Differential Regulation of Hemichannels and Gap Junction Channels by RhoA GTPase and Actin Cytoskeleton: A Comparative Analysis of Cx43 and Cx26.

Connexins (Cxs) are transmembrane proteins that assemble into gap junction channels (GJCs) and hemichannels (HCs). Previous researches support the involvement of Rho GTPases and actin microfilaments in the trafficking of Cxs, formation of GJCs plaques, and regulation of channel activity. Nonetheless, it remains uncertain whether distinct types of Cxs HCs and GJCs respond differently to Rho GTPases or changes in actin polymerization/depolymerization dynamics. Our investigation revealed that inhibiting RhoA, a small GTPase that controls actin polymerization, or disrupting actin microfilaments with cytochalasin B (Cyto-B), resulted in reduced GJCs plaque size at appositional membranes and increased transport of HCs to non-appositional plasma membrane regions. Notably, these effects were consistent across different Cx types, since Cx26 and Cx43 exhibited similar responses, despite having distinct trafficking routes to the plasma membrane. Functional assessments showed that RhoA inhibition and actin depolymerization decreased the activity of Cx43 GJCs while significantly increasing HC activity. However, the functional status of GJCs and HCs composed of Cx26 remained unaffected. These results support the hypothesis that RhoA, through its control of the actin cytoskeleton, facilitates the transport of HCs to appositional cell membranes for GJCs formation while simultaneously limiting the positioning of free HCs at non-appositional cell membranes, independently of Cx type. This dynamic regulation promotes intercellular communications and reduces non-selective plasma membrane permeability through a Cx-type dependent mechanism, whereby the activity of Cx43 HCs and GJCs are differentially affected but Cx26 channels remain unchanged.

Corrigendum: Contribution of Connexin Hemichannels to the Decreases in Cell Viability Induced by Linoleic Acid in the Human Lens Epithelial Cells (HLE-B3).

[This corrects the article DOI: 10.3389/fphys.2019.01574.].

Influence of distance, area, and cultural context in active commuting: Continental and insular children.

Commuting by walking or cycling is a way to increase physical activity levels. The objective of this article was to determine the modes of commuting to school and the distance and time of the way to school among children from Easter Island and from the mainland (Valparaíso), in Chile. A total of 666 children and adolescents aged 10 to 18 years old (208 from Easter Island and 458 from Valparaíso) participated and completed a valid questionnaire including data about age, gender, usual commuting mode to and from school, distance, and travel time. There are important differences in the mode of commuting between students of Valparaíso and Easter Island. Private transport is more commonly used in Valparaíso than in Easter Island (p<0.001). Furthermore, it was observed that cycling and public transportation are not used as mode of commuting in Valparaíso and Easter Island respectively. Students from Easter Island, who travel more distance and during more time, are more active than students from Valparaíso (going 24.8% and 17.6%; from: 61% and 28.8% respectively). This situation is influenced by the geographic context of the island, the distances from home to school, and the type of commuting, which fosters the level of active commuting. On the other hand, the passive modes of commuting to school are higher in the mainland urban setting of Valparaíso. It is necessary to study the diverse contexts of the Easter Island population, but, for now, the rural setting of Easter Island seems to be associated with a greater level of active commuting to school.

Contribution of Connexin Hemichannels to the Decreases in Cell Viability Induced by Linoleic Acid in the Human Lens Epithelial Cells (HLE-B3).

Connexin (Cx) proteins form gap junction channels (GJC) and hemichannels that a allow bidirectional flow of ions and metabolites between the cytoplasm and extracellular space, respectively. Under physiological conditions, hemichannels have a very low probability of opening, but in certain pathologies, hemichannels activity can increase and induce and/or accelerate cell death. Several mechanisms control hemichannels activity, including phosphorylation and oxidation (i.e., S-nitrosylation). Recently, the effect of polyunsaturated fatty acids (PUFAs) such as linoleic acid (LA), were found to modulate Cxs. It has been seen that LA increase cell death in bovine and human lens cells. The lens is a structure allocated in the eye that highly depends on Cx for the metabolic coupling between its cells, a condition necessary for its transparency. Therefore, we hypothesized that LA induces lens cells death by modulating hemichannel activity. In this work, we characterized the effect of LA on hemichannel activity and survival of HLE-B3 cells (a human lens epithelial cell line). We found that HLE-B3 cells expresses Cx43, Cx46, and Cx50 and can form functional hemichannels in their plasma membrane. The extracellular exposure to 10-50 µM of LA increases hemichannels activity (dye uptake) in a concentration-dependent manner, which was reduced by Cx-channel blockers, such as the Cx-mimetic peptide Gap27 and TATGap19, La3+, carbenoxolone (CBX) and the Akt kinase inhibitor. Additionally, LA increases intracellular calcium, which is attenuated in the presence of TATGap19, a specific Cx43-hemichannel inhibitor. Finally, the long exposure of HLE-B3 cells to LA 20 and 50 µM, reduced cell viability, which was prevented by CBX. Moreover, LA increased the proportion of apoptotic HLE-B3 cells, effect that was prevented by the Cx-mimetic peptide TAT-Gap19 but not by Akt inhibitor. Altogether, these findings strongly suggest a contribution of hemichannels opening in the cell death induced by LA in HLE-B3 cells. These cells can be an excellent tool to develop pharmacological studies in vitro.

Connexinopathies: a structural and functional glimpse.

Mutations in human connexin (Cx) genes have been related to diseases, which we termed connexinopathies. Such hereditary disorders include nonsyndromic or syndromic deafness (Cx26, Cx30), Charcot Marie Tooth disease (Cx32), occulodentodigital dysplasia and cardiopathies (Cx43), and cataracts (Cx46, Cx50). Despite the clinical phenotypes of connexinopathies have been well documented, their pathogenic molecular determinants remain elusive. The purpose of this work is to identify common/uncommon patterns in channels function among Cx mutations linked to human diseases. To this end, we compiled and discussed the effect of mutations associated to Cx26, Cx32, Cx43, and Cx50 over gap junction channels and hemichannels, highlighting the function of the structural channel domains in which mutations are located and their possible role affecting oligomerization, gating and perm/selectivity processes.

From Hyperactive Connexin26 Hemichannels to Impairments in Epidermal Calcium Gradient and Permeability Barrier in the Keratitis-Ichthyosis-Deafness Syndrome.

The keratitis-ichthyosis-deafness (KID) syndrome is characterized by corneal, skin, and hearing abnormalities. KID has been linked to heterozygous dominant missense mutations in the GJB2 and GJB6 genes, encoding connexin26 and 30, respectively. In vitro evidence indicates that KID mutations lead to hyperactive (open) hemichannels, which in some cases is accompanied by abnormal function of gap junction channels. Transgenic mouse models expressing connexin26 KID mutations reproduce human phenotypes and present impaired epidermal calcium homeostasis and abnormal lipid composition of the stratum corneum affecting the water barrier. Here we have compiled relevant data regarding the KID syndrome and propose a mechanism for the epidermal aspects of the disease.

Keratitis-ichthyosis-deafness syndrome-associated Cx26 mutants produce nonfunctional gap junctions but hyperactive hemichannels when co-expressed with wild type Cx43.

Mutations in Cx26 gene are found in most cases of human genetic deafness. Some mutations produce syndromic deafness associated with skin disorders, like the Keratitis-Ichthyosis-Deafness syndrome (KID). Because in the human skin connexin 26 (Cx26) is co-expressed with other connexins, like Cx43 and Cx30, and as the KID syndrome is inherited as autosomal dominant condition, it is possible that KID mutations change the way Cx26 interacts with other co-expressed connexins. Indeed, some Cx26 syndromic mutations showed gap junction dominant negative effect when co-expressed with wild-type connexins, including Cx26 and Cx43. The nature of these interactions and the consequences on hemichannels and gap junction channel (GJC) functions remain unknown. In this study, we demonstrate that syndromic mutations, at the N terminus segment of Cx26, change connexin oligomerization compatibility, allowing aberrant interactions with Cx43. Strikingly, heteromeric oligomer formed by Cx43/Cx26 (syndromic mutants) shows exacerbated hemichannel activity but nonfunctional GJCs; this also occurs for those Cx26 KID mutants that do not show functional homomeric hemichannels. Heterologous expression of these hyperactive heteromeric hemichannels increases cell membrane permeability, favoring ATP release and Ca(2+) overload. The functional paradox produced by oligomerization of Cx43 and Cx26 KID mutants could underlie the severe syndromic phenotype in human skin.

Extracellular gentamicin reduces the activity of connexin hemichannels and interferes with purinergic Ca(2+) signaling in HeLa cells.

Gap junction channels (GJCs) and hemichannels (HCs) are composed of protein subunits termed connexins (Cxs) and are permeable to ions and small molecules. In most organs, GJCs communicate the cytoplasm of adjacent cells, while HCs communicate the intra and extracellular compartments. In this way, both channel types coordinate physiological responses of cell communities. Cx mutations explain several genetic diseases, including about 50% of autosomal recessive non-syndromic hearing loss. However, the possible involvement of Cxs in the etiology of acquired hearing loss remains virtually unknown. Factors that induce post-lingual hearing loss are diverse, exposure to gentamicin an aminoglycoside antibiotic, being the most common. Gentamicin has been proposed to block GJCs, but its effect on HCs remains unknown. In this work, the effect of gentamicin on the functional state of HCs was studied and its effect on GJCs was reevaluated in HeLa cells stably transfected with Cxs. We focused on Cx26 because it is the main Cx expressed in the cochlea of mammals where it participates in purinergic signaling pathways. We found that gentamicin applied extracellularly reduces the activity of HCs, while dye transfer across GJCs was not affected. HCs were also blocked by streptomycin, another aminoglycoside antibiotic. Gentamicin also reduced the adenosine triphosphate release and the HC-dependent oscillations of cytosolic free-Ca(2+) signal. Moreover, gentamicin drastically reduced the Cx26 HC-mediated membrane currents in Xenopus laevis oocytes. Therefore, the extracellular gentamicin-induced inhibition of Cx HCs may adversely affect autocrine and paracrine signaling, including the purinergic one, which might partially explain its ototoxic effects.

Linoleic acid induces opening of connexin26 hemichannels through a PI3K/Akt/Ca(2+)-dependent pathway.

Connexin hemichannel (Cx HC) opening is involved in physiological and pathological processes, allowing the cellular release of autocrine/paracrine signaling molecules. Linoleic acid (LA) is known to modulate the functional state of connexin46 (Cx46) HCs. However, the molecular mechanism involved in this effect, or whether LA affects HCs constituted of other connexins, remains unknown. Here, we report the effects of LA on HCs in HeLa cells that express Cx26, one of the main Cxs in the cochlear sensory epithelium. Cx26 HC activity (dye uptake) was increased in a concentration-dependent manner by bath application of LA and inhibited by HC blockers. Moreover, intracellular BAPTA, a Ca(2+) chelator, and PI3K/AKT inhibitors were found to reduce the LA-induced Cx26 HC opening, suggesting that the LA effect is mediated by an increase of free intracellular Ca(2+) concentration and activation of the PI3K/Akt-dependent pathway. The LA-induced increase in free intracellular Ca(2+) concentration was mainly due to Ca(2+) influx through Cx26 HCs. In addition, the involvement of SH groups was ruled out, because dithiothreitol (DTT) did not block the LA-induced dye uptake. LA also increased the membrane current mediated by Cx26 HCs expressed in Xenopus oocytes and the dye uptake in HeLa cells expressing Cxs 32, 43 or 45. Since LA is an essential polyunsaturated fatty acid, its effect on HCs might be relevant to cell growth as well as to cellular functions of differentiated cells such as audition.

Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function.

To identify motifs involved in oligomerization of the gap junction protein Cx26, we studied individual transmembrane (TM) domains and the full-length protein. Using the TOXCAT assay for interactions of isolated TM α-helices, we found that TM1, a Cx26 pore domain, had a strong propensity to homodimerize. We identified amino acids Val-37-Ala-40 (VVAA) as the TM1 motif required for homodimerization. Two deafness-associated Cx26 mutations localized in this region, Cx26V37I and Cx26A40G, differentially affected dimerization. TM1-V37I dimerized only weakly, whereas TM1-A40G did not dimerize. When the full-length mutants were expressed in HeLa cells, both Cx26V37I and Cx26A40G formed oligomers less efficiently than wild-type Cx26. A Cx26 cysteine substitution mutant, Cx26V37C formed dithiothreitol-sensitive dimers. Substitution mutants of Val-37 formed intercellular channels with reduced function, while mutants of Ala-40 did not form functional gap junction channels. Unlike wild-type Cx26, neither Cx26V37I nor Cx26A40G formed functional hemichannels in low extracellular calcium. Thus the VVAA motif of Cx26 is critical for TM1 dimerization, hexamer formation, and channel function. The differential effects of VVAA mutants on hemichannels and gap junction channels imply that inter-TM interactions can differ in unapposed and docked hemichannels. Moreover, Cx26 oligomerization appears dependent on transient TM1 dimerization as an intermediate step.

Aportes para una Historia de la educación popular en el Perú / History of popular education

Este libro se ha estructurado en tres partes que se interrelacionan y brindan una visión compleja del proceso de la Educación Popular. El primer título "El proceso peruano de la educación popular", ofrece una periodización global sobre el tema, así como una evolución histórica del concepto. El siguiente título sobre "Los centros en el proceso peruano de la educación popular", son reflexiones que plantean miradas históricas hacia la educación popular pero muy en función del rol de las organizacines no gubernamentales. El tercer título sobre "El proceso latinoamericano de la educación popular", nos permite un contexto más amplio para comprender las diversas influencias y diferencias del proceso latinoamericano y peruano

Educacion popular y cultura popular

Contiene: Educacion popular y cultura popular - "Cultura del pueblo" y "cultura polpular" - La "cultura del pueblo": expresion de dominacion - La "cultura popular": expresion de resistencia de ofensiva - La "recuperacion critica y devolucion sistematica" de la cultura popular - La estrategia, la tectica y las armas de la educacion popular - La tecnicas de comunicacion y analisis: armas de la educacion popular - Los metodos de trabajo: practicas de la educacion popular - La autoformacion: objetivo tactico de estrategia educativa - Algunas consideraciones sobre la elaboracion de material didactico en la educacion popular.