Expression of LRRC8/VRAC currents in Xenopus oocytes: advantages and caveats

Volume-regulated anion channels (VRACs) play a role in controlling cell volume by opening upon cell swelling. Apart from controlling cell volume, their function is important in many other physiological processes, such as transport of metabolites or drugs, and extracellular signal transduction. VRACs are formed by heteromers of the pannexin homologous protein LRRC8A (also named Swell1) with other LRRC8 members (B, C, D, and E). LRRC8 proteins are difficult to study, since they are expressed in all cells of our body, and the channel stoichiometry can be changed by overexpression, resulting in non-functional heteromers. Two different strategies have been developed to overcome this issue: complementation by transient transfection of LRRC8 genome-edited cell lines, and reconstitution in lipid bilayers. Alternatively, we have used Xenopus oocytes as a simple system to study LRRC8 proteins. Here, we have reviewed all previous experiments that have been performed with VRAC and LRRC8 proteins in Xenopus oocytes. We also discuss future strategies that may be used to perform structure-function analysis of the VRAC in oocytes and other systems, in order to understand its role in controlling multiple physiological functions

Chloride channels in astrocytes: structure, roles in brain homeostasis and implications in disease

Astrocytes are the most abundant cell type in the CNS (central nervous system). They exert multiple functions during development and in the adult CNS that are essential for brain homeostasis. Both cation and anion channel activities have been identified in astrocytes and it is believed that they play key roles in astrocyte function. Whereas the proteins and the physiological roles assigned to cation channels are becoming very clear, the study of astrocytic chloride channels is in its early stages. In recent years, we have moved from the identification of chloride channel activities present in astrocyte primary culture to the identification of the proteins involved in these activities, the determination of their 3D structure and attempts to gain insights about their physiological role. Here, we review the recent findings related to the main chloride channels identified in astrocytes: the voltage-dependent ClC-2, the calcium-activated bestrophin, the volume-activated VRAC (volume-regulated anion channel) and the stress-activated Maxi-Cl−. We discuss key aspects of channel biophysics and structure with a focus on their role in glial physiology and human disease

Bubble-laden thermals in supersaturated water

Bubble-laden thermals provide a formidable gas transport mechanism responsible, for instance, for the explosive foaming-up process during the beer tapping prank, or the infamous gas eruption of Lake Nyos in 1986. In this work we investigate experimentally the growth and motion of laser-induced turbulent thermals in a carbonated water solution with surfactants. One of the novelties of this study is that we are able to quantify with high temporal resolution the rate at which the gas volume contained in the bubbles grows. After an initial transient stage, the gas bubble and entrained liquid volumes of the thermal both grow as a cubic power of time. The buoyancy generation rate is well explained by the mass transfer scaling expected for individual bubbles. In contrast, the thermal rise velocity does not adhere to any particular scaling law. These facts lie in qualitative agreement with a phenomenological model, based on classical models for turbulent thermals, that takes into account buoyancy generation.We acknowledge the support of the Spanish Ministry of Economy and Competitiveness through grants DPI2017-88201-C3-3-R and DPI2018-102829-REDT, partly funded through European Funds. This work was supported by the Netherlands Center for Multiscale Catalytic Energy Conversion (MCEC), an NWO Gravitation programme funded by the Ministry of Education, Culture and Science of the government of the Netherlands

Identification and characterization of the zebrafish ClC-2 chloride channel orthologs

ClC-2 is a Cl− channel that belongs to the CLC family of chloride channel/transport proteins. ClC-2 molecular role is not clear, and Clcn2 knockout mice develop blindness, sterility, and leukodystrophy by unknown reasons. ClC-2 is associated in the brain with the adhesion molecule GlialCAM, which is defective in a type of leukodystrophy, involving ClC-2 in the homeostasis of myelin. To get more insight into the functions of ClC-2, we have identified in this work the three ClC-2 orthologs in zebrafish. clcn2a and clcn2b resulted from the teleost-specific whole genome duplication, while clcn2c arose from a gene duplication from clcn2b. The expression patterns in adult tissues and embryos of zebrafish clcn2 paralogs support their subfunctionalization after the duplications, with clcn2a being enriched in excitable tissues and clcn2c in ionocytes. All three zebrafish clc-2 proteins interact with human GLIALCAM, that is able to target them to cell junctions, as it does with mammalian ClC-2. We could detect clc-2a and clc-2b inward rectified chloride currents with different voltage-dependence and kinetics in Xenopus oocytes, while clc-2c remained inactive. Interestingly, GlialCAM proteins did not modify clc-2b inward rectification. Then, our work extends the repertoire of ClC-2 proteins and provides new tools for structure-function and physiology studies

Estudio sobre un problema de la XXIV Olimpiada Matemática Thales

En el presente artículo nos proponemos analizar uno de los problemas propuestos al alumnado de 2º de E.S.O. que participó en la vigésimo cuarta edición de la fase regional de la Olimpiada Matemática Thales, la cual ha tenido lugar en Jaén en 2008

Shaky life of a water drop in an anise oil-rich environment

This paper is associated with a video winner of a 2018 APS/DFD Milton van Dyke Award for work presented at the DFD Gallery of Fluid Motion. The original video is available online at the Gallery of Fluid Motion, https://doi.org/10.1103/APS.DFD.2018.GFM.V0054This paper is associated with a video winner of a 2018 APS/DFD Milton van Dyke Award for work presented at the DFD Gallery of Fluid Motion.We acknowledge the support of the Spanish FEDER/Ministerio de Ciencia, Innovación y Universidades – Agencia Estatal de Investigación through Grants No. DPI2014-59292-C3-1-P, No. DPI2015-71901-REDT, and No. DPI2017-88201-C3-3-R

Drosophila ClC-a is required in glia of the stem cell niche for proper neurogenesis and wiring of neural circuits

Glial cells form part of the neural stem cell niche and express a wide variety of ion channels; however, the contribution of these channels to nervous system develop ment is poorly understood. We explored the function of the Drosophila ClC-a chloride channel, since its mammalian ortholog CLCN2 is expressed in glial cells, and defective channel function results in leukodystrophies, which in humans are accompanied by cognitive impairment. We found that ClC-a was expressed in the niche in cortex glia, which are closely associated with neurogenic tissues. Characterization of loss-of function ClC-a mutants revealed that these animals had smaller brains and widespread wiring defects. We showed that ClC-a is required in cortex glia for neurogenesis in neuroepithelia and neuroblasts, and identified defects in a neuroblast lineage that generates guidepost glial cells essential for photoreceptor axon guidance. We propose that glia-mediated ionic homeostasis could nonautonomously affect neurogenesis, and consequently, the correct assembly of neural circuits

Reduced current density and surface expression of a CLCN1 mutation causing dominant or recessive myotonia in Costa Rica

Mutations in CLCN1 coding for the muscle ClC-1 Cl- channel lead to dominant (Thomsen's disease) of recessive (Becker's disease) myotonia. Here we characterized several CLCN1 mutations identified in Costa Rica myotonia patients: Q412P (Morales 2008. RevBiolTrop 56:1), R105C, Q154R, and F167L (R105C and F167L have been described earlier in German families (Meyer-Kleine 1995. AmJHumGenet57:1325, Zhang 2000. Neurology54:937). In different families, the Q412P mutation was found to be inherited both in recessive and dominant fashion. We studied the Cl- currents generated by these mutants in Xenopus oocytes using electrophysiological techniques, and for the Q412P mutant also surface membrane expression. Assaying for fast and common gating properties and single channel conductance, we found that none of the mutations exhibited significant alterations of gating parameters or conductance. In fact, mutations R105C, Q154R, and F167L were indistinguishable from WT ClC-1 (in agreement with earlier studies for R105C and F167L (Desaphy 2013, ExpNeurol 248:530). However, mutation Q412P displayed a dramatically reduced current density and a reduced surface expression. However, mimicking the heterozygous status of patients by co-injecting WT and mutant RNA in a 1:1 ratio did not reveal a significant reduction of current density compared to the injection of the half amount of WT. Our results indicate that Q412P does not exert a dominant negative effect on WT and that the pathophysiology of the R105C, Q154R, and F167L mutations is not due to reduced open probability or single channel current, but that these mutations must affect mechanisms that are not well reproduced in a non-skeletal muscle expression systems. The lack of dominance of Q412P might be due to early protein degradation (before being able to interact with WT subunits) or to a silent phenotype in WT/Q412P heteromers.Telethon Italy (grant GGP 12008 to M.P.), the Jepa Limmatfoundation, the Compagnia San Paolo (M.P.); SAF2012-31486, 2012 SGR719 and ICREA Academia Prize (RE)Universidad de Costa Rica/[742-B4-318]/UCR/Costa RicaConsejo Nacional para Investigaciones Científicas y Tecnológicas/[]/CONICIT/Costa RicaMinisterio de Ciencia, Innovación, Tecnología y Telecomunicaciones/[]/MICITT/Costa RicaTelethon Italia/[GGP 12008]/ItaliaJepa-Limmat Foundation/[]//ItaliaCompagnia di San Paolo/[]//ItaliaCompagnia di San Paolo/[SAF2012-31486]//ItaliaCompagnia di San Paolo/[2012 SGR719]//ItaliaICREA Acadèmia Prize/[]/EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto de Investigaciones en Salud (INISA)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicin

Structural basis for the dominant or recessive character of GLIALCAM mutations found in leukodystrophies

Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is a type of leukodystrophy characterized by white matter edema, and it is caused mainly by recessive mutations in MLC1 and GLIALCAM genes. These variants are called MLC1 and MLC2A with both types of patients sharing the same clinical phenotype. In addition, dominant mutations in GLIALCAM have also been identified in a subtype of MLC patients with a remitting phenotype. This variant has been named MLC2B. GLIALCAM encodes for an adhesion protein containing two immunoglobulin (Ig) domains and it is needed for MLC1 targeting to astrocyte-astrocyte junctions. Most mutations identified in GLIALCAM abolish GlialCAM targeting to junctions. However, it is unclear why some mutations behave as recessive or dominant. Here, we used a combination of biochemistry methods with a new developed anti-GlialCAM nanobody, double-mutants and cysteine cross-links experiments, together with computer docking, to create a structural model of GlialCAM homo-interactions. Using this model, we suggest that dominant mutations affect different GlialCAM-GlialCAM interacting surfaces in the first Ig domain, which can occur between GlialCAM molecules present in the same cell (cis) or present in neighbouring cells (trans). Our results provide a framework that can be used to understand the molecular basis of pathogenesis of all identified GLIALCAM mutations

Effects of Adding an Online Exercise Program on Physical Function in Individuals Hospitalized by COVID-19: A Randomized Controlled Trial

The worldwide pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus has impacted all healthcare systems. One potential sequela experienced by hospitalized coronavirus disease 2019 (COVID-19) survivors includes muscle weakness with a reduction in strength and, consequently, a possible increase in frailty. The aim of this clinical trial was to evaluate the efficacy of adding an online therapeutic exercise program for 8 weeks to the medical prescriptions on functional variables in patients hospitalized due to COVID-19. A randomized controlled trial including 70 previously hospitalized COVID-19 survivors was conducted. Patients were randomly allocated to an experimental (n = 35) or control (n = 35) group. Both groups received regular prescriptions provided by their medical doctors. The experimental group also received a live online therapeutic exercise program for 8 weeks (3 sessions/week). Handgrip strength, gait speed, lower-extremity strength, balance, and frailty were assessed at baseline, at the end of the program, and one month after the end of the intervention. The repeated measures analysis of variance revealed significant Group*Time interactions for all the outcomes: (handgrip dominant: F = 17.395, p < 0.001, η2 = 0.24; handgrip non-dominant: F = 33.197, p < 0.001, η2 = 0.33; 4 m walk test (4WT): F = 13.039, p = 0.001, η2 = 0.16; short physical performance battery (SPPB): F = 26.421, p < 0.001, η2 = 0.28; the five chair-raise test (5CRT): F = 5.628, p = 0.004, η2 = 0.08; FRAIL scale: F = 11.249, p = 0.001, η2 = 0.14): patients in the experimental group experienced greater improvements in all outcomes than those assigned to the control group. This study revealed that the addition of an online exercise program for 8 weeks obtained greater improvements in handgrip strength, gait speed, lower-extremity strength, balance, and frailty in a sample of previously hospitalized COVID-19 survivors than application of just usual medical prescription