RESUMEN
Neuroimmune cross-talk participates in intestinal tissue homeostasis and host defense. However, the matrix of interactions between arrays of molecularly defined neuron subsets and of immunocyte lineages remains unclear. We used a chemogenetic approach to activate eight distinct neuronal subsets, assessing effects by deep immunophenotyping, microbiome profiling, and immunocyte transcriptomics in intestinal organs. Distinct immune perturbations followed neuronal activation: Nitrergic neurons regulated T helper 17 (TH17)-like cells, and cholinergic neurons regulated neutrophils. Nociceptor neurons, expressing Trpv1, elicited the broadest immunomodulation, inducing changes in innate lymphocytes, macrophages, and RORγ+ regulatory T (Treg) cells. Neuroanatomical, genetic, and pharmacological follow-up showed that Trpv1+ neurons in dorsal root ganglia decreased Treg cell numbers via the neuropeptide calcitonin gene-related peptide (CGRP). Given the role of these neurons in nociception, these data potentially link pain signaling with gut Treg cell function.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina , Ganglios Espinales , Neuroinmunomodulación , Nociceptores , Linfocitos T Reguladores , Canales Catiónicos TRPV , Células Th17 , Animales , Ratones , Péptido Relacionado con Gen de Calcitonina/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Neuronas Colinérgicas/metabolismo , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Microbioma Gastrointestinal , Intestinos/inmunología , Intestinos/citología , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Nocicepción , Nociceptores/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Células Th17/inmunología , Canales Catiónicos TRPV/metabolismo , Canales Catiónicos TRPV/genéticaRESUMEN
SNARE-mediated membrane fusion plays a crucial role in presynaptic vesicle exocytosis and also in postsynaptic receptor delivery. The latter is considered particularly important for synaptic plasticity and learning and memory, yet the identity of the key SNARE proteins remains elusive. Here, we investigate the role of neuronal synaptosomal-associated protein-23 (SNAP-23) by analyzing pyramidal-neuron specific SNAP-23 conditional knockout (cKO) mice. Electrophysiological analysis of SNAP-23 deficient neurons using acute hippocampal slices showed normal basal neurotransmission in CA3-CA1 synapses with unchanged AMPA and NMDA currents. Nevertheless, we found theta-burst stimulation-induced long-term potentiation (LTP) was vastly diminished in SNAP-23 cKO slices. Moreover, unlike syntaxin-4 cKO mice where both basal neurotransmission and LTP decrease manifested changes in a broad set of behavioral tasks, deficits of SNAP-23 cKO are more limited to spatial memory. Our data reveal that neuronal SNAP-23 is selectively crucial for synaptic plasticity and spatial memory without affecting basal glutamate receptor function.
RESUMEN
Pathogen infection causes a stereotyped state of sickness that involves neuronally orchestrated behavioural and physiological changes1,2. On infection, immune cells release a 'storm' of cytokines and other mediators, many of which are detected by neurons3,4; yet, the responding neural circuits and neuro-immune interaction mechanisms that evoke sickness behaviour during naturalistic infections remain unclear. Over-the-counter medications such as aspirin and ibuprofen are widely used to alleviate sickness and act by blocking prostaglandin E2 (PGE2) synthesis5. A leading model is that PGE2 crosses the blood-brain barrier and directly engages hypothalamic neurons2. Here, using genetic tools that broadly cover a peripheral sensory neuron atlas, we instead identified a small population of PGE2-detecting glossopharyngeal sensory neurons (petrosal GABRA1 neurons) that are essential for influenza-induced sickness behaviour in mice. Ablating petrosal GABRA1 neurons or targeted knockout of PGE2 receptor 3 (EP3) in these neurons eliminates influenza-induced decreases in food intake, water intake and mobility during early-stage infection and improves survival. Genetically guided anatomical mapping revealed that petrosal GABRA1 neurons project to mucosal regions of the nasopharynx with increased expression of cyclooxygenase-2 after infection, and also display a specific axonal targeting pattern in the brainstem. Together, these findings reveal a primary airway-to-brain sensory pathway that detects locally produced prostaglandins and mediates systemic sickness responses to respiratory virus infection.
Asunto(s)
Barrera Hematoencefálica , Encéfalo , Dinoprostona , Nasofaringe , Infecciones por Orthomyxoviridae , Células Receptoras Sensoriales , Animales , Humanos , Ratones , Conducta Animal , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Tronco Encefálico/fisiopatología , Dinoprostona/metabolismo , Ingestión de Líquidos , Ingestión de Alimentos , Gripe Humana/complicaciones , Gripe Humana/metabolismo , Movimiento , Nasofaringe/inervación , Orthomyxoviridae/patogenicidad , Infecciones por Orthomyxoviridae/complicaciones , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Células Receptoras Sensoriales/metabolismo , Tasa de SupervivenciaRESUMEN
Recent evidence suggests that SNARE fusion machinery play critical roles in postsynaptic neurotransmitter receptor trafficking, which is essential for synaptic plasticity. However, the key SNAREs involved remain highly controversial; syntaxin-3 and syntaxin-4 are leading candidates for the syntaxin isoform underlying postsynaptic plasticity. In a previous study, we showed that pyramidal-neuron specific conditional knockout (cKO) of syntaxin-4 significantly reduces basal transmission, synaptic plasticity and impairs postsynaptic receptor trafficking. However, this does not exclude a role for syntaxin-3 in such processes. Here, we generated and analyzed syntaxin-3 cKO mice. Extracellular field recordings in hippocampal slices showed that syntaxin-3 cKO did not exhibit significant changes in CA1 basal neurotransmission or in paired-pulse ratios. Importantly, there were no observed differences during LTP in comparison to control mice. Syntaxin-3 cKO mice performed similarly as the controls in spatial and contextual learning tasks. Consistent with the minimal effects of syntaxin-3 cKO, syntaxin-3 mRNA level was very low in hippocampal and cortex pyramidal neurons, but strongly expressed in the corpus callosum and caudate axon fibers. Together, our data suggest that syntaxin-3 is dispensable for hippocampal basal neurotransmission and synaptic plasticity, and further supports the notion that syntaxin-4 is the major isoform mediating these processes.
Asunto(s)
Región CA1 Hipocampal/fisiología , Plasticidad Neuronal/genética , Plasticidad Neuronal/fisiología , Proteínas Qa-SNARE/fisiología , Transmisión Sináptica/genética , Transmisión Sináptica/fisiología , Animales , Región CA1 Hipocampal/metabolismo , Cuerpo Calloso/metabolismo , Expresión Génica , Técnicas In Vitro , Potenciación a Largo Plazo/fisiología , Ratones Noqueados , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , ARN Mensajero/metabolismoRESUMEN
Voa protein is a subunit of V-ATPase proton pump which is essential to acidify intracellular organelles including synaptic vesicles. Voa1 is one of the four isoforms of Voa family with strong expression in neurons. Our present study was aimed to examine the role of Voa1 protein in mammalian brain neurons. To circumvent embryonic lethality, we generated conditional Voa1 knockout mice in which Voa1 was selectively deleted from forebrain pyramidal neurons. We performed experiments in the Voa1 knockout mice of ages 5-6 months to assess the persistent effects of Voa1 deletion. We found that the Voa1 knockout mice exhibited poor performance in the Morris water maze test compared to control mice. In addition, synaptic field potentials of the hippocampal CA1 region were greatly diminished in the Voa1 knockout mice when examined in brain slices in vitro. Furthermore, brain histological experiments showed severe degeneration of dorsal hippocampal CA1 neurons while CA3 neurons were largely preserved. The CA1 neurodegeneration was associated with general brain atrophy as overall hemispheric areas were reduced in the Voa1 cKO mice. Despite the CA1 degeneration and dysfunction, electroencephalographic recordings from the hippocampal CA3 area revealed aberrant spikes and non-convulsive discharges in the Voa1 knockout mice but not in control mice. These hippocampal spikes were suppressed by single intra-peritoneal injection of diazepam which is a benzodiazepine GABAA receptor enhancer. Together these results suggest that Voa1 related activities are essential for the survival of the targeted neurons in the dorsal hippocampal CA1 as well as other forebrain areas. We postulate that the Voa1 knockout mice may serve as a valuable model for further investigation of V-ATPase dysfunction related neuronal degeneration and functional abnormalities in forebrain areas particularly the hippocampus.
RESUMEN
Trafficking or delivery of neurotransmitter receptors on postsynaptic membranes is critical for basal neurotransmission and synaptic plasticity. Importantly, dysfunction of such postsynaptic receptor trafficking can lead to severe brain diseases such as Alzheimer's Disease, autism spectrum disorder, and intellectual disability, yet underlying mechanisms remain elusive. One attractive hypothesis is that postsynaptic SNARE proteins play key roles in the delivery of receptors by mediating membrane fusion at postsynaptic neurons. However, the identities of the critical SNARE proteins mediating the delivery remain controversial. The lack of consensus in previous studies is partly due to differences in preparations and methodologies. In this review, we propose to employ a pyramidal-neuron specific conditional knockout (cKO) model to study the roles of candidate SNARE proteins in postsynaptic receptor trafficking. We highlight our recent results which we obtained from such approaches to syntaxin-4 protein. These results provide clear evidence on the critical role of syntaxin-4 in trafficking of ionotropic glutamate receptors which are essential for basal neurotransmission, synaptic plasticity and spatial memory.
Asunto(s)
Plasticidad Neuronal/fisiología , Transporte de Proteínas/fisiología , Células Piramidales/metabolismo , Receptores de Glutamato/metabolismo , Proteínas SNARE/metabolismo , Animales , Ratones , Ratones Noqueados , Modelos AnimalesRESUMEN
Trafficking of neurotransmitter receptors on postsynaptic membranes is critical for basal neurotransmission and synaptic plasticity, yet the underlying mechanisms remain elusive. Here, we investigated the role of syntaxin 4 in postsynaptic hippocampal CA1 neurons by analyzing conditional knockout (syntaxin 4 cKO) mice. We show that syntaxin 4 cKO resulted in reduction of basal neurotransmission without changes in paired-pulse ratios. Both α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptor-mediated charge transfers were diminished. Patch-clamp experiments revealed that amplitudes, but not frequencies, of spontaneous excitatory postsynaptic currents are reduced. Syntaxin 4 knockout (KO) caused drastic reduction in expression of surface α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and N-methyl-d-aspartic acid (NMDA) receptors in cultured hippocampal neurons. Furthermore, cKO caused defects in theta-burst stimulation induced long-term potentiation and spatial learning as assessed by a water maze task, indicating that synaptic plasticity was altered. Our data reveal a crucial role of syntaxin 4 in trafficking of ionotropic glutamate receptors that are essential for basal neurotransmission, synaptic plasticity, and spatial memory.
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Región CA1 Hipocampal/fisiología , Plasticidad Neuronal , Neuronas/fisiología , Proteínas Qa-SNARE/metabolismo , Sinapsis/metabolismo , Transmisión Sináptica , Animales , Células Cultivadas , Potenciales Postsinápticos Excitadores/fisiología , Eliminación de Gen , Potenciación a Largo Plazo/fisiología , Ratones Noqueados , Especificidad de Órganos , Receptores AMPA/metabolismo , Receptores de Glutamato/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Memoria EspacialRESUMEN
In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.
Asunto(s)
Células Asesinas Naturales/inmunología , Linfohistiocitosis Hemofagocítica/inmunología , Mastocitos/inmunología , Proteínas de la Membrana/metabolismo , Vesículas Secretoras/metabolismo , Animales , Ácido Aspártico/genética , Señalización del Calcio , Degranulación de la Célula , Células Cultivadas , Citotoxicidad Inmunológica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Mutación/genética , Dominios Proteicos/genética , RatasRESUMEN
Mast cells play pivotal roles in innate and adaptive immunities but are also culprits in allergy, autoimmunity, and cardiovascular diseases. Mast cells respond to environmental changes by initiating regulated exocytosis/secretion of various biologically active compounds called mediators (e.g. proteases, amines, and cytokines). Many of these mediators are stored in granules/lysosomes and rely on intricate degranulation processes for release. Mast cell stabilizers (e.g. sodium cromoglicate), which prevent such degranulation processes, have therefore been clinically employed to treat asthma and allergic rhinitis. However, it has become increasingly clear that different mast cell diseases often involve multiple mediators that rely on overlapping but distinct mechanisms for release. This review illustrates existing evidence that highlights the diverse exocytic pathways in mast cells. We also discuss strategies to delineate these pathways so as to identify unique molecular components which could serve as new drug targets for more effective and specific treatments against mast cell-related diseases.
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Exocitosis , Mastocitos/inmunología , Aminas/metabolismo , Animales , Degranulación de la Célula/efectos de los fármacos , Fusión Celular , Citocinas/metabolismo , Humanos , Lisosomas/metabolismo , Mastocitos/efectos de los fármacos , Mastocitos/metabolismo , Péptido Hidrolasas/metabolismoRESUMEN
We describe here a simple, cost-effective apparatus for continuous tethered electroencephalographic (EEG) monitoring of spontaneous recurrent seizures in mice. We used a small, low torque slip ring as an EEG commutator, mounted the slip ring onto a standard mouse cage and connected rotary wires of the slip ring directly to animal's implanted headset. Modifications were made in the cage to allow for a convenient installation of the slip ring and accommodation of animal ambient activity. We tested the apparatus for hippocampal EEG recordings in adult C57 black mice. Spontaneous recurrent seizures were induced using extended hippocampal kindling (≥95 daily stimulation). Control animals underwent similar hippocampal electrode implantations but no stimulations were given. Combined EEG and webcam monitoring were performed for 24 h daily for 5-9 consecutive days. During the monitoring periods, the animals moved and accessed water and food freely and showed no apparent restriction in ambient cage activities. Ictal-like hippocampal EEG discharges and concurrent convulsive behaviors that are characteristics of spontaneous recurrent seizures were reliably recorded in a majority of the monitoring experiments in extendedly kindled but not in control animals. However, 1-2 rotary wires were disconnected from the implanted headset in some animals after continuous recordings for ≥5 days. The key features and main limitations of our recording apparatus are discussed.
RESUMEN
Munc18-1/UNC-18 is believed to prime SNARE-mediated membrane fusion, yet the underlying mechanisms remain enigmatic. Here, we examine how potential gain-of-function mutations of Munc18-1/UNC-18 affect locomotory behavior and synaptic transmission, and how Munc18-1-mediated priming is related to Munc13-1/UNC-13 and Tomosyn/TOM-1, positive and negative SNARE regulators, respectively. We show that a Munc18-1(P335A)/UNC-18(P334A) mutation leads to significantly increased locomotory activity and acetylcholine release in Caenorhabditis elegans, as well as enhanced synaptic neurotransmission in cultured mammalian neurons. Importantly, similar to tom-1 null mutants, unc-18(P334A) mutants partially bypass the requirement of UNC-13. Moreover, unc-18(P334A) and tom-1 null mutations confer a strong synergy in suppressing the phenotypes of unc-13 mutants. Through biochemical experiments, we demonstrate that Munc18-1(P335A) exhibits enhanced activity in SNARE complex formation as well as in binding to the preformed SNARE complex, and partially bypasses the Munc13-1 requirement in liposome fusion assays. Our results indicate that Munc18-1/UNC-18 primes vesicle fusion downstream of Munc13-1/UNC-13 by templating SNARE complex assembly and acts antagonistically with Tomosyn/TOM-1.SIGNIFICANCE STATEMENT At presynaptic sites, SNARE-mediated membrane fusion is tightly regulated by several key proteins including Munc18/UNC-18, Munc13/UNC-13, and Tomosyn/TOM-1. However, how these proteins interact with each other to achieve the precise regulation of neurotransmitter release remains largely unclear. Using Caenorhabditis elegans as an in vivo model, we found that a gain-of-function mutant of UNC-18 increases locomotory activity and synaptic acetylcholine release, that it partially bypasses the requirement of UNC-13 for release, and that this bypass is synergistically augmented by the lack of TOM-1. We also elucidated the biochemical basis for the gain-of-function caused by this mutation. Thus, our study provides novel mechanistic insights into how Munc18/UNC-18 primes synaptic vesicle release and how this protein interacts functionally with Munc13/UNC-13 and Tomosyn/TOM-1.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Proteínas Portadoras/metabolismo , Locomoción/fisiología , Fosfoproteínas/metabolismo , Proteínas SNARE/metabolismo , Transmisión Sináptica/fisiología , Proteínas de Transporte Vesicular/metabolismo , Animales , Proteínas de Caenorhabditis elegans/genética , Proteínas Portadoras/genética , Mutación/genética , Neuronas , Fosfoproteínas/genética , Vesículas Sinápticas/metabolismo , Proteínas de Transporte Vesicular/genéticaRESUMEN
Syntaxin-1 is the central SNARE protein for neuronal exocytosis. It interacts with Munc18-1 through its cytoplasmic domains, including the N-terminal peptide (N-peptide). Here we examine the role of the N-peptide binding in two conformational states ("closed" vs. "open") of syntaxin-1 using PC12 cells and Caenorhabditis elegans. We show that expression of "closed" syntaxin-1A carrying N-terminal single point mutations (D3R, L8A) that perturb interaction with the hydrophobic pocket of Munc18-1 rescues impaired secretion in syntaxin-1-depleted PC12 cells and the lethality and lethargy of unc-64 (C. elegans orthologue of syntaxin-1)-null mutants. Conversely, expression of the "open" syntaxin-1A harboring the same mutations fails to rescue the impairments. Biochemically, the L8A mutation alone slightly weakens the binding between "closed" syntaxin-1A and Munc18-1, whereas the same mutation in the "open" syntaxin-1A disrupts it. Our results reveal a striking interplay between the syntaxin-1 N-peptide and the conformational state of the protein. We propose that the N-peptide plays a critical role in intracellular trafficking of syntaxin-1, which is dependent on the conformational state of this protein. Surprisingly, however, the N-peptide binding mode seems dispensable for SNARE-mediated exocytosis per se, as long as the protein is trafficked to the plasma membrane.
Asunto(s)
Proteínas de Caenorhabditis elegans/química , Exocitosis , Proteínas Munc18/metabolismo , Neuronas/fisiología , Sintaxina 1/química , Secuencia de Aminoácidos , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Técnicas de Silenciamiento del Gen , Datos de Secuencia Molecular , Neuronas/metabolismo , Células PC12 , Péptidos/química , Péptidos/metabolismo , Mutación Puntual , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Ratas , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation whose levels and intracellular localization are regulated by Munc18-2 remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.
Asunto(s)
Mastocitos/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Unión Proteica/genética , Proteínas Qa-SNARE/metabolismo , Animales , Exocitosis , Humanos , RatasRESUMEN
Munc18-1 plays essential dual roles in exocytosis: (i) stabilizing and trafficking the central SNARE protein, syntaxin-1 (i.e. chaperoning function), by its domain-1; and (ii) priming/stimulating exocytosis by its domain-3a. Here, we examine whether or not domain-3a also plays a significant role in the chaperoning of syntaxin-1 and, if so, how these dual functions of domain-3a are regulated. We demonstrate that introduction of quintuple mutations (K332E/K333E/P335A/Q336A/Y337L) in domain-3a of Munc18-1 abolishes its ability to bind syntaxin-1 and fails to rescue the level and trafficking of syntaxin-1 as well as to restore exocytosis in Munc18-1/2 double knockdown cells. By contrast, a quadruple mutant (K332E/K333E/Q336A/Y337L) sparing the Pro-335 residue retains all of these capabilities. A single point mutant of P335A reduces the ability to bind syntaxin-1 and rescue syntaxin-1 levels. Nonetheless, it surprisingly outperforms the wild type in the rescue of exocytosis. However, when additional mutations in the neighboring residues are combined with P335A mutation (K332E/K333E/P335A, P335A/Q336A/Y337L), the ability of the Munc18-1 variants to chaperone syntaxin-1 and to rescue exocytosis is strongly impaired. Our results indicate that residues from Lys-332 to Tyr-337 of domain-3a are intimately tied to the chaperoning function of Munc18-1. We also propose that Pro-335 plays a pivotal role in regulating the balance between the dual functions of domain-3a. The hinged conformation of the α-helix containing Pro-335 promotes the syntaxin-1 chaperoning function, whereas the P335A mutation promotes its priming function by facilitating the α-helix to adopt an extended conformation.
Asunto(s)
Exocitosis/fisiología , Chaperonas Moleculares/metabolismo , Proteínas Munc18/metabolismo , Sustitución de Aminoácidos , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Chaperonas Moleculares/genética , Proteínas Munc18/genética , Mutación Missense , Prolina/genética , Prolina/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Sintaxina 1/genética , Sintaxina 1/metabolismoRESUMEN
The function of the Munc18-1 protein hydrophobic pocket, which interacts with the syntaxin-1 N-terminal peptide, has been highly controversial in neurosecretion. Recent analysis of patients with familial hemophagocytic lymphohistiocytosis type 5 has identified the E132A mutation in the hydrophobic pocket of Munc18-2, prompting us to examine the role of this region in the context of immune cell secretion. Double knockdown of Munc18-1 and Munc18-2 in RBL-2H3 mast cells eliminates both IgE-dependent and ionomycin-induced degranulation and causes a significant reduction in syntaxin-11 without altering expressions of the other syntaxin isoforms examined. These phenotypes were effectively rescued on reexpression of wild-type Munc18-1 or Munc18-2 but not the mutants (F115E, E132A, and F115E/E132A) in the hydrophobic pocket of Munc18. In addition, these mutants show that they are unable to directly interact with syntaxin-11, as tested through protein interaction experiments. Our results demonstrate the crucial roles of the hydrophobic pocket of Munc18 in mast cell degranulation, which include the regulation of syntaxin-11. We also suggest that the functional importance of this region is significantly different between neuronal and immune cell exocytosis.
Asunto(s)
Degranulación de la Célula , Linfohistiocitosis Hemofagocítica/metabolismo , Mastocitos/metabolismo , Proteínas Munc18/metabolismo , Sustitución de Aminoácidos , Animales , Ionóforos de Calcio/farmacología , Células HEK293 , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoglobulina E/metabolismo , Inmunoglobulina E/farmacología , Ionomicina/farmacología , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/patología , Mastocitos/patología , Ratones , Proteínas Munc18/genética , Mutación Missense , Células PC12 , Estructura Terciaria de Proteína , Proteínas Qa-SNARE/genética , Proteínas Qa-SNARE/metabolismo , RatasRESUMEN
Munc18-1 is believed to prime or stimulate SNARE-mediated membrane fusion/exocytosis through binding to the SNARE complex, in addition to chaperoning its cognate syntaxins. Nevertheless, a Munc18-1 mutant that selectively loses the priming function while retaining the syntaxin chaperoning activity has not been identified. As a consequence, the mechanism that mediates Munc18-1-dependent priming remains unclear. In the course of analyzing the functional outcomes of a variety of point mutations in domain 3a of Munc18-1, we discovered insertion mutants (K332E/K333E with insertions of 5 or 39 residues). These mutants completely lose their ability to rescue secretion whereas they effectively restore syntaxin-1 expression at the plasma membrane as well as dense-core vesicle docking in Munc18-1 and Munc18-2 double-knockdown PC12 cells. The mutants can bind syntaxin-1A in a stoichiometric manner. However, binding to the SNARE complex is impaired compared with the wild type or the hydrophobic pocket mutant (F115E). Our results suggest that the domain 3a of Munc18-1 plays a crucial role in priming of exocytosis, which is independent of its syntaxin-1 chaperoning activity and is downstream of dense-core vesicle docking. We also suggest that the priming mechanism of Munc18-1 involves its domain-3a-dependent interaction with the SNARE complex.