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Background and Objectives: Brolucizumab (IVBr) is a recently introduced anti-vascular endothelial growth factor (anti-VEGF) which has been found to be very effective in treating neovascular age-related macular degeneration (nAMD). We reported our findings in a case of nAMD that developed intraocular inflammation (IOI) after IVBr injections. Materials and Methods: A 79-year-old man was referred to our hospital complaining of reduced vision in both eyes of one-month's duration. His decimal best-corrected visual acuity (BCVA) was 0.9 in the right eye and 1.0 in the left eye. He was diagnosed with nAMD in the left eye and was treated with intravitreal aflibercept (IVA). Despite the three-monthly IVA injections, the serous retinal pigment epithelial detachment (PED) and subretinal fluid (SRF) remained, and the VA gradually decreased to 0.1. Because of the patient being refractory to aflibercept treatment, we switched to 3-monthly IVBr injections. The BCVA gradually improved to 0.3 and optical coherence tomography (OCT) showed an absence of the serous PED and SRF. Three weeks after his third IVBr, he returned to our hospital with a complaint of reduced vision in his left eye that he first noted two weeks earlier. Our examination of the left eye showed signs of IOI mainly in the anterior chamber. The inflammation improved with topical steroids but the treatment of the IOI was delayed for two weeks. The patient was instructed that it was important to begin the treatment as soon as the symptoms of IOI developed. We then performed the Mini-Mental State Examination (MMSE), and his score indicated that he had cognitive impairment. Conclusions: We concluded that before beginning IVBr treatment in nAMD patients, a careful assessment must be made of the cognitive status of the patient.
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Degeneración Macular , Desprendimiento de Retina , Baja Visión , Masculino , Humanos , Anciano , Inhibidores de la Angiogénesis/efectos adversos , Inflamación , Desprendimiento de Retina/diagnóstico , Desprendimiento de Retina/tratamiento farmacológico , Tomografía de Coherencia Óptica/métodos , Degeneración Macular/complicaciones , Degeneración Macular/tratamiento farmacológicoRESUMEN
PURPOSE: To identify the eyes with macular edema (ME) due to a branch retinal vein occlusion (BRVO) that have good visual acuity during the continuous anti-vascular endothelial growth factor (anti-VEGF) treatment based on the patients' clinical information and optical coherence tomography (OCT) images by using machine learning. METHODS: Sixty-six eyes of 66 patients received 1 anti-VEGF injection followed by repeated injections in the pro re nata (PRN) regimen for 12 months. The patients were divided into two groups: those with and those without good vision during the 1-year experimental period. Handcraft features were defined from the OCT images at the time of the first resolution of the ME. Variables with a significant difference between the groups were used as explanatory variables. A classifier was created with handcrafted features based on a support vector machine (SVM) that adjusted parameters for increasing maximal precision. RESULTS: The age, best-corrected visual acuity (BCVA) at the baseline, BCVA at the first resolution of the ME, integrity and reflectivity of the external limiting membrane (ELM), the ellipsoid zone (EZ), and area of the outer segments of the photoreceptors were selected as explanatory variables. The classification performance was 0.806 for accuracy, 0.768 for precision, 0.772 for recall, and 0.752 for the F-measure. CONCLUSION: The use of the SVM of the patient's clinical information and OCT images can be helpful for determining the prognosis of the BCVA during continued pro re nata anti-VEGF treatment in eyes with ME associated with BRVO.
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Edema Macular , Oclusión de la Vena Retiniana , Inhibidores de la Angiogénesis , Humanos , Inyecciones Intravítreas , Edema Macular/diagnóstico , Edema Macular/tratamiento farmacológico , Edema Macular/etiología , Oclusión de la Vena Retiniana/complicaciones , Oclusión de la Vena Retiniana/diagnóstico , Oclusión de la Vena Retiniana/tratamiento farmacológico , Estudios Retrospectivos , Máquina de Vectores de Soporte , Tomografía de Coherencia Óptica/métodos , Resultado del Tratamiento , Agudeza VisualRESUMEN
BACKGROUND: Acquired color anomalies caused by cerebral trauma are classified as either achromatopsias or dyschromatopsias (Zeki, Brain 113:1721-1777, 1990). The three main brain regions stimulated by color are V1, the lingual gyrus, which was designated as human V4 (hV4), and the fusiform gyrus, designated as V4α. (Zeki, Brain 113:1721-1777, 1990). An acquired cerebral color anomaly is often accompanied by visual field loss (hemi- and quadrantanopia), facial agnosia, prosopagnosia, visual agnosia, and anosognosia depending on the underlying pathology (Bartels and Zeki, Eur J Neurosci 12:172-193, 2000), (Meadows, Brain 97:615-632, 1974), (Pearman et al., Ann Neurol 5:253-261, 1979). The purpose of this study was to determine the characteristics of a patient who developed dyschromatopsia following a traumatic injury to her brain. CASE PRESENTATION: The patient was a 24-year-old woman who had a contusion to her right anterior temporal lobe. After the injury, she noticed color distortion and that blue objects appeared green in the left half of the visual field. Although conventional color vision tests did not detect any color vision abnormalities, short wavelength automated perimetry (SWAP) showed a decrease in sensitivity consistent with a left hemi-dyschromatopsia. Magnetic resonance imaging (MRI) detected abnormalities in the right fusiform gyrus, a part of the anterior temporal lobe. At follow-up 14 months later, subjective symptoms had disappeared, but the SWAP abnormalities persisted and a thinning of the sectorial ganglion cell complex (GCC) was detected. CONCLUSION: The results indicate that although the subjective symptoms resolved early, a reduced sensitivity of SWAP remained and the optical coherence tomography (OCT) showed GCC thinning. We conclude that local abnormalities in the anterior section of fusiform gyrus can cause mild cerebral dyschromatopsia without other symptoms. These findings indicate that it is important to listen to the symptoms of the patient and perform appropriate tests including the SWAP and OCT at the early stage to objectively prove the presence of acquired cerebral color anomaly.
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Defectos de la Visión Cromática , Prosopagnosia , Adulto , Defectos de la Visión Cromática/diagnóstico , Defectos de la Visión Cromática/etiología , Femenino , Humanos , Imagen por Resonancia Magnética , Lóbulo Occipital , Campos Visuales , Adulto JovenRESUMEN
Ethanol consumption leads to a wide range of pharmacological effects by acting on the signaling proteins in the human nervous system, such as ion channels. Despite its familiarity and biological importance, very little is known about the molecular mechanisms underlying the ethanol action, due to extremely weak binding affinity and the dynamic nature of the ethanol interaction. In this research, we focused on the primary in vivo target of ethanol, G-protein-activated inwardly rectifying potassium channel (GIRK), which is responsible for the ethanol-induced analgesia. By utilizing solution NMR spectroscopy, we characterized the changes in the structure and dynamics of GIRK induced by ethanol binding. We demonstrated here that ethanol binds to GIRK with an apparent dissociation constant of 1.0 M and that the actual physiological binding site of ethanol is located on the cavity formed between the neighboring cytoplasmic regions of the GIRK tetramer. From the methyl-based NMR relaxation analyses, we revealed that ethanol activates GIRK by shifting the conformational equilibrium processes, which are responsible for the gating of GIRK, to stabilize an open conformation of the cytoplasmic ion gate. We suggest that the dynamic molecular mechanism of the ethanol-induced activation of GIRK represents a general model of the ethanol action on signaling proteins in the human nervous system.
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Etanol/metabolismo , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Regulación Alostérica , Animales , Sitios de Unión , Etanol/química , Cinética , Espectroscopía de Resonancia Magnética , Ratones , Conformación Proteica , Dominios ProteicosRESUMEN
In electroretinographic (ERG) recordings of zebrafish, the light stimulus is usually delivered by a fiber optic cable. The purpose of this study was to determine whether the angle of incidence of the stimulus light from the fiber optic cable will affect the amplitudes and implicit times of the ERGs of zebrafish larvae. The larvae were positioned on their side with the right eye pointed upward. The light stimuli were delivered by a fiber optic cable from three directions of the larvae: frontal 0° (F0°), dorsal 30°(D30°), and ventral 30°(V30°). Photopic ERGs were recorded from 16 larvae at age 5-6 days post-fertilization. Our results showed that the mean amplitude of the b-wave elicited at D30° and V30° stimulation was significantly smaller than that elicited at F0° stimulation (P = 0.014 and P = 0.019, respectively). In addition, the mean amplitude of the d-wave elicited at D30° and V30° stimulation was significantly smaller than that elicited at F0° stimulation (P < 0.0001 and P = 0.015, respectively). However, the difference between the b-wave amplitudes elicited at D30° and V30° stimuli were not significant (P = 0.98), and the d-wave amplitudes were also not significantly different (P = 0.20). The average b-wave amplitudes elicited at D30° stimulation was 84.6 ± 15.7% and V30° stimulation was 84.8 ± 17.4% relative to that of F0° stimulation. The average d-wave amplitudes elicited by D30° stimulation was 85.5 ± 15.2% and by V30° stimulation was 79.0 ± 11.0% relative to that of F0° stimulation. The differences in the implicit times of the b- and d-wave elicited by the different directions of stimulation were not significant (P = 0.52 and P = 0.14, respectively). We conclude that the amplitude of the photopic ERGs is affected by the angle of the incident light. Thus, it would be better to use ganzfeld stimuli to elicit maximum b- and d-wave amplitudes of the photopic ERGs of zebrafish larvae.
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Electrorretinografía , Larva , Luz , Estimulación Luminosa , Pez Cebra , Animales , Pez Cebra/fisiología , Larva/fisiología , Retina/fisiologíaRESUMEN
Purpose: To determine the recurrence rate of neovascular age-related macular degeneration (nAMD) during a 5-year period after the suspension of anti-vascular endothelial growth factor (anti-VEGF) treatments. Methods: Thirty-four eyes of 34 nAMD patients who met the inclusion criteria and were treated by anti-VEGF drugs were studied. All met the treatment suspension criteria and were followed for 5 years after the suspension of the anti-VEGF treatment. Patients with a recurrence within one year were placed in Group A, and patients with a recurrence between 1 and 5 years were placed in Group B. The rate and time of a recurrence were analyzed using the Kaplan-Meier method. We also examined whether there were differences in the baseline factors of age, sex, subtype, treatment period, and treatment interval between Groups A and B. Results: Twenty-five of 34 eyes (73.5%) had a recurrence within 5 years of stopping the anti-VEGF treatments. Thirteen (52.0%) of the 25 eyes had a recurrence within 1 year, 4 (16.0%) eyes between 1 and 2 years, 4 (16.0%) eyes between 2 and 3 years, 2 (8%) between 3 and 4 years, and 2 eyes (8%) between 4 and 5 years. The baseline factors were not significantly different between Groups A and B. Conclusions: The results showed that the recurrence rate was highest within one year after the suspension of the anti-VEGF treatments, with a number of recurrences one year after the suspension. Clinicians should remember that nAMD may recur several years after the suspension of anti-VEGF treatments.
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BACKGROUND: The purpose of this study was to develop a model that can predict the postoperative visual acuity in eyes that had undergone vitrectomy for an epiretinal membrane (ERM). The Light Gradient Boosting Machine (LightGBM) was used to evaluate the accuracy of the prediction and the contribution of the explanatory variables. Two models were designed to predict the postoperative visual acuity in 67 ERM patients. Model 1 used the age, sex, affected eye, axial length, preoperative visual acuity, Govetto's classification stage, and OCT-derived vector information as features to predict the visual acuity at 1, 3, and 6 months postoperatively. Model 2 incorporated the early postoperative visual acuity as an additional variable to predict the visual acuity at 3, and 6 months postoperatively. LightGBM with 100 iterations of 5-fold cross-validation was used to tune the hyperparameters and train the model. This involved addressing multicollinearity and selecting the explanatory variables. The generalized performance of these models was evaluated using the root mean squared error (RMSE) in a 5-fold cross-validation, and the contributions of the explanatory variables were visualized using the average Shapley Additive exPlanations (SHAP) values. RESULTS: The RMSEs for the predicted visual acuity of Model 1 were 0.14 ± 0.02 logMAR units at 1 month, 0.12 ± 0.03 logMAR units at 3 months, and 0.13 ± 0.04 logMAR units at 6 months. High SHAP values were observed for the preoperative visual acuity and the ectopic inner foveal layer (EIFL) area with significant and positive correlations across all models. Model 2 that incorporated the postoperative visual acuity was used to predict the visual acuity at 3 and 6 months, and it had superior accuracy with RMSEs of 0.10 ± 0.02 logMAR units at 3 months and 0.10 ± 0.04 logMAR units at 6 months. High SHAP values were observed for the postoperative visual acuity in Model 2. CONCLUSION: Predicting the postoperative visual acuity in ERM patients is possible using the preoperative clinical data and OCT images with LightGBM. The contribution of the explanatory variables can be visualized using the SHAP values, and the accuracy of the prediction models improved when the postoperative visual acuity is included as an explanatory variable. Our data-driven machine learning models reveal that preoperative visual acuity and the size of the EIFL significantly influence postoperative visual acuity. Early intervention may be crucial for achieving favorable visual outcomes in eyes with an ERM.
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Membrana Epirretinal , Aprendizaje Automático , Agudeza Visual , Vitrectomía , Humanos , Membrana Epirretinal/cirugía , Membrana Epirretinal/diagnóstico por imagen , Membrana Epirretinal/fisiopatología , Agudeza Visual/fisiología , Masculino , Femenino , Anciano , Persona de Mediana Edad , Periodo Posoperatorio , Tomografía de Coherencia Óptica/métodosRESUMEN
G protein-gated inwardly rectifying potassium channel (GIRK) plays a crucial role in regulating heart rate and neuronal excitability. The gating of GIRK is regulated by the association and dissociation of G protein ßγ subunits (Gßγ), which are released from pertussis toxin-sensitive G protein α subunit (Gα(i/o)) upon GPCR activation in vivo. Several lines of evidence indicate that Gα(i/o) also interacts directly with GIRK, playing functional roles in the signaling efficiency and the modulation of the channel activity. However, the underlying mechanism for GIRK regulation by Gα(i/o) remains to be elucidated. Here, we performed NMR analyses of the interaction between the cytoplasmic region of GIRK1 and Gα(i3) in the GTP-bound state. The NMR spectral changes of Gα upon the addition of GIRK as well as the transferred cross-saturation (TCS) results indicated their direct binding mode, where the K(d) value was estimated as â¼1 mm. The TCS experiments identified the direct binding sites on Gα and GIRK as the α2/α3 helices on the GTPase domain of Gα and the αA helix of GIRK. In addition, the TCS and paramagnetic relaxation enhancement results suggested that the helical domain of Gα transiently interacts with the αA helix of GIRK. Based on these results, we built a docking model of Gα and GIRK, suggesting the molecular basis for efficient GIRK deactivation by Gα(i/o).
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Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Modelos Moleculares , Animales , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ratones , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
G protein-activated inwardly rectifying potassium channel (GIRK) plays crucial roles in regulating heart rate and neuronal excitability in eukaryotic cells. GIRK is activated by the direct binding of heterotrimeric G protein ßγ subunits (Gßγ) upon stimulation of G protein-coupled receptors, such as M2 acetylcholine receptor. The binding of Gßγ to the cytoplasmic pore (CP) region of GIRK causes structural rearrangements, which are assumed to open the transmembrane ion gate. However, the crucial residues involved in the Gßγ binding and the structural mechanism of GIRK gating have not been fully elucidated. Here, we have characterized the interaction between the CP region of GIRK and Gßγ, by ITC and NMR. The ITC analyses indicated that four Gßγ molecules bind to a tetramer of the CP region of GIRK with a dissociation constant of 250 µM. The NMR analyses revealed that the Gßγ binding site spans two neighboring subunits of the GIRK tetramer, which causes conformational rearrangements between subunits. A possible binding mode and mechanism of GIRK gating are proposed.
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Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Activación del Canal Iónico/fisiología , Modelos Biológicos , Animales , Sitios de Unión , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Subunidades beta de la Proteína de Unión al GTP/química , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades gamma de la Proteína de Unión al GTP/química , Subunidades gamma de la Proteína de Unión al GTP/genética , Humanos , Ratones , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de ProteínaRESUMEN
BACKGROUND: IgG4-related diseases (IgG4-RDs) are known to disrupt the functioning of multiple organs and are usually associated with mass lesions. Periaortitis, an inflammation of the adventitia and tissues surrounding the aorta, is an example of an IgG4-RD. In ophthalmology, an enlargement of the lacrimal gland is a well-known IgG4-RD, and scleritis has also been reported to be an IgG4-RD although it is rare. We report our findings in a case with periaortitis and posterior scleritis that were present at the same time, and they responded well to systemic steroid therapy. PATIENTS CONCERNS: A 79-year-old man with dementia and Lewy bodies was referred to our hospital because of uveitis in both eyes that did not respond to topical steroid therapy. DIAGNOSIS: We found anterior scleritis in the right eye and uveitis with shallow anterior chambers in both eyes. B-mode echography showed choroidal detachments (CDs) and a T sign in the right eye. The CDs were assumed to have progressed to the posterior scleritis which then caused the severe vision reduction. The patient was referred to the Internal Medicine Department because the systemic inflammatory disease was suspected due to the high levels of C-reactive protein (CRP) and the fast erythrocyte sedimentation rate. Systemic CT scans showed periaortitis only at the lumbar region. Because of the high levels of IgG4, the patient was diagnosed with IgG4-RD. INTERVENTIONS: The patient received intravenous and oral steroid therapy. The first 125 mg of methylprednisolone (mPSL) for 3 days was intravenous, after which it was switched to oral prednisolone (PSL) therapy and the dosage was gradually reduced. OUTCOMES: The posterior scleritis and periaortitis responded well to the systemic steroid therapy. One year and a half after the onset of the disease, the patient is still taking 5 mg of PSL. CONCLUSIONS: Scleritis with multiple CDs and periaortitis were strongly suspected to be due to IgG4-RD although no definitive diagnosis was made by biopsy of the lesions. Clinicians should be aware that IgG4-RD should be considered as one of the causes of posterior scleritis.
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Efusiones Coroideas , Enfermedad Relacionada con Inmunoglobulina G4 , Escleritis , Uveítis , Anciano , Humanos , Inmunoglobulina G , Enfermedad Relacionada con Inmunoglobulina G4/complicaciones , Masculino , Prednisolona , Escleritis/complicaciones , Escleritis/diagnóstico , Escleritis/tratamiento farmacológico , Uveítis/complicacionesRESUMEN
This study's goal is to determine the accuracy of a linear classifier that predicts the prognosis of patients with macular edema (ME) due to a branch retinal vein occlusion during the maintenance phase of antivascular endothelial growth factor (anti-VEGF) therapy. The classifier was created using the clinical information and optical coherence tomographic (OCT) findings obtained up to the time of the first resolution of ME. In total, 66 eyes of 66 patients received an initial intravitreal injection of anti-VEGF followed by repeated injections with the pro re nata (PRN) regimen for 12 months. The patients were divided into two groups: those with and those without good vision during the PRN phase. The mean AUC of the classifier was 0.93, and the coefficients of the explanatory variables were: best-corrected visual acuity (BCVA) at baseline was 0.66, BCVA at first resolution of ME was 0.51, age was 0.21, the average brightness of the ellipsoid zone (EZ) was -0.12, the intactness of the external limiting membrane (ELM) was -0.14, the average brightness of the ELM was -0.17, the brightness value of EZ was -0.17, the area of the outer segments of the photoreceptors was -0.20, and the intactness of the EZ was -0.24. This algorithm predicted the prognosis over time for individual patients during the PRN phase.
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The inwardly rectifying potassium channel (Kir) regulates resting membrane potential, K+ homeostasis, heart rate, and hormone secretion. The outward current is blocked in a voltage-dependent manner, upon the binding of intracellular polyamines or Mg2+ to the transmembrane pore domain. Meanwhile, electrophysiological studies have shown that mutations of several acidic residues in the intracellular regions affected the inward rectification. Although these acidic residues are assumed to bind polyamines, the functional role of the binding of polyamines and Mg2+ to the intracellular regions of Kirs remains unclear. Here, we report thermodynamic and structural studies of the interaction between polyamines and the cytoplasmic pore of mouse Kir3.1/GIRK1, which is gated by binding of G-protein betagamma-subunit (Gbetagamma). ITC analyses showed that two spermine molecules bind to a tetramer of Kir3.1/GIRK1 with a dissociation constant of 26 microM, which is lower than other blockers. NMR analyses revealed that the spermine binding site is Asp-260 and its surrounding area. Small but significant chemical shift perturbations upon spermine binding were observed in the subunit-subunit interface of the tetramer, suggesting that spermine binding alters the relative orientations of the four subunits. Our ITC and NMR results postulated a spermine binding mode, where one spermine molecule bridges two Asp-260 side chains from adjacent subunits, with rearrangement of the subunit orientations. This suggests the functional roles of spermine binding to the cytoplasmic pore: stabilization of the resting state conformation of the channel, and instant translocation to the transmembrane pore upon activation through the Gbetagamma-induced conformational rearrangement.
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Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Espermina/química , Animales , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/genética , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Magnesio/química , Magnesio/metabolismo , Ratones , Resonancia Magnética Nuclear Biomolecular/métodos , Bloqueadores de los Canales de Potasio/química , Bloqueadores de los Canales de Potasio/metabolismo , Unión Proteica/fisiología , Estructura Cuaternaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Espermina/metabolismoRESUMEN
CC-chemokine receptor 5 (CCR5) belongs to the G protein-coupled receptor (GPCR) family and plays important roles in the inflammatory response. In addition, its ligands inhibit the HIV infection. Structural analyses of CCR5 have been hampered by its instability in the detergent-solubilized form. Here, CCR5 was reconstituted into high density lipoprotein (rHDL), which enabled CCR5 to maintain its functions for >24 h and to be suitable for structural analyses. By applying the methyl-directed transferred cross-saturation (TCS) method to the complex between rHDL-reconstituted CCR5 and its ligand MIP-1alpha, we demonstrated that valine 59 and valine 63 of MIP-1alpha are in close proximity to CCR5 in the complex. Furthermore, these results suggest that the protective influence on HIV-1 infection of a SNP of MIP-1alpha is due to its change of affinity for CCR5. This method will be useful for investigating the various and complex signaling mediated by GPCR, and will also provide structural information about the interactions of other GPCRs with lipids, ligands, G-proteins, and effector molecules.
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Membrana Dobles de Lípidos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Receptores CCR5/química , Receptores CCR5/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Quimiocina CCL3/química , Quimiocina CCL3/genética , Quimiocina CCL3/metabolismo , Variación Genética , Humanos , Ligandos , Membrana Dobles de Lípidos/química , Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Maltosa/análogos & derivados , Maltosa/química , Micelas , Modelos Moleculares , Unión Proteica , Conformación Proteica , Estabilidad Proteica , SolucionesRESUMEN
G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.
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Canales de Potasio Rectificados Internamente Asociados a la Proteína G/ultraestructura , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/ultraestructura , Subunidades beta de la Proteína de Unión al GTP/ultraestructura , Subunidades gamma de la Proteína de Unión al GTP/ultraestructura , Activación del Canal Iónico/fisiología , Transferencia de Energía por Resonancia de Bioluminiscencia , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/aislamiento & purificación , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Subunidades beta de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Subunidades gamma de la Proteína de Unión al GTP/aislamiento & purificación , Subunidades gamma de la Proteína de Unión al GTP/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructuraRESUMEN
The gating of potassium ion (K+) channels is regulated by various kinds of protein-protein interactions (PPIs). Structural investigations of these PPIs provide useful information not only for understanding the gating mechanisms of K+ channels, but also for developing the pharmaceutical compounds targeting K+ channels. Here, we describe a nuclear magnetic resonance spectroscopic method, termed the cross saturation (CS) method, to accurately determine the binding surfaces of protein complexes, and its application to the investigation of the interaction between a G protein-coupled inwardly rectifying K+ channel and a G protein α subunit.
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Canales de Potasio Rectificados Internamente Asociados a la Proteína G/química , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Subunidades alfa de la Proteína de Unión al GTP/química , Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Animales , Sitios de Unión , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Dominios y Motivos de Interacción de ProteínasRESUMEN
Heterotrimeric guanine-nucleotide-binding proteins (G proteins) serve as molecular switches in signalling pathways, by coupling the activation of cell surface receptors to intracellular responses. Mutations in the G protein α-subunit (Gα) that accelerate guanosine diphosphate (GDP) dissociation cause hyperactivation of the downstream effector proteins, leading to oncogenesis. However, the structural mechanism of the accelerated GDP dissociation has remained unclear. Here, we use magnetic field-dependent nuclear magnetic resonance relaxation analyses to investigate the structural and dynamic properties of GDP bound Gα on a microsecond timescale. We show that Gα rapidly exchanges between a ground-state conformation, which tightly binds to GDP and an excited conformation with reduced GDP affinity. The oncogenic D150N mutation accelerates GDP dissociation by shifting the equilibrium towards the excited conformation.
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Subunidades alfa de la Proteína de Unión al GTP/metabolismo , Guanosina Difosfato/metabolismo , Campos Magnéticos , Espectroscopía de Resonancia Magnética , Subunidades alfa de la Proteína de Unión al GTP/química , Humanos , Magnesio/farmacología , Proteínas Mutantes/metabolismo , Péptidos/metabolismo , Conformación ProteicaRESUMEN
Guanine-nucleotide binding proteins (G proteins) serve as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of the G protein α subunit with GDP (Gα·GDP) and the G protein ßγ subunit (Gßγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα·GTP) and Gßγ. Then, Gα·GTP and Gßγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gßγ. The G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Here, we established the backbone resonance assignments of human Gαi3, a member of the i/o family with a molecular weight of 41 K, in complex with GDP. The chemical shifts were compared with those of Gα(i3) in complex with a GTP-analogue, GTPγS, which we recently reported, indicating that the residues with significant chemical shift differences are mostly consistent with the regions with the structural differences between the GDP- and GTPγS-bound states, as indicated in the crystal structures. The assignments of Gα(i3)·GDP would be useful for the analyses of the dynamics of Gα(i3) and its interactions with various target molecules.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP Gi-Go/química , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Guanosina Difosfato/metabolismo , Resonancia Magnética Nuclear Biomolecular , Subunidades de Proteína/química , Subunidades de Proteína/metabolismo , Guanosina 5'-O-(3-Tiotrifosfato)/química , Guanosina 5'-O-(3-Tiotrifosfato)/metabolismo , Humanos , Modelos Moleculares , Unión Proteica , Conformación ProteicaRESUMEN
Guanine-nucleotide binding proteins (G proteins) act as molecular switches in signaling pathways, by coupling the activation of G protein-coupled receptors (GPCRs) at the cell surface to intracellular responses. In the resting state, G protein forms a heterotrimer, consisting of GDP-bound form of the G protein α subunit (Gα(GDP)) and G protein ßγ subunit (Gßγ). Ligand binding to GPCRs promotes the GDP-GTP exchange on Gα, leading to the dissociation of the GTP-bound form of Gα (Gα(GTP)) and Gßγ. Then, Gα(GTP) and Gßγ bind to their downstream effector enzymes or ion channels and regulate their activities, leading to a variety of cellular responses. Finally, Gα hydrolyzes the bound GTP to GDP and returns to the resting state by re-associating with Gßγ. G proteins are classified with four major families based on the amino acid sequences of Gα: i/o, s, q/11, and 12/13. Each family transduces the signaling from different GPCRs to the specific effectors. Here, we established the backbone resonance assignments of human Gα(i3), a member of the i/o family, with a molecular weight of 41 K in complex with a GTP analogue, GTPγS.