RESUMEN
BACKGROUND: The ability to accurately distinguish bacterial from viral infection would help clinicians better target antimicrobial therapy during suspected lower respiratory tract infections (LRTI). Although technological developments make it feasible to rapidly generate patient-specific microbiota profiles, evidence is required to show the clinical value of using microbiota data for infection diagnosis. In this study, we investigated whether adding nasal cavity microbiota profiles to readily available clinical information could improve machine learning classifiers to distinguish bacterial from viral infection in patients with LRTI. RESULTS: Various multi-parametric Random Forests classifiers were evaluated on the clinical and microbiota data of 293 LRTI patients for their prediction accuracies to differentiate bacterial from viral infection. The most predictive variable was C-reactive protein (CRP). We observed a marginal prediction improvement when 7 most prevalent nasal microbiota genera were added to the CRP model. In contrast, adding three clinical variables, absolute neutrophil count, consolidation on X-ray, and age group to the CRP model significantly improved the prediction. The best model correctly predicted 85% of the 'bacterial' patients and 82% of the 'viral' patients using 13 clinical and 3 nasal cavity microbiota genera (Staphylococcus, Moraxella, and Streptococcus). CONCLUSIONS: We developed high-accuracy multi-parametric machine learning classifiers to differentiate bacterial from viral infections in LRTI patients of various ages. We demonstrated the predictive value of four easy-to-collect clinical variables which facilitate personalized and accurate clinical decision-making. We observed that nasal cavity microbiota correlate with the clinical variables and thus may not add significant value to diagnostic algorithms that aim to differentiate bacterial from viral infections.
Asunto(s)
Infecciones Bacterianas , Microbiota , Infecciones del Sistema Respiratorio , Virosis , Infecciones Bacterianas/tratamiento farmacológico , Proteína C-Reactiva/metabolismo , Humanos , Nariz/microbiología , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Virosis/diagnósticoRESUMEN
OBJECTIVES: Identifying infection aetiology is essential for appropriate antibiotic use. Previous studies have shown that a host-protein signature consisting of TNF-related apoptosis-induced ligand (TRAIL), interferon-γ-induced protein-10 (IP-10), and C-reactive protein (CRP) can accurately differentiate bacterial from viral infections. METHODS: This prospective, multicentre cohort study, entitled AutoPilot-Dx, aimed to validate signature performance and to estimate its potential impact on antibiotic use across a broad paediatric population (>90 days to 18 years) with respiratory tract infections, or fever without source, at emergency departments and wards in Italy and Germany. Infection aetiology was adjudicated by experts based on clinical and laboratory investigations, including multiplex PCR and follow-up data. RESULTS: In total, 1140 patients were recruited (February 2017-December 2018), of which 1008 met the eligibility criteria (mean age 3.5 years, 41.9% female). Viral and bacterial infections were adjudicated for 628 (85.8%) and 104 (14.2%) children, respectively; 276 patients were assigned an indeterminate reference standard outcome. For the 732 children with reference standard aetiology, the signature discriminated bacterial from viral infections with a sensitivity of 93.7% (95%CI 88.7-98.7), a specificity of 94.2% (92.2-96.1), positive predictive value of 73.0% (65.0-81.0), and negative predictive value of 98.9% (98.0-99.8); in 9.8% the test results were equivocal. The signature performed consistently across different patient subgroups and detected bacterial immune responses in viral PCR-positive patients. CONCLUSIONS: The findings validate the high diagnostic performance of the TRAIL/IP-10/CRP signature in a broad paediatric cohort, and support its potential to reduce antibiotic overuse in children with viral infections.
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Infecciones Bacterianas , Virosis , Antibacterianos/uso terapéutico , Apoptosis , Infecciones Bacterianas/microbiología , Biomarcadores , Proteína C-Reactiva/análisis , Quimiocina CXCL10 , Niño , Preescolar , Estudios de Cohortes , Diagnóstico Diferencial , Femenino , Humanos , Ligandos , Masculino , Estudios Prospectivos , Virosis/diagnósticoRESUMEN
OBJECTIVE: If a gold standard is lacking in a diagnostic test accuracy study, expert diagnosis is frequently used as reference standard. However, interobserver and intraobserver agreements are imperfect. The aim of this study was to quantify the reproducibility of a panel diagnosis for pediatric infectious diseases. STUDY DESIGN AND SETTING: Pediatricians from six countries adjudicated a diagnosis (i.e., bacterial infection, viral infection, or indeterminate) for febrile children. Diagnosis was reached when the majority of panel members came to the same diagnosis, leaving others inconclusive. We evaluated intraobserver and intrapanel agreement with 6 weeks and 3 years' time intervals. We calculated the proportion of inconclusive diagnosis for a three-, five-, and seven-expert panel. RESULTS: For both time intervals (i.e., 6 weeks and 3 years), intrapanel agreement was higher (kappa 0.88, 95%CI: 0.81-0.94 and 0.80, 95%CI: NA) compared to intraobserver agreement (kappa 0.77, 95%CI: 0.71-0.83 and 0.65, 95%CI: 0.52-0.78). After expanding the three-expert panel to five or seven experts, the proportion of inconclusive diagnoses (11%) remained the same. CONCLUSION: A panel consisting of three experts provides more reproducible diagnoses than an individual expert in children with lower respiratory tract infection or fever without source. Increasing the size of a panel beyond three experts has no major advantage for diagnosis reproducibility.
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Toma de Decisiones Clínicas/métodos , Fiebre de Origen Desconocido/diagnóstico , Pediatría , Infecciones del Sistema Respiratorio/diagnóstico , Preescolar , Diagnóstico Diferencial , Pruebas Diagnósticas de Rutina , Testimonio de Experto/métodos , Testimonio de Experto/normas , Femenino , Humanos , Lactante , Masculino , Pediatría/métodos , Pediatría/normas , Estándares de Referencia , Reproducibilidad de los Resultados , Nivel de AtenciónRESUMEN
Respiratory tract infections (RTI) are more commonly caused by viral pathogens in children than in adults. Surprisingly, little is known about antibiotic use in children as compared to adults with RTI. This prospective study aimed to determine antibiotic misuse in children and adults with RTI, using an expert panel reference standard, in order to prioritise the target age population for antibiotic stewardship interventions. We recruited children and adults who presented at the emergency department or were hospitalised with clinical presentation of RTI in The Netherlands and Israel. A panel of three experienced physicians adjudicated a reference standard diagnosis (i.e. bacterial or viral infection) for all the patients using all available clinical and laboratory information, including a 28-day follow-up assessment. The cohort included 284 children and 232 adults with RTI (median age, 1.3 years and 64.5 years, respectively). The proportion of viral infections was larger in children than in adults (209(74%) versus 89(38%), p < 0.001). In case of viral RTI, antibiotics were prescribed (i.e. overuse) less frequently in children than in adults (77/209 (37%) versus 74/89 (83%), p < 0.001). One (1%) child and three (2%) adults with bacterial infection were not treated with antibiotics (i.e. underuse); all were mild cases. This international, prospective study confirms major antibiotic overuse in patients with RTI. Viral infection is more common in children, but antibiotic overuse is more frequent in adults with viral RTI. Together, these findings support the need for effective interventions to decrease antibiotic overuse in RTI patients of all ages.
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Antibacterianos/uso terapéutico , Programas de Optimización del Uso de los Antimicrobianos/normas , Prescripción Inadecuada/estadística & datos numéricos , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Anciano , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/tratamiento farmacológico , Infecciones Bacterianas/epidemiología , Preescolar , Femenino , Humanos , Lactante , Israel/epidemiología , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Estudios Prospectivos , Estándares de Referencia , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/epidemiología , Virosis/diagnóstico , Virosis/tratamiento farmacológico , Virosis/epidemiologíaRESUMEN
Distinguishing bacterial from viral infections is often challenging, leading to antibiotic misuse, and detrimental ramifications for the patient, the healthcare system and society. A novel ELISA-based assay that integrates the circulating levels of three host-response proteins (TRAIL, IP-10 and CRP) was developed to assist in differentiation between bacterial and viral etiologies. We developed a new protocol for measuring the host-based assay biomarkers using an automated ELISA workstation. The automated protocol was validated and was able to reduce technician hands-on time by 76%, while maintaining high analytical performance. Following automation, the assay has been incorporated into the routine workflow at a pediatric department, and is performed daily on admitted and emergency department patients. The automation protocol reduces the overall burden on the hospital laboratory performing the assay. This benefit has potential to promote adoption of the host-based assay, facilitating timely triage of febrile patients and prudent use of antibiotics.
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Infecciones Bacterianas/diagnóstico , Quimiocina CXCL10/sangre , Ensayo de Inmunoadsorción Enzimática/métodos , Ligando Inductor de Apoptosis Relacionado con TNF/sangre , Virosis/diagnóstico , Infecciones Bacterianas/sangre , Quimiocina CXCL10/análisis , Ensayo de Inmunoadsorción Enzimática/economía , Interacciones Huésped-Patógeno , Humanos , Límite de Detección , Ligando Inductor de Apoptosis Relacionado con TNF/análisis , Factores de Tiempo , Virosis/sangreRESUMEN
Bacterial and viral infections often present with similar symptoms. Etiologic misdiagnosis can alter the trajectory of patient care, including antibiotic overuse. A host-protein signature comprising tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), interferon gamma-induced protein-10 (IP-10), and C-reactive protein (CRP) was validated recently for differentiating bacterial from viral disease. However, a focused head-to-head comparison of its diagnostic performance against other biomarker candidates for this indication was lacking in patients with respiratory infection and fever without source. We compared the signature to other biomarkers and prediction rules using specimens collected prospectively at two secondary medical centers from children and adults. Inclusion criteria included fever > 37.5 °C, symptom duration ≤ 12 days, and presentation with respiratory infection or fever without source. Comparator method was based on expert panel adjudication. Signature and biomarker cutoffs and prediction rules were predefined. Of 493 potentially eligible patients, 314 were assigned unanimous expert panel diagnosis and also had sufficient specimen volume. The resulting cohort comprised 175 (56%) viral and 139 (44%) bacterial infections. Signature sensitivity 93.5% (95% CI 89.1-97.9%), specificity 94.3% (95% CI 90.7-98.0%), or both were significantly higher (all p values < 0.01) than for CRP, procalcitonin, interleukin-6, human neutrophil lipocalin, white blood cell count, absolute neutrophil count, and prediction rules. Signature identified as viral 50/57 viral patients prescribed antibiotics, suggesting potential to reduce antibiotic overuse by 88%. The host-protein signature demonstrated superior diagnostic performance in differentiating viral from bacterial respiratory infections and fever without source. Future utility studies are warranted to validate potential to reduce antibiotic overuse.
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Infecciones Bacterianas/diagnóstico , Proteína C-Reactiva/análisis , Quimiocina CXCL10/sangre , Infecciones del Sistema Respiratorio/diagnóstico , Ligando Inductor de Apoptosis Relacionado con TNF/sangre , Virosis/diagnóstico , Adolescente , Adulto , Biomarcadores/sangre , Calcitonina/sangre , Niño , Diagnóstico Diferencial , Femenino , Humanos , Interleucina-6/sangre , Recuento de Leucocitos , Lipocalinas/sangre , Masculino , Estudios Prospectivos , Adulto JovenRESUMEN
Bacterial and viral lower respiratory tract infections (LRTIs) are often clinically indistinguishable, leading to antibiotic overuse. We compared the diagnostic accuracy of a new assay that combines 3 host-biomarkers (TRAIL, IP-10, CRP) with parameters in routine use to distinguish bacterial from viral LRTIs. Study cohort included 184 potentially eligible pediatric and adult patients. Reference standard diagnosis was based on adjudication by an expert panel following comprehensive clinical and laboratory investigation (including respiratory PCRs). Experts were blinded to assay results and assay performers were blinded to reference standard outcomes. Evaluated cohort included 88 bacterial and 36 viral patients (23 did not fulfill inclusion criteria; 37 had indeterminate reference standard outcome). Assay distinguished bacterial from viral LRTI patients with sensitivity of 0.93±0.06 and specificity of 0.91±0.09, outperforming routine parameters, including WBC, CRP and chest x-ray signs. These findings support the assay's potential to help clinicians avoid missing bacterial LRTIs or overusing antibiotics.
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Infecciones Bacterianas/diagnóstico , Infecciones del Sistema Respiratorio/diagnóstico , Virosis/diagnóstico , Adolescente , Adulto , Biomarcadores/análisis , Proteína C-Reactiva/análisis , Quimiocina CXCL10/análisis , Niño , Preescolar , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Infecciones del Sistema Respiratorio/microbiología , Infecciones del Sistema Respiratorio/virología , Sensibilidad y Especificidad , Ligando Inductor de Apoptosis Relacionado con TNF/análisis , Adulto JovenRESUMEN
Bacterial and viral infections are often clinically indistinguishable, leading to inappropriate patient management and antibiotic misuse. Bacterial-induced host proteins such as procalcitonin, C-reactive protein (CRP), and Interleukin-6, are routinely used to support diagnosis of infection. However, their performance is negatively affected by inter-patient variability, including time from symptom onset, clinical syndrome, and pathogens. Our aim was to identify novel viral-induced host proteins that can complement bacterial-induced proteins to increase diagnostic accuracy. Initially, we conducted a bioinformatic screen to identify putative circulating host immune response proteins. The resulting 600 candidates were then quantitatively screened for diagnostic potential using blood samples from 1002 prospectively recruited patients with suspected acute infectious disease and controls with no apparent infection. For each patient, three independent physicians assigned a diagnosis based on comprehensive clinical and laboratory investigation including PCR for 21 pathogens yielding 319 bacterial, 334 viral, 112 control and 98 indeterminate diagnoses; 139 patients were excluded based on predetermined criteria. The best performing host-protein was TNF-related apoptosis-inducing ligand (TRAIL) (area under the curve [AUC] of 0.89; 95% confidence interval [CI], 0.86 to 0.91), which was consistently up-regulated in viral infected patients. We further developed a multi-protein signature using logistic-regression on half of the patients and validated it on the remaining half. The signature with the highest precision included both viral- and bacterial-induced proteins: TRAIL, Interferon gamma-induced protein-10, and CRP (AUC of 0.94; 95% CI, 0.92 to 0.96). The signature was superior to any of the individual proteins (P<0.001), as well as routinely used clinical parameters and their combinations (P<0.001). It remained robust across different physiological systems, times from symptom onset, and pathogens (AUCs 0.87-1.0). The accurate differential diagnosis provided by this novel combination of viral- and bacterial-induced proteins has the potential to improve management of patients with acute infections and reduce antibiotic misuse.
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Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/metabolismo , Proteómica , Virosis/diagnóstico , Virosis/metabolismo , Adolescente , Adulto , Biomarcadores/metabolismo , Niño , Preescolar , Estudios de Cohortes , Diagnóstico Diferencial , Femenino , Humanos , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Adulto JovenRESUMEN
The inappropriate use of antibiotics has severe global health and economic consequences, including the emergence of antibiotic-resistant bacteria. A major driver of antibiotic misuse is the inability to accurately distinguish between bacterial and viral infections based on currently available diagnostic solutions. A multifaceted 'omics' approach that integrates personalized patient data such as genetic predisposition to infections (genomics), natural microbiota composition and immune response to infection (proteomics and transcriptomics) together with comprehensive pathogen profiling has the potential to help physicians improve their antimicrobial prescribing practices. In this respect, the EU has funded a multidisciplinary project (TAILORED-Treatment) that will develop novel omics-based personalized treatment schemes that have the potential to reduce antibiotic consumption, and help limiting the spread of antibiotic resistance.
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Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Infecciones Bacterianas/diagnóstico , Infecciones Bacterianas/tratamiento farmacológico , Farmacorresistencia Bacteriana , Genómica , Proteómica , Antibacterianos/uso terapéutico , Bacterias/aislamiento & purificación , Infecciones Bacterianas/microbiología , Biología Computacional , Diagnóstico Diferencial , Utilización de Medicamentos , Humanos , Microbiota , Medicina de Precisión/métodos , Virosis/diagnósticoRESUMEN
The activity of background K(2P) channels adjusts the resting membrane potential to enable plasticity of excitable cells. Here we have studied the regulation of neuronal K(2P)2.1 (KCNK2, TREK-1) channel activity by resting membrane potential. When heterologously expressed, K(2P)2.1 currents gradually increased at hyperpolarizing potentials and declined at depolarizing potentials, with a midpoint potential of -60 mV. As K(2P) channels are not equipped with an integral voltage sensor, we sought extrinsic cellular components that could convert changes in the membrane electrical field to cellular activity that would indirectly modify K(2P)2.1 currents. We propose that membrane depolarization activated the Gq protein-coupled receptor pathway, in the apparent absence of ligand, resulting in phosphatidylinositol-4,5-bisphosphate (PIP(2)) depletion through the action of phospholipase C. Our results suggest a novel mechanism in which an indirect pathway confers membrane potential regulation onto channels that are not intrinsically voltage sensitive to enhance regulation of neuronal excitability levels.
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Potenciales de la Membrana/fisiología , Neuronas/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Secuencia de Aminoácidos , Animales , Inhibidores Enzimáticos/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gq-G11/metabolismo , Silenciador del Gen , Humanos , Activación del Canal Iónico/fisiología , Datos de Secuencia Molecular , Neuronas/citología , Oocitos/citología , Oocitos/fisiología , Técnicas de Placa-Clamp , Fosfatidilinositol 4,5-Difosfato/metabolismo , Canales de Potasio de Dominio Poro en Tándem/genética , Receptores Acoplados a Proteínas G/metabolismo , Alineación de Secuencia , Fosfolipasas de Tipo C/metabolismo , Xenopus laevisRESUMEN
A key feature of potassium channel function is the ability to switch between conducting and non-conducting states by undergoing conformational changes in response to cellular or extracellular signals. Such switching is facilitated by the mechanical coupling of gating domain movements to pore opening and closing. Two-pore domain potassium channels (K(2P)) conduct leak or background potassium-selective currents that are mostly time- and voltage-independent. These channels play a significant role in setting the cell resting membrane potential and, therefore modulate cell responsiveness and excitability. Thus, K(2P) channels are key players in numerous physiological processes and were recently shown to also be involved in human pathologies. It is well established that K(2P) channel conductance, open probability and cell surface expression are significantly modulated by various physical and chemical stimuli. However, in understanding how such signals are translated into conformational changes that open or close the channels gate, there remain more open questions than answers. A growing line of evidence suggests that the outer pore area assumes a critical role in gating K(2P) channels, in a manner reminiscent of C-type inactivation of voltage-gated potassium channels. In some K(2P) channels, this gating mechanism is facilitated in response to external pH levels. Recently, it was suggested that K(2P) channels also possess a lower activation gate that is positively coupled to the outer pore gate. The purpose of this review is to present an up-to-date summary of research describing the conformational changes and gating events that take place at the K(2P) channel ion-conducting pathway during the channel regulation.
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Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Humanos , Concentración de Iones de HidrógenoRESUMEN
Pain is a physiological state promoting protective responses to harmful episodes. However, pain can become pathophysiological and become a chronic disruptive condition, damaging quality of life. The mammalian K(2P)2.1 (KCNK2, TREK-1) channel, expressed in sensory neurons of the dorsal root ganglia, was previously identified as a polymodal molecular sensor involved in pain perception. Here, we report that two pain-associated signals, external acidosis and lysophosphatidic acid (LPA), known to rise during injury, inflammation and cancer, profoundly down-modulate human K(2P)2.1 activity. The pH regulatory effect was mediated by activation of proton-sensitive G-protein coupled receptors and phospholipase C. Physiological concentrations of LPA overcame the effects of known K(2P)2.1 activators, such as arachidonic acid, lysophosphatidylcholine and temperature, by activating cell-surface receptors stimulating the G(q) pathway. Furthermore, we identified three K(2P)2.1 carboxy-terminal residues that mediate both pH and LPA regulatory effects. Our results highlight the important role of K(2P)2.1 channels as receptors for mediators known to cause nociception.
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Acidosis/metabolismo , Lisofosfolípidos/metabolismo , Neuronas/metabolismo , Dolor/metabolismo , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Animales , Activación Enzimática , Proteínas de Unión al GTP/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Oocitos/citología , Oocitos/fisiología , Fosfolipasas de Tipo C/metabolismo , Xenopus laevisRESUMEN
We describe a maternally transmitted genomic-imprinting syndrome of mental retardation, hypotonia, and unique dysmorphism with elongated face. We mapped the disease-associated locus to approximately 7.27 Mb on chromosome 8q24 and demonstrated that the disease is caused by a missense mutation in the maternal copy of KCNK9 within this locus. KCNK9 is maternally transmitted (imprinted with paternal silencing) and encodes K(2P)9.1, a member of the two pore-domain potassium channel (K(2P)) subfamily. The mutation fully abolishes the channel's currents--both when functioning as a homodimer or as a heterodimer with K(2P)3.1.
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Impresión Genómica , Discapacidad Intelectual/genética , Mutación , Polimorfismo Genético , Canales de Potasio de Dominio Poro en Tándem/genética , Animales , Cromosomas Humanos Par 8 , Femenino , Ligamiento Genético , Humanos , Masculino , Repeticiones de Microsatélite , Madres , Síndrome , Xenopus laevisRESUMEN
The mammalian K2P2.1 potassium channel (TREK-1, KCNK2) is highly expressed in excitable tissues, where it plays a key role in the cellular mechanisms of neuroprotection, anesthesia, pain perception, and depression. Here, we report that external acidification, within the physiological range, strongly inhibits the human K2P2.1 channel by inducing "C-type" closure. We have identified two histidine residues (i.e. His-87 and His-141), located in the first external loop of the channel, that govern the response of the channel to external pH. We demonstrate that these residues are within physical proximity to glutamate 84, homologous to Shaker Glu-418, KcsA Glu-51, and KCNK0 Glu-28 residues, all previously argued to stabilize the outer pore gate in the open conformation by forming hydrogen bonds with pore-adjacent residues. We thus propose a novel mechanism for pH sensing in which protonation of His-141 and His-87 generates a local positive charge that serves to draw Glu-84 away from its natural interactions, facilitating the collapse of the selectivity filter region. In accordance with this proposed mechanism, low pH modified K2P2.1 selectivity toward potassium. Moreover, the proton-mediated effect was inhibited by external potassium ions and was enhanced by a mutation (S164Y) known to accelerate C-type gating. Furthermore, proton-induced current inhibition was more pronounced at negative potentials. Thus, voltage-dependent C-type gating acceleration by protons represents a novel mechanism for K2P2.1 outward rectification.
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Histidina/metabolismo , Activación del Canal Iónico , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Protones , Animales , Depresión/genética , Depresión/metabolismo , Femenino , Regulación de la Expresión Génica , Histidina/química , Histidina/genética , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Especificidad de Órganos , Dolor/genética , Dolor/metabolismo , Canales de Potasio de Dominio Poro en Tándem/química , Canales de Potasio de Dominio Poro en Tándem/genética , Estructura Secundaria de Proteína/genética , Xenopus laevisRESUMEN
The biophysical and pharmacological properties of ion channels and transporters are often studied in exogenous expression systems using either the two-electrode voltage clamp (TEVC) in Xenopus oocytes or the patch clamp techniques. Cells machinery is trusted to produce active proteins that are correctly phosphorylated and glycosylated. However, native physiological cellular processes that might be altered during the course of the experiment are often ignored. Here, we detected and quantified the effects of various electrophysiological recording conditions on the phosphorylation levels of Xenopus oocytes proteins, including membrane proteins, as phosphorylation/dephosphorylation events modulate ion channels gating and cell surface expression. Two strategies were chosen to determine relative protein phosphorylation levels: a direct detection with a phospho-Ser/Thr PKA substrate antibody, and a functional method employing two different leak potassium channels as indicators, chosen based on their opposite responses to protein kinase phosphorylation. We report that holding potential, and bath solution properties such as pH, osmolarity, temperature and ion composition, dramatically affect protein phosphorylation levels in Xenopus oocytes. Our results might explain some of the fluctuations in the biophysical properties of expressed channels, often observed during electrophysiological measurements. Minimizing possible misinterpretations could be achieved using either mutated, kinase insensitive, channels or kinases/phosphatases modulators.