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The two-pore potassium channel TASK-3 has been shown to localize to both the plasma membrane and the mitochondrial inner membrane. TASK-3 is highly expressed in melanoma and breast cancer cells and has been proposed to promote tumor formation. Here we investigated whether pharmacological modulation of TASK-3, and specifically of mitochondrial TASK-3 (mitoTASK-3), had any effect on cancer cell survival and mitochondrial physiology. A novel, mitochondriotropic version of the specific TASK-3 inhibitor IN-THPP has been synthesized by addition of a positively charged triphenylphosphonium moiety. While IN-THPP was unable to induce apoptosis, mitoIN-THPP decreased survival of breast cancer cells and efficiently killed melanoma lines, which we show to express mitoTASK-3. Cell death was accompanied by mitochondrial membrane depolarization and fragmentation of the mitochondrial network, suggesting a role of the channel in the maintenance of the correct function of this organelle. In accordance, cells treated with mitoIN-THPP became rapidly depleted of mitochondrial ATP which resulted in activation of the AMP-dependent kinase AMPK. Importantly, cell survival was not affected in mouse embryonic fibroblasts and the effect of mitoIN-THPP was less pronounced in human melanoma cells stably knocked down for TASK-3 expression, indicating a certain degree of selectivity of the drug both for pathological cells and for the channel. In addition, mitoIN-THPP inhibited cancer cell migration to a higher extent than IN-THPP in two melanoma cell lines. In summary, our results point to the importance of mitoTASK-3 for melanoma cell survival and migration.
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Mitocondrias/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Pirimidinas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Mitocondrias/fisiología , Bloqueadores de los Canales de Potasio/síntesis química , Pirimidinas/síntesis química , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The permeability transition (PT) is an increased permeation of the inner mitochondrial membrane due to the opening of the PT pore (PTP), a Ca2+-activated high conductance channel involved in Ca2+ homeostasis and cell death. Alterations of the PTP have been associated with many pathological conditions and its targeting represents an incessant challenge in the field. Although the modulation of the PTP has been extensively explored, the lack of a clear picture of its molecular nature increases the degree of complexity for any target-based approach. Recent advances suggest the existence of at least two mitochondrial permeability pathways mediated by the F-ATP synthase and the ANT, although the exact molecular mechanism leading to channel formation remains elusive for both. A full comprehension of this to-pore conversion will help to assist in drug design and to develop pharmacological treatments for a fine-tuned PT regulation. Here, we will focus on regulatory mechanisms that impinge on the PTP and discuss the relevant literature of PTP targeting compounds with particular attention to F-ATP synthase and ANT.
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Translocador 1 del Nucleótido Adenina/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Animales , HumanosRESUMEN
The mitochondrial permeability transition, an established mechanism for heart diseases, is a long-standing mystery of mitochondrial biology and a prime drug target for cardioprotection. Several hypotheses about its molecular nature have been put forward over the years, and the prevailing view is that permeabilization of the inner mitochondrial membrane follows opening of a high-conductance channel, the permeability transition pore, which is also called mitochondrial megachannel or multiconductance channel. The permeability transition strictly requires matrix Ca2+ and is favored by the matrix protein cyclophilin D, which mediates the inhibitory effects of cyclosporin A. Here we provide a review of the field, with specific emphasis on the possible role of the adenine nucleotide translocator and of the F-ATP synthase in channel formation, and on currently available small molecule inhibitors. While the possible mechanisms through which the adenine nucleotide translocator and the F-ATP synthase might form high-conductance channels remain unknown, reconstitution experiments and site-directed mutagenesis combined to electrophysiology have provided important clues. The hypothesis that more than one protein may act as a permeability transition pore provides a reasonable explanation for current controversies in the field, and holds great promise for the solution of the mystery of the permeability transition.
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Cardiotónicos/farmacología , Mitocondrias Cardíacas/efectos de los fármacos , Mitocondrias Cardíacas/fisiología , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , Animales , Biomarcadores , Descubrimiento de Drogas , Ratones , Ratones Noqueados , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/genética , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Transportadores de Anión Orgánico/química , Transportadores de Anión Orgánico/genética , Transportadores de Anión Orgánico/metabolismo , Permeabilidad/efectos de los fármacos , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Multimerización de ProteínaRESUMEN
The mitochondrial F-ATP synthase is a complex molecular motor arranged in V-shaped dimers that is responsible for most cellular ATP synthesis in aerobic conditions. In the yeast F-ATP synthase, subunits e and g of the FO sector constitute a lateral domain, which is required for dimer stability and cristae formation. Here, by using site-directed mutagenesis, we identified Arg-8 of subunit e as a critical residue in mediating interactions between subunits e and g, most likely through an interaction with Glu-83 of subunit g. Consistent with this hypothesis, (i) the substitution of Arg-8 in subunit e (eArg-8) with Ala or Glu or of Glu-83 in subunit g (gGlu-83) with Ala or Lys destabilized the digitonin-extracted F-ATP synthase, resulting in decreased dimer formation as revealed by blue-native electrophoresis; and (ii) simultaneous substitution of eArg-8 with Glu and of gGlu-83 with Lys rescued digitonin-stable F-ATP synthase dimers. When tested in lipid bilayers for generation of Ca2+-dependent channels, WT dimers displayed the high-conductance channel activity expected for the mitochondrial megachannel/permeability transition pore, whereas dimers obtained at low digitonin concentrations from the Arg-8 variants displayed currents of strikingly small conductance. Remarkably, double replacement of eArg-8 with Glu and of gGlu-83 with Lys restored high-conductance channels indistinguishable from those seen in WT enzymes. These findings suggest that the interaction of subunit e with subunit g is important for generation of the full-conductance megachannel from F-ATP synthase.
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Mitocondrias/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Dimerización , Potencial de la Membrana Mitocondrial , Poro de Transición de la Permeabilidad Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/genética , Mutagénesis Sitio-Dirigida , Estabilidad Proteica , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Mutations of the GJB1 gene encoding connexin 32 (Cx32) cause the X-linked form of Charcot-Marie-Tooth disease (CMTX1), a demyelinating peripheral neuropathy for which there is no cure. A growing body of evidence indicates that ATP release through Cx32 hemichannels in Schwann cells could be critical for nerve myelination, but it is unknown if CMTX1 mutations alter the cytosolic Ca2+-dependent gating mechanism that controls Cx32 hemichannel opening and ATP release. The current study uncovered that loss of the C-terminus in Cx32 (R220X mutation), which causes a severe CMTX1 phenotype, inhibits hemichannel opening during a canonical IP3-mediated increase in cytosolic Ca2+ in HeLa cells. Interestingly, the gating function of R220X hemichannels was completely restored by both the intracellular and extracellular application of a peptide that mimics the Cx32 cytoplasmic loop. All-atom molecular dynamics simulations suggest that loss of the C-terminus in the mutant hemichannel triggers abnormal fluctuations of the cytoplasmic loop which are prevented by binding to the mimetic peptide. Experiments that stimulated R220X hemichannel opening by cell depolarization displayed reduced voltage sensitivity with respect to wild-type hemichannels which was explained by loss of subconductance states at the single channel level. Finally, experiments of intercellular diffusion mediated by wild-type or R220X gap junction channels revealed similar unitary permeabilities to ions, signalling molecules (cAMP) or larger solutes (Lucifer yellow). Taken together, our findings support the hypothesis that paracrine signalling alteration due to Cx32 hemichannel dysfunction underlies CMTX1 pathogenesis and suggest a candidate molecule for novel studies investigating a therapeutic approach.
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Canales de Calcio/metabolismo , Calcio/metabolismo , Enfermedad de Charcot-Marie-Tooth/metabolismo , Conexinas/genética , Conexinas/metabolismo , Mutación , Adenosina Trifosfato/metabolismo , Canales de Calcio/genética , Enfermedad de Charcot-Marie-Tooth/genética , Conexinas/antagonistas & inhibidores , Conexinas/química , Citosol/metabolismo , Uniones Comunicantes/genética , Uniones Comunicantes/metabolismo , Células HeLa , Humanos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/fisiología , Modelos Moleculares , Técnicas de Placa-Clamp , Células de Schwann/metabolismo , Transfección , Proteína beta1 de Unión ComunicanteAsunto(s)
Angiotensina II/fisiología , Betacoronavirus , Infecciones por Coronavirus/sangre , Endotelio Vascular/fisiopatología , Factor de Crecimiento Placentario/sangre , Neumonía Viral/sangre , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Anciano , Enzima Convertidora de Angiotensina 2 , Betacoronavirus/metabolismo , Biomarcadores/sangre , COVID-19 , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Peptidil-Dipeptidasa A/metabolismo , SARS-CoV-2Asunto(s)
Deferasirox/uso terapéutico , Quelantes del Hierro/uso terapéutico , Sobrecarga de Hierro/tratamiento farmacológico , Mielofibrosis Primaria/tratamiento farmacológico , Anciano , Deferasirox/farmacología , Femenino , Humanos , Quelantes del Hierro/farmacología , Masculino , Mielofibrosis Primaria/complicaciones , Estudios RetrospectivosRESUMEN
COVID-19 and preeclampsia (preE) share the ANG-II mediated endothelial dysfunction, resulting from a significant dysregulation of RAS and an imbalanced proportion of anti-angiogenic and pro-angiogenic soluble plasmatic factors. Of note, an increased incidence of preE has been reported among COVID-19-infected mothers compared to the general pregnant population. The two most promising angiogenic markers are the soluble fms-like tyrosine kinase receptor-1 (sFlt-1), the major antiangiogenic factor, and the placental growth factor (PlGF), a powerful angiogenic factor. Since these markers have proven useful in the prediction, diagnosis, and severity of preE, this study aimed to evaluate their maternal serum levels in pregnancies complicated by SARS-CoV-2 infection and to assess their potential use to guide the management of these women. A retrospective analysis of SARS-CoV-2-positive pregnant women was performed. The serum levels of sFlt-1 and PlGF were collected at the diagnosis of SARS-CoV-2 infection at the hospital, before the beginning of steroid/hydroxychloroquine and/or antithrombotic therapy. The sFlt-1/PlGF ratio was stratified using cut-off values clinically utilized in the diagnosis and prediction of preE (low < 38, intermediate 38−85/110* and high >85/110*, * if before or after the 34th week of gestation). A total of 57 women were included, of whom 20 (35%) had signs and symptoms of COVID-19 at hospital presentation and 37 (65%) were asymptomatic. None were vaccinated. The mean gestational age at diagnosis of SARS-CoV-2 infection was 32 weeks in symptomatic patients and 37 weeks and 5 days in asymptomatic ones (p = 0.089). sFlt-1 serum levels were higher in SARS-CoV-2 positive asymptomatic patients compared to women with COVID-19 related symptoms (4899 ± 4357 pg/mL vs. 3187 ± 2426 pg/mL, p = 0.005). sFlt-1/PlGF at admission was <38 in 18 of the 20 symptomatic women (90%) compared to 22 (59%) of the asymptomatic patients (p = 0.018). Of note, two of the three women admitted to the intensive care unit had a very low ratio (<2). In turn, rates of patients with sFlt-1/PlGF at admission > 85/110 were not significantly different between the two groups: 11% in asymptomatic patients (4/37) vs. none of the symptomatic patients (p = 0.286), and all of them presented a placental dysfunction, like preE (n = 1) and FGR (n = 3). Of note, there were no stillbirths or maternal or neonatal deaths among symptomatic patients; also, no cases of preE, FGR, or small for gestational age neonates were diagnosed. In conclusion, our data suggest that SARS-CoV-2 infection during pregnancy could influence the angiogenic balance. A significant pathological alteration of the sFlt-1/PlGF ratio cannot be identified during the symptomatic phase; however, if left untreated, SARS-CoV-2 infection could potentially trigger placental dysfunction.
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COVID-19 , Preeclampsia , Recién Nacido , Femenino , Humanos , Embarazo , Factor de Crecimiento Placentario , Receptor 1 de Factores de Crecimiento Endotelial Vascular , COVID-19/diagnóstico , Estudios Retrospectivos , Inductores de la Angiogénesis , Hidroxicloroquina , Fibrinolíticos , Placenta , SARS-CoV-2 , Preeclampsia/diagnóstico , Mortinato , BiomarcadoresRESUMEN
F-ATP synthase is a leading candidate as the mitochondrial permeability transition pore (PTP) but the mechanism(s) leading to channel formation remain undefined. Here, to shed light on the structural requirements for PTP formation, we test cells ablated for g, OSCP and b subunits, and ρ0 cells lacking subunits a and A6L. Δg cells (that also lack subunit e) do not show PTP channel opening in intact cells or patch-clamped mitoplasts unless atractylate is added. Δb and ΔOSCP cells display currents insensitive to cyclosporin A but inhibited by bongkrekate, suggesting that the adenine nucleotide translocator (ANT) can contribute to channel formation in the absence of an assembled F-ATP synthase. Mitoplasts from ρ0 mitochondria display PTP currents indistinguishable from their wild-type counterparts. In this work, we show that peripheral stalk subunits are essential to turn the F-ATP synthase into the PTP and that the ANT provides mitochondria with a distinct permeability pathway.
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Calcio/metabolismo , Mitocondrias/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Calcio/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Línea Celular Tumoral , Células HeLa , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Potencial de la Membrana Mitocondrial/fisiología , Mitocondrias/efectos de los fármacos , ATPasas de Translocación de Protón Mitocondriales/genética , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ionóforos de Protónes/farmacologíaRESUMEN
We read with interest the work by Espino-y-Sosa and colleagues [...].
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COVID-19 , Preeclampsia , Biomarcadores , Femenino , Humanos , Embarazo , SARS-CoV-2 , Receptor 1 de Factores de Crecimiento Endotelial VascularRESUMEN
Mitochondria are bioenergetic organelles with a plethora of fundamental functions ranging from metabolism and ATP production to modulation of signaling events leading to cell survival or cell death. Ion channels located in the outer and inner mitochondrial membranes critically control mitochondrial function and, as a consequence, also cell fate. Opening or closure of mitochondrial ion channels allow the fine-tuning of mitochondrial membrane potential, ROS production, and function of the respiratory chain complexes. In this review, we critically discuss the intracellular regulatory factors that affect channel activity in the inner membrane of mitochondria and, indirectly, contribute to cell death. These factors include various ligands, kinases, second messengers, and lipids. Comprehension of mitochondrial ion channels regulation in cell death pathways might reveal new therapeutic targets in mitochondria-linked pathologies like cancer, ischemia, reperfusion injury, and neurological disorders.
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The potassium channel Kv1.3, involved in several important pathologies, is the target of a family of psoralen-based drugs whose mechanism of action is not fully understood. Here we provide evidence for a physical interaction of the mitochondria-located Kv1.3 (mtKv1.3) and Complex I of the respiratory chain and show that this proximity underlies the death-inducing ability of psoralenic Kv1.3 inhibitors. The effects of PAP-1-MHEG (PAP-1, a Kv1.3 inhibitor, with six monomeric ethylene glycol units attached to the phenyl ring of PAP-1), a more soluble novel derivative of PAP-1 and of its various portions on mitochondrial physiology indicate that the psoralenic moiety of PAP-1 bound to mtKv1.3 facilitates the diversion of electrons from Complex I to molecular oxygen. The resulting massive production of toxic Reactive Oxygen Species leads to death of cancer cells expressing Kv1.3. In vivo, PAP-1-MHEG significantly decreased melanoma volume. In summary, PAP-1-MHEG offers insights into the mechanisms of cytotoxicity of this family of compounds and may represent a valuable clinical tool.
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Canal de Potasio Kv1.3 , Mitocondrias , Animales , Línea Celular Tumoral , Disección , Humanos , Canal de Potasio Kv1.3/antagonistas & inhibidores , Canal de Potasio Kv1.3/genética , Ratones Endogámicos C57BL , Especies Reactivas de OxígenoRESUMEN
Connexin hemichannels, which are plasma membrane hexameric channels (connexons) composed of connexin protein protomers, have been implicated in a host of physiological processes and pathological conditions. A number of single point pathological mutations impart a "leaky" character to the affected hemichannels, i.e., make them more active or hyperactive, suggesting that normal physiological condition could be recovered using selective hemichannel inhibitors. Recently, a human-derived monoclonal antibody named abEC1.1 has been shown to inhibit both wild type and hyperactive hemichannels composed of human (h) connexin 26 (hCx26) subunits. The aims of this work were (1) to characterize further the ability of abEC1.1 to selectively modulate connexin hemichannel function and (2) to assess its in vitro stability in view of future translational applications. In silico analysis of abEC1.1 interaction with the hCx26 hemichannel identified critically important extracellular domain amino acids that are conserved in connexin 30 (hCx30) and connexin 32 (hCx32). Patch clamp experiments performed in HeLa DH cells confirmed the inhibition efficiency of abEC1.1 was comparable for hCx26, hCx30 and hCx32 hemichannels. Of note, even a single amino acid difference in the putative binding region reduced drastically the inhibitory effects of the antibody on all the other tested hemichannels, namely hCx30.2/31.3, hCx30.3, hCx31, hCx31.1, hCx37, hCx43 and hCx45. Plasma membrane channels composed of pannexin 1 were not affected by abEC1.1. Finally, size exclusion chromatography assays showed the antibody does not aggregate appreciably in vitro. Altogether, these results indicate abEC1.1 is a promising tool for further translational studies.
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The molecular identity of the mitochondrial megachannel (MMC)/permeability transition pore (PTP), a key effector of cell death, remains controversial. By combining highly purified, fully active bovine F-ATP synthase with preformed liposomes we show that Ca2+ dissipates the H+ gradient generated by ATP hydrolysis. After incorporation of the same preparation into planar lipid bilayers Ca2+ elicits currents matching those of the MMC/PTP. Currents were fully reversible, were stabilized by benzodiazepine 423, a ligand of the OSCP subunit of F-ATP synthase that activates the MMC/PTP, and were inhibited by Mg2+ and adenine nucleotides, which also inhibit the PTP. Channel activity was insensitive to inhibitors of the adenine nucleotide translocase (ANT) and of the voltage-dependent anion channel (VDAC). Native gel-purified oligomers and dimers, but not monomers, gave rise to channel activity. These findings resolve the long-standing mystery of the MMC/PTP and demonstrate that Ca2+ can transform the energy-conserving F-ATP synthase into an energy-dissipating device.
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Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/metabolismo , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Animales , Bovinos , Microscopía por Crioelectrón , Hidrólisis , Magnesio/metabolismo , Potencial de la Membrana Mitocondrial , Mitocondrias Cardíacas/enzimología , Mitocondrias Cardíacas/metabolismo , Poro de Transición de la Permeabilidad Mitocondrial , ATPasas de Translocación de Protón Mitocondriales/química , ATPasas de Translocación de Protón Mitocondriales/ultraestructura , Necrosis por Permeabilidad de la Transmembrana Mitocondrial , Multimerización de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/metabolismoRESUMEN
BACKGROUND: Vitamin B12 deficiency-induced thrombotic microangiopathy, known as pseudothrombotic microangiopathy, is a rare condition which resembles the clinical features of thrombotic thrombocytopenic purpura but requires a markedly different treatment. Most cases of vitamin B12 deficiency have only mild hematological findings, but in approximately 10% of patients life-threatening conditions have been reported. CASE PRESENTATION: We report a case of a 46-year-old Moroccan man presenting with severe hemolytic anemia, thrombocytopenia, and renal failure in absence of macrocytosis, thus mimicking a genuine thrombotic thrombocytopenic purpura. Rapid improvement of renal function observed with only hydration and transfusions of packed red blood cells and the presence of pancytopenia suggested a bone marrow deficiency associated to a hemolytic component of unclear origin. Detection of low levels of vitamin B12 and rapid restitutio ad integrum with its replacement supported the diagnosis of pseudothrombotic thrombocytopenic purpura caused by vitamin B12 deficiency. CONCLUSIONS: Diagnosis of pseudothrombotic thrombocytopenic purpura caused by vitamin B12 deficiency might be difficult. Awareness of clinicians toward this differential diagnosis might spare patients from unnecessary therapeutic plasma exchange that is burdened by morbidity and mortality.
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Lesión Renal Aguda , Transfusión de Eritrocitos/métodos , Púrpura Trombocitopénica Trombótica/diagnóstico , Microangiopatías Trombóticas , Deficiencia de Vitamina B 12 , Vitamina B 12 , Lesión Renal Aguda/diagnóstico , Lesión Renal Aguda/etiología , Lesión Renal Aguda/fisiopatología , Lesión Renal Aguda/terapia , Diagnóstico Diferencial , Fluidoterapia/métodos , Humanos , Pruebas de Función Renal/métodos , Masculino , Persona de Mediana Edad , Microangiopatías Trombóticas/diagnóstico , Microangiopatías Trombóticas/etiología , Microangiopatías Trombóticas/terapia , Resultado del Tratamiento , Vitamina B 12/administración & dosificación , Vitamina B 12/sangre , Deficiencia de Vitamina B 12/complicaciones , Deficiencia de Vitamina B 12/diagnóstico , Deficiencia de Vitamina B 12/terapia , Vitaminas/administración & dosificaciónRESUMEN
Connexin channels play numerous essential roles in virtually every organ by mediating solute exchange between adjacent cells, or between cytoplasm and extracellular milieu. Our understanding of the structure-function relationship of connexin channels relies on X-ray crystallographic data for human connexin 26 (hCx26) intercellular gap junction channels. Comparison of experimental data and molecular dynamics simulations suggests that the published structures represent neither fully-open nor closed configurations. To facilitate the search for alternative stable configurations, we developed a coarse grained (CG) molecular model of the hCx26 hemichannel and studied its responses to external electric fields. When challenged by a field of 0.06 V/nm, the hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second transmembrane helix (TM2) at the level of the conserved Pro87. A point mutation that inhibited such transition in our simulations impeded hemichannel opening in electrophysiology and dye uptake experiments conducted on HeLa tranfectants. These results suggest that the hCx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among the whole connexin family.
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Panx1 forms plasma membrane channels in brain and several other organs, including the inner ear. Biophysical properties, activation mechanisms and modulators of Panx1 channels have been characterized in detail, however the impact of Panx1 on auditory function is unclear due to conflicts in published results. To address this issue, hearing performance and cochlear function of the Panx1-/- mouse strain, the first with a reported global ablation of Panx1, were scrutinized. Male and female homozygous (Panx1-/-), hemizygous (Panx1+/-) and their wild type (WT) siblings (Panx1+/+) were used for this study. Successful ablation of Panx1 was confirmed by RT-PCR and Western immunoblotting in the cochlea and brain of Panx1-/- mice. Furthermore, a previously validated Panx1-selective antibody revealed strong immunoreactivity in WT but not in Panx1-/- cochleae. Hearing sensitivity, outer hair cell-based "cochlear amplifier" and cochlear nerve function, analyzed by auditory brainstem response (ABR) and distortion product otoacoustic emission (DPOAE) recordings, were normal in Panx1+/- and Panx1-/- mice. In addition, we determined that global deletion of Panx1 impacts neither on connexin expression, nor on gap-junction coupling in the developing organ of Corti. Finally, spontaneous intercellular Ca2+ signal (ICS) activity in organotypic cochlear cultures, which is key to postnatal development of the organ of Corti and essential for hearing acquisition, was not affected by Panx1 ablation. Therefore, our results provide strong evidence that, in mice, Panx1 is dispensable for hearing acquisition and auditory function.
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Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies.
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RESUMO O estudo teve como objetivo avaliar a efetividade da atenção primária, identificando as distinções entre os modelos de serviços com estratégia saúde da família em comparação a unidades básicas tradicionais, com e sem profissionais do programa mais médicos.Pesquisa quantitativa, avaliativa, realizada com 128 profissionais - médicos, enfermeiros e coordenadores de unidades de atenção primária, por meio do instrumento Primary Care Assessment Tool (PCATool - Brasil), versão profissionais de saúde, em 2015 e 2016eadotou análise estatística inferencial. Os resultados indicaram escores acima de 6,6 naquelas unidades que adotam a estratégia saúde da família e abaixo do ponto de corte nas unidades básicas de saúde tradicionais.Coordenação - sistemas de informações (5,8) e longitudinalidade (6,2) mostraram baixa efetividade dos serviços na unidade tradicional. O acesso obteve baixo escore (4,0), em ambos os modelos.A avaliação da APS nos distintos modelos de atenção revelou que as ações e serviços em saúde oferecidos no município foram mais efetivos quando executados em unidades de ESF, em comparação a UBS tradicional, independentemente da presença de profissionais do programa mais médicos.
RESUMEN El estudio tuvo como objetivo evaluar la efectividad de la atención primaria, identificando las distinciones entre los modelos de servicios con estrategia salud de la familia (ESF) en comparación a unidades básicas (UBS) tradicionales, con y sin profesionales del programa "mais médicos". Investigación cuantitativa, evaluativa, realizada con 128 profesionales - médicos, enfermeros y coordinadores de unidades de atención primaria (APS), por medio del instrumento Primary Care Assessment Tool (PCATool - Brasil), versión profesionales de salud, en 2015 y 2016 y adoptó el análisis estadístico inferencial. Los resultados indicaron puntuaciones arriba de 6,6 en aquellas unidades que adoptan la estrategia salud de la familia y abajo del punto de corte en las unidades básicas de salud tradicionales. Coordinación - sistemas de informaciones (5,8) y longitudinalidad (6,2) demostraron baja efectividad de los servicios en la unidad tradicional. El acceso obtuvo baja puntuación (4,0) en ambos los modelos. La evaluación de la APS en los distintos modelos de atención reveló que las acciones y los servicios en salud ofrecidos en el municipio fueron más efectivos cuando ejecutados en unidades de ESF, en comparación a UBS tradicional, independientemente de la presencia de profesionales del programa "mais médicos".
ABSTRACT The study had as objective to evaluate the effectiveness of primary care, identifying the distinctions between the models of services with the family health strategy in comparison to traditional basic units, with and without professionals of the program more medical. Quantitative research, evaluative, performed with 128professionals -physicians, nurses and coordinators of primary care units, by means of the instrument Primary Care Assessment Tool (PCATool - Brazil) version to health professionals, in 2015 and 2016 and adopted inferential statistical analysis. The results indicated scores above 6.6 on those units that adopt the family health strategy and below the cut-off point in the basic units of traditional health. Coordination- information systems (5.8) and longitudinally (6.2) showed low effectiveness of services in traditional health units. The access obtained a low score (4.0), in both models. The evaluation of the primary care in different models of services revealed that the health actions and services offered in the municipality were more effective when performed in family health units, in comparison to traditional ones, regardless of the presence of professionals in the program more medical.