RESUMEN
BACKGROUND: This retrospective study analyzed the effectiveness of intrathoracic negative pressure therapy for debilitated patients with empyema and compared the short-term and long-term outcomes of three different intrapleural vacuum-assisted closure (VAC) techniques. METHODS: We investigated 43 consecutive (pre)septic patients with poor general condition (Karnofsky index ≤ 50 %) and multimorbidity (≥ 3 organ diseases) or immunosuppression, who had been treated for primary, postoperative, or recurrent pleural empyema with VAC in combination with open window thoracostomy (OWT-VAC) with minimally invasive technique (Mini-VAC), and instillation (Mini-VAC-Instill). RESULTS: The overall duration of intrathoracic vacuum therapy was 14 days (5-48 days). Vacuum duration in the Mini-VAC and Mini-VAC-Instill groups (12.4 ± 5.7 and 10.4 ± 5.4 days) was significantly shorter (p = 0.001) than in the group treated with open window thoracostomy (OWT)-VAC (20.3 ± 9.4 days). No major complication was related to intrathoracic VAC therapy. Chest wall closure rates were significantly higher in the Mini-VAC and Mini-VAC-Instill groups than in the OWT-VAC group (p = 0.034 and p = 0.026). Overall, the mean postoperative length of stay in hospital (LOS) was 21 days (median 18, 6-51 days). LOS was significantly shorter (p = 0.027) in the Mini-VAC-Instill group (15.1 ± 4.8) than in the other two groups (23.8 ± 12.3 and 22.7 ± 1.5). Overall, the 30-day and 60-day mortality rates were 4.7 % (2/43) and 9.3 % (4/43), and none of the deaths was related to infection. CONCLUSIONS: For debilitated patients, immediate minimally invasive intrathoracic vacuum therapy is a safe and viable alternative to OWT. Mini-VAC-Instill may have the fastest clearance and healing rates of empyema.
Asunto(s)
Empiema Pleural/cirugía , Terapia de Presión Negativa para Heridas/métodos , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Quirúrgicos Mínimamente Invasivos , Estudios Retrospectivos , Toracostomía/métodos , Resultado del Tratamiento , Cicatrización de HeridasRESUMEN
OBJECTIVES: This prospective study is an evaluation of the mini-open vacuum-assisted closure with instillation (Mini-VAC-Instill) therapy for the treatment of complicated pleural empyema. METHODS: We investigated septic patients in poor general physical condition (Karnofsky index ≤50%) with multimorbidity and/or immunosuppression who were treated by minimally invasive intrathoracic VAC-Instill therapy without the insertion of an open-window thoracostomy (OWT) between December 2012 and November 2014. All patients underwent mini-thoracotomy with position of a tissue retractor, surgical debridement and local decortication. Surgery was followed by intrathoracic vacuum therapy including periodic instillation using antiseptics. The VAC dressings were changed under general anaesthesia and the chest wall was closed during the same hospital stay. All patients received systemic antibiotic therapy. RESULTS: Fifteen patients (13 males, median age: 71 years) underwent intrathoracic Mini-VAC-Instill dressings for the management of pleural empyema without bronchopleural fistula. The median length of vacuum therapy was 9 days (5-25 days) and the median number of VAC changes per patient was 1 (1-5). In-hospital mortality was 6.7% (n = 1) and was not related to Mini-VAC-Instill therapy or intrathoracic infection. Control of intrathoracic infection and closure of the chest cavity was achieved in 85.7% of surviving patients (12 of 14). After the follow-up at an average of 13.2 months (range, 3-25 months), we observed recurrence once, 21 days after discharge. Two patients died in the late postoperative period (Day 43 and Day 100 after discharge) of fulminant urosepsis and carcinoma-related multiorgan failure, respectively. Analysis of the follow-up interviews in the outpatient clinic showed a good quality of life and a subjectively good long-term aesthetic result. CONCLUSIONS: Mini-VAC-Instill therapy is an upgrade of Mini-VAC, which guarantees the advantage of an open treatment, including flushing but without OWT. This procedure is minimally invasive, highly compatible especially with patients in poor general condition and may be an alternative to the OWT in selected patients. Consequently, a very short course of therapy results in good patient acceptance.
Asunto(s)
Empiema Pleural/cirugía , Terapia de Presión Negativa para Heridas/métodos , Sepsis/cirugía , Adulto , Anciano , Anciano de 80 o más Años , Antibacterianos/administración & dosificación , Terapia Combinada , Desbridamiento/métodos , Empiema Pleural/tratamiento farmacológico , Estudios de Factibilidad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Estudios Prospectivos , Sepsis/tratamiento farmacológico , Toracotomía/métodos , Resultado del TratamientoRESUMEN
Signaling at nerve cell synapses is a key determinant of proper brain function, and synaptic defects--or synaptopathies--are at the basis of many neurological and psychiatric disorders. In key areas of the mammalian brain, such as the hippocampus or the basolateral amygdala, the clustering of the scaffolding protein Gephyrin and of γ-aminobutyric acid type A receptors at inhibitory neuronal synapses is critically dependent upon the brain-specific guanine nucleotide exchange factor Collybistin (Cb). Accordingly, it was discovered recently that an R290H missense mutation in the diffuse B-cell lymphoma homology domain of Cb, which carries the guanine nucleotide exchange factor activity, leads to epilepsy and intellectual disability in human patients. In the present study, we determined the mechanism by which the Cb(R290H) mutation perturbs inhibitory synapse formation and causes brain dysfunction. Based on a combination of biochemical, cell biological, and molecular dynamics simulation approaches, we demonstrate that the R290H mutation alters the strength of intramolecular interactions between the diffuse B-cell lymphoma homology domain and the pleckstrin homology domain of Cb. This defect reduces the phosphatidylinositol 3-phosphate binding affinity of Cb, which limits its normal synaptogenic activity. Our data indicate that impairment of the membrane lipid binding activity of Cb and a consequent defect in inhibitory synapse maturation represent a likely molecular pathomechanism of epilepsy and mental retardation in humans.
Asunto(s)
Epilepsia/genética , Discapacidad Intelectual/genética , Fosfatos de Fosfatidilinositol/química , Factores de Intercambio de Guanina Nucleótido Rho/genética , Sinapsis/fisiología , Secuencia de Aminoácidos , Animales , Células COS , Proteínas Portadoras/química , Membrana Celular/metabolismo , Chlorocebus aethiops , Estudios de Asociación Genética , Proteínas de la Membrana/química , Ratones , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación Missense , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Transporte de Proteínas , Ratas , Factores de Intercambio de Guanina Nucleótido Rho/química , Factores de Intercambio de Guanina Nucleótido Rho/metabolismo , Proteínas de Unión al GTP rho/químicaRESUMEN
Enthusiasm for minimally invasive thoracic surgery is increasing. Thoracoscopy plays a significant therapeutic role in the fibrinopurulent stage (stage II) of empyema, in which loculated fluid cannot often be adequately drained by chest tube alone. For some debilitated and septic patients, further procedures such as open-window thoracostomy (OWT) with daily wound care or vacuum-assisted closure (VAC) therapy are necessary. In the present article, we propose a new option of minimally invasive VAC therapy including a topical solution of the empyema without open-window thoracostomy (Mini-VAC-instill). Three patients who underwent surgery using this technique are also presented. The discussion is focused on the advantages and disadvantages of the approach.
Asunto(s)
Empiema Pleural/cirugía , Procedimientos Quirúrgicos Mínimamente Invasivos/métodos , Terapia de Presión Negativa para Heridas/métodos , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Toracoscopía , ToracostomíaRESUMEN
Glycine receptors (GlyRs) mediate inhibitory neurotransmission in spinal cord and brainstem. They are clustered at inhibitory postsynapses via a tight interaction of their ß subunits (GlyRß) with the scaffolding protein gephyrin. In an attempt to isolate additional proteins interacting with GlyRß, we performed pulldown experiments with rat brain extracts using a glutathione S-transferase fusion protein encompassing amino acids 378-455 of the large intracellular loop of GlyRß as bait. This identified syndapin I (SdpI) as a novel interaction partner of GlyRß that coimmunoprecipitates with native GlyRs from brainstem extracts. Both SdpI and SdpII bound efficiently to the intracellular loop of GlyRß in vitro and colocalized with GlyRß upon coexpression in COS-7 cells. The SdpI-binding site was mapped to a proline-rich sequence of 22 amino acids within the intracellular loop of GlyRß. Deletion and point mutation analysis disclosed that SdpI binding to GlyRß is Src homology 3 domain-dependent. In cultured rat spinal cord neurons, SdpI immunoreactivity was found to partially colocalize with marker proteins of inhibitory and excitatory synapses. When SdpI was acutely knocked down in cultured spinal cord neurons by viral miRNA expression, postsynaptic GlyR clusters were significantly reduced in both size and number. Similar changes in GlyR cluster properties were found in spinal cultures from SdpI-deficient mice. Our results are consistent with a role of SdpI in the trafficking and/or cytoskeletal anchoring of synaptic GlyRs.
Asunto(s)
Encéfalo/metabolismo , Proteínas Portadoras/metabolismo , Neuropéptidos/metabolismo , Fosfoproteínas/metabolismo , Receptores de Glicina/metabolismo , Médula Espinal/metabolismo , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Encéfalo/citología , Células COS , Proteínas Portadoras/genética , Chlorocebus aethiops , Proteínas del Citoesqueleto , Humanos , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Mutantes , Neuronas/citología , Neuronas/metabolismo , Neuropéptidos/genética , Fosfoproteínas/genética , Mutación Puntual , Unión Proteica , Estructura Secundaria de Proteína , Transporte de Proteínas/fisiología , Proteómica , Ratas , Ratas Wistar , Receptores de Glicina/genética , Médula Espinal/citología , Sinapsis/genéticaRESUMEN
BACKGROUND: Chylothorax is characterized by the presence of chyle in the pleural cavity. The healing rate of non-operative treatment varies enormously; the maximum success rate in series is 70%. We investigate the efficacy and outcomes of radiotherapy for postoperative chylothorax. METHODS: Chylothorax was identified based on the quantity and quality of the drainage fluid. Radiation was indicated if the daily chyle flow exceeded 450 ml after complete cessation of oral intake. Radiotherapy consisted of opposed isocentric portals to the mediastinum using 15 MV photon beams from a linear accelerator, a single dose of 1-1.5 Gy, and a maximum of five fractions per week. The radiation target area was the anatomical region between TH3 and TH10 depending on the localization of the resected lobe. The mean doses of the ionizing energy was 8.5 Gy ± 3.5 Gy. RESULTS: The median start date of the radiation was the fourth day after chylothorax diagnosis. The patients' mediastinum was radiated an average of six times. Radiotherapy, in combination with dietary restrictions, was successful in all patients. The median time between the end of the radiation and the removal of the chest tube was one day. One patient underwent wound healing by secondary intention. The median time between the end of radiation and discharge was three days, and the overall hospital stay between the chylothorax diagnosis and discharge was 18 days (range: 11-30 days). After a follow-up of six months, no patient experienced chylothorax recurrence. CONCLUSIONS: Our results suggest that radiotherapy in combination with dietary restriction in the treatment of postoperative chylothorax is very safe, rapid and successful. This novel interventional procedure can obviate repeat major thoracic surgery and shorten hospital stays and could be the first choice in the treatment of postthoracotomy chylothorax.
Asunto(s)
Quilotórax/radioterapia , Adulto , Anciano , Tubos Torácicos , Quilotórax/etiología , Quilotórax/cirugía , Drenaje , Femenino , Humanos , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/etiología , Complicaciones Posoperatorias/radioterapia , Complicaciones Posoperatorias/cirugía , Estudios Retrospectivos , Toracotomía/efectos adversos , Resultado del TratamientoRESUMEN
OBJECTIVES: The pool of potential candidates for pleural empyema is expanding. In a previous technical report, we tested the feasibility of the minimally invasive insertion of a vacuum-assisted closure (Mini-VAC) system without the insertion of an open-window thoracostomy (OWT). In this study, we describe a consecutive case series of complex pleural empyemas that were managed by this Mini-VAC therapy. METHODS: In this retrospective study, we investigated 6 patients with multimorbidity (Karnofsky index ≤ 50%) who were consecutively treated with Mini-VAC for a primary, postoperative or recurrent pleural empyema between January 2011 and February 2012. RESULTS: Local control of the infection and control of sepsis were satisfactory in all 6 of the patients treated by Mini-VAC therapy. The suction used did not create any air leaks or bleeding from the lung or mediastinal structures. Mini-VAC therapy allowed a reduction of the empyema cavity and improved the re-expansion of the residual lung. Mini-VAC therapy resulted in a rapid eradication of the empyema. The chest wall was closed in all patients during the first hospital stay. All patients left the hospital in good health (Karnofsky index >70%) and with a non-infected pleural cavity at a mean of 22 ± 11 days after Mini-VAC installation. Pleural empyema was not detected in any of the 6 patients at the 3-month follow-up appointment. CONCLUSIONS: The Mini-VAC procedure with the abdication of an OWT offers a rapid treatment for complex pleural empyema with minimal surgical effort and the opportunity for a primary closure of the empyema cavity.
Asunto(s)
Empiema Pleural/terapia , Terapia de Presión Negativa para Heridas , Adulto , Anciano , Empiema Pleural/etiología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Terapia de Presión Negativa para Heridas/efectos adversos , Recurrencia , Estudios Retrospectivos , Factores de Tiempo , Resultado del TratamientoRESUMEN
α-Conotoxins are subtype-selective nicotinic acetylcholine receptor (nAChR) antagonists. Although potent α3ß2 nAChR-selective α-conotoxins have been identified, currently characterized α-conotoxins show no or only weak affinity for α4ß2 nAChRs, which are, besides α7 receptors, the most abundant nAChRs in the mammalian brain. To identify the determinants responsible for this difference, we substituted selected amino acid residues in the ligand-binding domain of the α4 subunit by the corresponding residues in the α3 subunit. Two-electrode voltage clamp analysis of these mutants revealed increased affinity of α-conotoxins MII, TxIA, and [A10L]TxIA at the α4(R185I)ß2 receptor. Conversely, α-conotoxin potency was reduced at the reverse α3(I186R)ß2 mutant. Replacement of α4Arg185 by alanine, glutamate, and lysine demonstrated that a positive charge in this position prevents α-conotoxin binding. Combination of the R185I mutation with a P195Q mutation outside the binding site but in loop C completely transferred high α-conotoxin potency to the α4ß2 receptor. Molecular dynamics simulations of homology models with docked α-conotoxin indicate that these residues control access to the α-conotoxin binding site.
Asunto(s)
Conotoxinas/metabolismo , Receptores Nicotínicos/metabolismo , Acetilcolina/farmacología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Sitios de Unión , Femenino , Simulación del Acoplamiento Molecular , Datos de Secuencia Molecular , Mutación , Agonistas Nicotínicos/farmacología , Oocitos/fisiología , Técnicas de Placa-Clamp , Unión Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Receptores Nicotínicos/genética , Alineación de Secuencia , Xenopus laevisRESUMEN
A 64-year-old man was diagnosed with complex empyema after a second course of palliative chemotherapy for metastatic lung cancer. Because of the poor general condition of the patient, the decision was made to proceed with vacuum-assisted closure (VAC) therapy of the empyema without Eloesser or Clagett open-window thoracostomy (OWT). Installation and changing of the VAC sponge were performed using the ALEXIS Wound Protector/Retractor (Applied Medical, Rancho Santa Margarita, CA), a flexible polymer membrane tube. After 10 days of VAC treatment, the pleural cavity was sterile and was closed with single stitches. Chemotherapy was resumed 1 week later.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/cirugía , Empiema Pleural/terapia , Neoplasias Pulmonares/cirugía , Terapia de Presión Negativa para Heridas , Cuidados Paliativos/métodos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Quimioterapia Adyuvante , Enfermedad Crítica/terapia , Empiema Pleural/diagnóstico por imagen , Empiema Pleural/etiología , Estudios de Seguimiento , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Invasividad Neoplásica/patología , Estadificación de Neoplasias , Complicaciones Posoperatorias/diagnóstico , Complicaciones Posoperatorias/terapia , Medición de Riesgo , Toracostomía , Tomografía Computarizada por Rayos X/métodos , Resultado del TratamientoRESUMEN
OBJECTIVE: For patients with postoperative pleural empyema, open window thoracostomy (OWT) is often necessary to prevent sepsis. Vacuum-assisted closure (VAC) is a well-known therapeutic option in wound treatment. The efficacy and safety of intrathoracal VAC therapy, especially in patients with pleural empyema with bronchial stump insufficiency or remain lung, has not yet been investigated. METHODS: Between October 2009 and July 2010, eight consecutive patients (mean age of 66.1 years) with multimorbidity received an OWT with VAC for the treatment of postoperative or recurrent pleural empyema. Two of them had a bronchial stump insufficiency (BPF). RESULTS: VAC therapy ensured local control of the empyema and control of sepsis. The continuous suction up to 125 mm Hg cleaned the wound and thoracic cavity and supported the rapid healing. Additionally, installation of a stable vacuum was possible in the two patients with BPF. The smaller bronchus stump fistula closed spontaneously due to the VAC therapy, but the larger remained open. The direct contact of the VAC sponge did not create any air leak or bleeding from the lung or the mediastinal structures. The VAC therapy allowed a better re-expansion of remaining lung. One patient died in the late postoperative period (day 47 p.o.) of multiorgan failure. In three cases, VAC therapy was continued in an outpatient service, and in four patients, the OWT was treated with conventional wound care. After a mean time of three months, the chest wall was closed in five of seven cases. However, two patients rejected the closure of the OWT. After a follow-up at 7.7 months, neither recurrent pleural empyema nor BPF was observed. CONCLUSION: VAC therapy was effective and safe in the treatment of complicated pleural empyema. The presence of smaller bronchial stump fistula and of residual lung tissue are not a contraindication for VAC therapy.
Asunto(s)
Empiema Pleural/cirugía , Terapia de Presión Negativa para Heridas , Anciano , Comorbilidad , Empiema Pleural/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Recurrencia , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Mitochondrial dysfunction is well documented in presymptomatic brain tissue with Parkinson's disease (PD). Identification of the autosomal recessive variant PARK6 caused by loss-of-function mutations in the mitochondrial kinase PINK1 provides an opportunity to dissect pathogenesis. Although PARK6 shows clinical differences to PD, the induction of alpha-synuclein "Lewy" pathology by PINK1-deficiency proves that mitochondrial pathomechanisms are relevant for old-age PD. Mitochondrial dysfunction is induced by PINK1 deficiency even in peripheral tissues unaffected by disease, consistent with the ubiquitous expression of PINK1. It remains unclear whether this dysfunction is due to PINK1-mediated phosphorylation of proteins inside or outside mitochondria. Although PINK1 deficiency affects the mitochondrial fission/fusion balance, cell stress is required in mammals to alter mitochondrial dynamics and provoke apoptosis. Clearance of damaged mitochondria depends on pathways including PINK1 and Parkin and is critical for postmitotic neurons with high energy demand and cumulative stress, providing a mechanistic concept for the tissue specificity of disease.
Asunto(s)
Enfermedad de Parkinson/patología , Proteínas Quinasas/deficiencia , Animales , Humanos , Mitocondrias/patología , Proteínas Mitocondriales/fisiología , Estrés Oxidativo , Enfermedad de Parkinson/etiología , Enfermedad de Parkinson/metabolismo , Proteínas Quinasas/fisiología , Ubiquitina-Proteína Ligasas/fisiologíaRESUMEN
Tropeines are bidirectional modulators of native and recombinant glycine receptors (GlyRs) and promising leads for the development of novel modulatory agents. Tropisetron potentiates and inhibits agonist-triggered GlyR currents at femto- to nanomolar and micromolar concentrations respectively. Here, the potentiating and inhibitory effects of another tropeine, 3alpha-(3'-methoxy-benzoyloxy)nortropane (MBN) were examined by voltage-clamp electrophysiology at wild type and mutant alpha1 GlyRs expressed in Xenopus laevis oocytes. Several substitutions around the agonist-binding cavity of the alpha1 subunit interface (N46C, F63A, N102A, R119K, R131A, E157C, K200A, Y202L and F207A) were found to reduce or eliminate MBN inhibition of glycine activation. In contrast, the binding site mutations Q67A, R119A and S129A which did not affect MBN inhibition abolished the potentiation of chloride currents elicited by low concentrations of the partial agonist taurine following pre-incubation with MBN. Thus, potentiation and inhibition involve distinct binding modes of MBN in the inter-subunit agonist-binding pocket of alpha1 GlyRs. Homology modelling and molecular dynamics simulations disclosed two distinct docking modes for MBN, which are consistent with the differential effects of individual binding site substitutions on MBN inhibition and potentiation respectively. Together these results suggest that distinct binding modes at adjacent binding sites located within the agonist-binding pocket of the GlyR mediate the bidirectional modulatory effects of tropeines.
Asunto(s)
Receptores de Glicina/antagonistas & inhibidores , Receptores de Glicina/fisiología , Animales , Sitios de Unión/efectos de los fármacos , Sitios de Unión/genética , Biofisica , Relación Dosis-Respuesta a Droga , Estimulación Eléctrica , Humanos , Indoles/química , Indoles/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microinyecciones/métodos , Modelos Moleculares , Mutagénesis Sitio-Dirigida/métodos , Mutación , Nortropanos/farmacología , Oocitos , Técnicas de Placa-Clamp/métodos , Unión Proteica/efectos de los fármacos , Unión Proteica/genética , Estructura Terciaria de Proteína , Receptores de Glicina/genética , Tropisetrón , Xenopus laevisRESUMEN
The divalent cation Zn2+ has been shown to regulate inhibitory neurotransmission in the mammalian CNS by affecting the activation of the strychnine-sensitive glycine receptor (GlyR). In spinal neurons and cells expressing recombinant GlyRs, low micromolar (<10 microM) concentrations of Zn2+ enhance glycine currents, whereas higher concentrations (>10 microM) have an inhibitory effect. Mutational studies have localized the Zn2+ binding sites mediating allosteric potentiation and inhibition of GlyRs in distinct regions of the N-terminal extracellular domain of the GlyR alpha-subunits. Here, we examined the Zn2+ sensitivity of different mutations within the agonist binding site of the homomeric alpha(1)-subunit GlyR upon heterologous expression in Xenopus oocytes. This revealed that six substitutions within the ligand-binding pocket result in a total loss of Zn2+ inhibition. Furthermore, substitution of the positively charged residues arginine 65 and arginine 131 by alanine (alpha(1)(R65A), alpha(1)(R131A), or of the aromatic residue phenylalanine 207 by histidine (alpha(1)(F207H)), converted the alpha(1) GlyR into a chloride channel that was activated by Zn2+ alone. Dose-response analysis of the alpha(1)(F207H) GlyR disclosed an EC(50) value of 1.2 microM for Zn2+ activation; concomitantly the apparent glycine affinity was 1000-fold reduced. Thus, single point mutations within the agonist-binding site of the alpha(1) subunit convert the inhibitory GlyR from a glycine-gated into a selectively Zn2+-activated chloride channel. This might be exploited for the design of metal-specific biosensors by modeling-assisted mutagenesis.
Asunto(s)
Canales de Cloruro/química , Mutación , Receptores de Glicina/metabolismo , Zinc/química , Animales , Sitios de Unión , Técnicas Biosensibles , Cationes , Femenino , Humanos , Canales Iónicos/química , Ligandos , Oocitos/metabolismo , Estructura Terciaria de Proteína , Xenopus laevisRESUMEN
Calmodulin (CaM) binds in a Ca2+-dependent manner to the intracellular C-terminal domains of most group III metabotropic glutamate receptors (mGluRs). Here we combined mutational and biophysical approaches to define the structural basis of CaM binding to mGluR 7A. Ca2+/CaM was found to interact with mGluR 7A primarily via its C-lobe at a 1:1 CaM:C-tail stoichiometry. Pulldown experiments with mutant CaM and mGluR 7A C-tail constructs and high resolution NMR with peptides corresponding to the CaM binding region of mGluR 7A allowed us to define hydrophobic and ionic interactions required for Ca2+/CaM binding and identified a 1-8-14 CaM-binding motif. The Ca2+/CaM.mGluR 7A peptide complex displays a classical wraparound structure that closely resembles that formed by Ca2+/CaM upon binding to smooth muscle myosin light chain kinase. Our data provide insight into how Ca2+/CaM regulates group III mGluR signaling via competition with intracellular proteins for receptor-binding sites.
Asunto(s)
Calmodulina/química , Complejos Multiproteicos/química , Receptores de Glutamato Metabotrópico/química , Animales , Calcio/química , Calcio/metabolismo , Señalización del Calcio/fisiología , Calmodulina/genética , Calmodulina/metabolismo , Línea Celular , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Quinasa de Cadena Ligera de Miosina/química , Quinasa de Cadena Ligera de Miosina/genética , Quinasa de Cadena Ligera de Miosina/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Ratas , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo , Homología Estructural de Proteína , Relación Estructura-ActividadRESUMEN
In ionotropic glutamate receptors, agonist binding occurs in a conserved clam shell-like domain composed of the two lobes D1 and D2. Docking of glutamate into the binding cleft promotes rotation in the hinge region of the two lobes, resulting in closure of the binding pocket, which is thought to represent a prerequisite for channel gating. Here, we disrupted D1D2 interlobe interactions in the NR2A subunit of N-methyl-d-aspartate (NMDA) receptors through systematic mutation of individual residues and studied the influence on the activation kinetics of currents from NR1/NR2 NMDA receptors heterologously expressed in HEK cells. We show that the mutations affect differentially glutamate binding and channel gating, depending on their location within the binding domain, mainly by altering k(off) and k(cl), respectively. Whereas impaired stability of glutamate in its binding site is the only effect of mutations on one side of the ligand binding pocket, close to the hinge region, alterations in gating are the predominant consequence of mutations on the opposite side, at the entrance of the binding pocket. A mutation increasing D1D2 interaction at the entrance of the pocket resulted in an NMDA receptor with an increased open probability as demonstrated by single channel and whole cell kinetic analysis. Thus, the results indicate that agonist-induced binding domain closure is itself a complex process, certain aspects of which are coupled either to binding or to gating. Specifically, we propose that late steps of domain closure, in kinetic terms, represent part of channel gating.
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Ácido Glutámico/química , Receptores de N-Metil-D-Aspartato/química , Secuencia de Aminoácidos , Sitios de Unión , Humanos , Cinética , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Receptores de N-Metil-D-Aspartato/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Inhibitory glycine receptors (GlyRs) regulate motor coordination and sensory signal processing in spinal cord and other brain regions. GlyRs are pentameric proteins composed of membrane-spanning alpha and beta subunits. Here, site-directed mutagenesis combined with homology modeling based on the crystal structure of the acetylcholine binding protein identified key ligand binding residues of recombinant homooligomeric alpha1 and heterooligomeric alpha1beta GlyRs. This disclosed two highly conserved, oppositely charged residues located on adjacent subunit interfaces as being crucial for agonist binding. In addition, the beta subunit was found to determine the ligand binding properties of heterooligomeric GlyRs. Expression of an alpha1beta tandem construct and affinity purification of metabolically labeled GlyRs confirmed a subunit stoichiometry of 2alpha3beta. Because the beta subunit anchors GlyRs at synaptic sites, our results have important implications for the biosynthesis, clustering, and pharmacology of synaptic GlyRs.
Asunto(s)
Receptores de Glicina/metabolismo , Sinapsis/metabolismo , Secuencia de Aminoácidos , Animales , Relación Dosis-Respuesta a Droga , Femenino , Glicina/metabolismo , Glicina/farmacología , Humanos , Ligandos , Datos de Secuencia Molecular , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores de Glicina/química , Receptores de Glicina/genética , Estricnina/metabolismo , Estricnina/farmacología , Sinapsis/efectos de los fármacos , Sinapsis/genéticaRESUMEN
Binding of glutamate to ionotropic glutamate receptors occurs within a bilobate binding pocket built from conserved S1 and S2 domains. Using the crystal structure of the binding region of the (RS)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid (AMPA)-selective GluR2 subunit, we identified determinants of ligand selectivity and efficacy within the glutamate-binding pocket of the NR2B subunit of the N-methyl-D-aspartate (NMDA) receptor by site-directed mutagenesis. Electrophysiological analyses of mutated NR2B polypeptides revealed drastic effects on the affinity of L-glutamate but not of the co-agonist glycine. With seven out of 19 substitutions, we found differences in the potency of the full agonist L-glutamate and the partial agonist NMDA. In particular, substitutions located at the interface between the S1 and S2 domains resulted in changes of agonist efficacy, suggesting a role in transducing the ligand-binding signal. Inhibition by the competitive antagonist D-AP5 was highly sensitive to replacement of residues involved in stabilization of the closed conformation of the binding pocket, consistent with antagonists preventing closure of the binding pocket. In addition, we identified residues predicted to be important for liganding the methyl group of NMDA. Collectively our data describe specific side chain interactions that determine ligand efficacy and pharmacology at the glutamate site of the NMDA receptor.
Asunto(s)
Ácido Glutámico/metabolismo , Receptores de N-Metil-D-Aspartato/química , 2-Amino-5-fosfonovalerato/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Unión Competitiva/efectos de los fármacos , Agonistas de Aminoácidos Excitadores/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Ratas , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Receptores de N-Metil-D-Aspartato/genéticaRESUMEN
The N-methyl-d-aspartate (NMDA) receptor is a ligand-gated ion channel that requires both glutamate and glycine for efficient activation. Here, a strategy combining cysteine scanning mutagenesis and affinity labeling was used to investigate the glycine binding site located on the NR1 subunit. Based on homology modeling to the crystal structure of the glutamate binding site of the 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)-propionic acid receptor GluR2, cysteines were introduced into the NR1 subunit as chemical sensors for three thiol-reactive derivatives of the competitive antagonist L-701324. After coexpressing the mutant NR1 with wild-type NR2B subunits in Xenopus oocytes, agonist-induced currents were recorded to monitor irreversible receptor inactivation by the reactive antagonists. For each derivative, glycine site-specific inactivations were observed with a distinct subset of cysteine-substituted receptors. Together these inactivating substitutions identified seven NR1 residues (Ile-385, Gln-387, Glu-388, Thr-500, Asn-502, Ala-696, and Val-717) that undergo proximity-induced covalent coupling with specific regions of the bound antagonist and disclose its mode of docking in the glycine binding pocket of the NMDA receptor. Our approach may help to unravel the structural basis of distinct NMDA receptor subtype pharmacologies.
Asunto(s)
Glicina , Receptores de N-Metil-D-Aspartato/química , Animales , Sitios de Unión , Electrofisiología , Antagonistas de Aminoácidos Excitadores/química , Ratones , Modelos Moleculares , Mutación , Oocitos , Ingeniería de Proteínas/métodos , Quinolonas/química , Ratas , XenopusRESUMEN
Transmitter-gated ion channels mediate rapid synaptic transmission in the CNS and constitute important targets for many neuroactive drugs. Inhibitory glycine receptors (GlyRs) are members of the nicotinic acetylcholine receptor superfamily and inhibit neuronal firing by opening Cl(-) channels following agonist binding. In this article, we discuss recent developments in GlyR pharmacology, delineate the receptor domains that are involved in binding of agonists and allosteric modulators, and present a molecular model of the extracellular architecture of the receptor. The recent discovery of compounds that act preferentially on specific GlyR isoforms and the differential expression of these isoforms in distinct regions of the developing and adult CNS show considerable promise towards the development of drugs that act in defined glycine-mediated pathways. In particular, compounds that can potentiate GlyR function should provide leads for novel muscle relaxants in addition to sedative and analgesic agents.