuli into neuronal activity. Although several cilia components are known, the functions of many remain uncharacterized. Furthermore, the basis of morphological and functional differences between cilia remains largely unexplored. To understand the molecular basis of cilia morphogenesis and function, we studied the Caenorhabditis elegans mutants che-12 and dyf-11. These mutants fail to concentrate lipophilic dyes from their surroundings in sensory neurons and are chemotaxis defective. In che-12 mutants, sensory neuron cilia lack distal segments, while in dyf-11 animals, medial and distal segments are absent. CHE-12 and DYF-11 are conserved ciliary proteins that function cell-autonomously and are continuously required for maintenance of cilium morphology and function. CHE-12, composed primarily of HEAT repeats, may not be part of the intraflagellar transport (IFT) complex and is not required for the localization of some IFT components. DYF-11 undergoes IFT-like movement and may function at an early stage of IFT-B particle assembly. Intriguingly, while DYF-11 is expressed in all C. elegans ciliated neurons, CHE-12 expression is restricted to some amphid sensory neurons, suggesting a specific role in these neurons. Our results provide insight into general and neuron-specific aspects of cilium development and function.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Cilios/fisiología , Cilios/ultraestructura , Neuronas Aferentes/fisiología , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Carbocianinas/farmacocinética , Mapeo Cromosómico , Clonación Molecular , Colorantes/farmacocinética , Secuencia Conservada , Trastornos del Desarrollo Sexual/genética , Fluoresceína-5-Isotiocianato/farmacocinética , Neuronas Aferentes/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , PlásmidosRESUMEN
Cell-specific promoters allow only spatial control of transgene expression in Caenorhabditis elegans. We describe a method, using cell-specific rescue of heat-shock factor-1 (hsf-1) mutants, that allows spatial and temporal regulation of transgene expression. We demonstrate the utility of this method for timed reporter gene expression and for temporal studies of gene function.
Asunto(s)
Caenorhabditis elegans/genética , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Regulación de la Expresión Génica , Genes de Helminto , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Mutación , Fenotipo , Regiones Promotoras Genéticas , Proteínas Recombinantes/genética , Factores de Tiempo , Factores de Transcripción/genéticaAsunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Fusión de Membrana , Fosfoproteínas/metabolismo , Proteínas SNARE/metabolismo , Sintaxina 1/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Modelos Moleculares , Complejos Multiproteicos/química , Complejos Multiproteicos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/genética , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas SNARE/química , Proteínas SNARE/genética , Sintaxina 1/química , Sintaxina 1/genética , Factores de Tiempo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genéticaRESUMEN
Extended synaptotagmins (ESyts) are endoplasmic reticulum (ER) proteins composed of an N-terminal transmembrane region, a central SMP-domain, and five (ESyt1) or three C-terminal cytoplasmic C2-domains (ESyt2 and ESyt3). ESyts bind phospholipids in a Ca2+-dependent manner via their C2-domains, are localized to ER-plasma membrane contact sites, and may catalyze lipid exchange between the plasma membrane and the ER via their SMP-domains. However, the overall function of ESyts has remained enigmatic. Here, we generated triple constitutive and conditional knock-out mice that lack all three ESyt isoforms; in addition, we produced knock-in mice that express mutant ESyt1 or ESyt2 carrying inactivating substitutions in the Ca2+-binding sites of their C2A-domains. Strikingly, all ESyt mutant mice, even those lacking all ESyts, were apparently normal and survived and bred in a manner indistinguishable from control mice. ESyt mutant mice displayed no major changes in brain morphology or synaptic protein composition, and exhibited no large alterations in stress responses. Thus, in mice ESyts do not perform an essential role in basic cellular functions, suggesting that these highly conserved proteins may perform a specialized role that may manifest only during specific, as yet untested challenges.
Asunto(s)
Retículo Endoplásmico/metabolismo , Fertilidad/genética , Fenotipo , Sinaptotagminas/deficiencia , Animales , Encéfalo/metabolismo , Calcio/metabolismo , Señalización del Calcio , Línea Celular , Supervivencia Celular/genética , Técnicas de Inactivación de Genes , Orden Génico , Marcación de Gen , Sitios Genéticos , Genotipo , Humanos , Ratones , Ratones Noqueados , Neuronas/metabolismo , Estrés Fisiológico , Sinaptotagminas/genéticaRESUMEN
Rapid neurotransmitter release depends on the Ca2+ sensor Synaptotagmin-1 (Syt1) and the SNARE complex formed by synaptobrevin, syntaxin-1 and SNAP-25. How Syt1 triggers release has been unclear, partly because elucidating high-resolution structures of Syt1-SNARE complexes has been challenging. An NMR approach based on lanthanide-induced pseudocontact shifts now reveals a dynamic binding mode in which basic residues in the concave side of the Syt1 C2B-domain ß-sandwich interact with a polyacidic region of the SNARE complex formed by syntaxin-1 and SNAP-25. The physiological relevance of this dynamic structural model is supported by mutations in basic residues of Syt1 that markedly impair SNARE-complex binding in vitro and Syt1 function in neurons. Mutations with milder effects on binding have correspondingly milder effects on Syt1 function. Our results support a model whereby dynamic interaction facilitates cooperation between Syt1 and the SNAREs in inducing membrane fusion.
Asunto(s)
Proteínas SNARE/metabolismo , Sinaptotagmina I/metabolismo , Animales , Células Cultivadas , Humanos , Ratones Endogámicos C57BL , Modelos Moleculares , Neuronas/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Estructura Terciaria de Proteína , Ratas , Proteínas SNARE/química , Sinaptotagmina I/químicaRESUMEN
Contacts between the endoplasmic reticulum and the plasma membrane involve extended synaptotagmins (E-Syts) in mammals or tricalbins in yeast, proteins with multiple C2 domains. One of the tandem C2 domains of E-Syt2 is predicted to bind Ca²âº, but no Ca²âº-dependent function has been attributed to this protein. We have determined the crystal structures of the tandem C2 domains of E-Syt2 in the absence and presence of Ca²âº and analyzed their Ca²âº-binding properties by nuclear magnetic resonance spectroscopy. Our data reveal an unexpected V-shaped structure with a rigid orientation between the two C2 domains that is not substantially altered by Ca²âº. The E-Syt2 C2A domain binds up to four Ca²âº ions, whereas the C2B domain does not bind Ca²âº. These results suggest that E-Syt2 performs an as yet unidentified Ca²âº-dependent function through its C2A domain and uncover fundamental differences between the properties of the tandem C2 domains of E-Syts and synaptotagmins.
Asunto(s)
Calcio/química , Sinaptotagminas/química , Sitios de Unión , Dicroismo Circular , ADN Complementario/metabolismo , Retículo Endoplásmico/metabolismo , Escherichia coli/metabolismo , Humanos , Iones , Espectroscopía de Resonancia Magnética , Mutación , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Transducción de Señal , Temperatura , Difracción de Rayos XRESUMEN
Synaptic vesicle fusion during neurotransmitter release is mediated by assembly of SNARE- and SM-protein complexes composed of syntaxin-1, SNAP-25, synaptobrevin-2/VAMP2, and Munc18-1. Current models suggest that SNARE-complex assembly catalyzes membrane fusion by pulling the transmembrane regions (TMRs) of SNARE proteins together, thus allowing their TMRs to form a fusion pore. These models are consistent with the requirement for TMRs in viral fusion proteins. However, the role of the SNARE TMRs in synaptic vesicle fusion has not yet been tested physiologically. Here, we examined whether synaptic SNAREs require TMRs for catalysis of synaptic vesicle fusion, which was monitored electrophysiologically at millisecond time resolution. Surprisingly, we find that both lipid-anchored syntaxin-1 and lipid-anchored synaptobrevin-2 lacking TMRs efficiently promoted spontaneous and Ca(2+)-triggered membrane fusion. Our data suggest that SNARE proteins function during fusion primarily as force generators, consistent with the notion that forcing lipid membranes close together suffices to induce membrane fusion.
Asunto(s)
Fusión de Membrana/fisiología , Lípidos de la Membrana/metabolismo , Neurotransmisores/metabolismo , Proteínas SNARE/metabolismo , Transmisión Sináptica/fisiología , Vesículas Sinápticas/metabolismo , Animales , Células Cultivadas , Potenciales Postsinápticos Inhibidores/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Neuronas/fisiología , Proteínas SNARE/química , Proteínas SNARE/genética , Sintaxina 1/genética , Sintaxina 1/fisiología , Proteína 2 de Membrana Asociada a Vesículas/genética , Proteína 2 de Membrana Asociada a Vesículas/fisiologíaRESUMEN
In forebrain neurons, knockout of synaptotagmin-1 blocks fast Ca(2+)-triggered synchronous neurotransmitter release but enables manifestation of slow Ca(2+)-triggered asynchronous release. Here, we show using single-cell PCR that individual hippocampal neurons abundantly coexpress two Ca(2+)-binding synaptotagmin isoforms, synaptotagmin-1 and synaptotagmin-7. In synaptotagmin-1-deficient synapses of excitatory and inhibitory neurons, loss of function of synaptotagmin-7 suppressed asynchronous release. This phenotype was rescued by wild-type but not mutant synaptotagmin-7 lacking functional Ca(2+)-binding sites. Even in synaptotagmin-1-containing neurons, synaptotagmin-7 ablation partly impaired asynchronous release induced by extended high-frequency stimulus trains. Synaptotagmins bind Ca(2+) via two C2 domains, the C2A and C2B domains. Surprisingly, synaptotagmin-7 function selectively required its C2A domain Ca(2+)-binding sites, whereas synaptotagmin-1 function required its C2B domain Ca(2+)-binding sites. Our data show that nearly all Ca(2+)-triggered release at a synapse is due to synaptotagmins, with synaptotagmin-7 mediating a slower form of Ca(2+)-triggered release that is normally occluded by faster synaptotagmin-1-induced release but becomes manifest upon synaptotagmin-1 deletion.
Asunto(s)
Neurotransmisores/metabolismo , Sinaptotagmina I/fisiología , Sinaptotagminas/fisiología , Animales , Calcio/fisiología , Células Cultivadas , Dependovirus/genética , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Hipocampo/fisiología , Lentivirus/genética , Ratones , Ratones Noqueados , Neuronas/metabolismo , Técnicas de Placa-Clamp , Interferencia de ARN , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores Sensibles al Calcio/fisiología , Sinapsis/metabolismo , Sinaptotagmina I/genética , Sinaptotagminas/genéticaRESUMEN
Two families of Ca(2+)-binding proteins have been proposed as Ca(2+) sensors for spontaneous release: synaptotagmins and Doc2s, with the intriguing possibility that Doc2s may represent high-affinity Ca(2+) sensors that are activated by deletion of synaptotagmins, thereby accounting for the increased spontaneous release in synaptotagmin-deficient synapses. Here, we use an shRNA-dependent quadruple knockdown of all four Ca(2+)-binding proteins of the Doc2 family to confirm that Doc2-deficient synapses exhibit a marked decrease in the frequency of spontaneous release events. Knockdown of Doc2s in synaptotagmin-1-deficient synapses, however, failed to reduce either the increased spontaneous release or the decreased evoked release of these synapses, suggesting that Doc2s do not constitute Ca(2+) sensors for asynchronous release. Moreover, rescue experiments revealed that the decrease in spontaneous release induced by the Doc2 knockdown in wild-type synapses is fully reversed by mutant Doc2B lacking Ca(2+)-binding sites. Thus, our data suggest that Doc2s are modulators of spontaneous synaptic transmission that act by a Ca(2+)-independent mechanism.
Asunto(s)
Proteínas de Unión al Calcio/fisiología , Calcio/metabolismo , Proteínas del Tejido Nervioso/fisiología , Neuronas/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Recién Nacidos , Biofisica , Proteínas de Unión al Calcio/deficiencia , Proteínas de Unión al Calcio/genética , Células Cultivadas , Corteza Cerebral/citología , Humanos , Proteínas de la Membrana/deficiencia , Ratones , Ratones Noqueados , Mutagénesis Sitio-Dirigida/métodos , Mutación/fisiología , Proteínas del Tejido Nervioso/deficiencia , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp/métodos , Interferencia de ARN/fisiología , Transmisión Sináptica/efectos de los fármacos , Transfección , Proteínas de Transporte VesicularRESUMEN
Sensory organs are composed of neurons, which convert environmental stimuli to electrical signals, and glia-like cells, whose functions are not well understood. To decipher glial roles in sensory organs, we ablated the sheath glial cell of the major sensory organ of Caenorhabditis elegans. We found that glia-ablated animals exhibit profound sensory deficits and that glia provide activities that affect neuronal morphology, behavior generation, and neuronal uptake of lipophilic dyes. To understand the molecular bases of these activities, we identified 298 genes whose messenger RNAs are glia-enriched. One gene, fig-1, encodes a labile protein with conserved thrombospondin TSP1 domains. FIG-1 protein functions extracellularly, is essential for neuronal dye uptake, and also affects behavior. Our results suggest that glia are required for multiple aspects of sensory organ function.