Unnexin is a protein subunit of a large-pore channel expressed by unicellular organisms.

Cells of vertebrate and invertebrate organisms express proteins specialized in membrane channel-based cell-cell communication that are absent in unicellular organisms. We recently described the prediction of some members of the large-pore channel family in kinetoplastids, consisting of proteins called unnexins, which share several structural features with innexin and pannexin proteins. Here, we demonstrated that the unnexin1 protein (Unx1) is delivered to the cell membrane, displaying a topology consisting of four transmembrane domains with C and N termini on the cytoplasmic side and form large-pore channels that are permeable to small molecules. Low extracellular Ca2+/Mg2+ levels or extracellular alkalinization, but not mechanical stretching, increases channel activity. The Unx1 channel mediates the influx of Ca2+ and does not form intercellular dye coupling between HeLa Unx1 transfected cells. Unx1 channel function was further evidenced by its ability to mediate ionic currents when expressed in Xenopus oocytes. Downregulation of Unx1 mRNA with morpholine contains Trypanosoma cruzi invasion. Phylogenetic analysis revealed the presence of Unx1 homologs in other protozoan parasites, suggesting a conserved function for these channel parasites in other protists. Our data demonstrate that Unx1 forms large-pore membrane channels, which may serve as a diffusional pathway for ions and small molecules that are likely to be metabolic substrates or waste products, and signaling autocrine and paracrine molecules that could be involved in cell invasion. As morpholinos-induced downregulation of Unx1 reduces the infectivity of trypomastigotes, the Unx1 channels might be an attractive target for developing trypanocide drugs.

Connexin channels and hemichannels are modulated differently by charge reversal at residues forming the intracellular pocket.

BACKGROUND: Members of the ß-subfamily of connexins contain an intracellular pocket surrounded by amino acid residues from the four transmembrane helices. The presence of this pocket has not previously been investigated in members of the α-, γ-, δ-, and ε-subfamilies. We studied connexin50 (Cx50) as a representative of the α-subfamily, because its structure has been determined and mutations of Cx50 are among the most common genetic causes of congenital cataracts. METHODS: To investigate the presence and function of the intracellular pocket in Cx50 we used molecular dynamics simulation, site-directed mutagenesis, gap junction tracer intercellular transfer, and hemichannel activity detected by electrophysiology and by permeation of charged molecules. RESULTS: Employing molecular dynamics, we determined the presence of the intracellular pocket in Cx50 hemichannels and identified the amino acids participating in its formation. We utilized site-directed mutagenesis to alter a salt-bridge interaction that supports the intracellular pocket and occurs between two residues highly conserved in the connexin family, R33 and E162. Substitution of opposite charges at either position decreased formation of gap junctional plaques and cell-cell communication and modestly reduced hemichannel currents. Simultaneous charge reversal at these positions produced plaque-forming non-functional gap junction channels with highly active hemichannels. CONCLUSIONS: These results show that interactions within the intracellular pocket influence both gap junction channel and hemichannel functions. Disruption of these interactions may be responsible for diseases associated with mutations at these positions.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII.

Pannexin1 (Panx1) channels are ubiquitously expressed in vertebrate cells and are widely accepted as adenosine triphosphate (ATP)-releasing membrane channels. Activation of Panx1 has been associated with phosphorylation in a specific tyrosine residue or cleavage of its C-terminal domains. In the present work, we identified a residue (S394) as a putative phosphorylation site by Ca2+/calmodulin-dependent kinase II (CaMKII). In HeLa cells transfected with rat Panx1 (rPanx1), membrane stretch (MS)-induced activation-measured by changes in DAPI uptake rate-was drastically reduced by either knockdown of Piezo1 or pharmacological inhibition of calmodulin or CaMKII. By site-directed mutagenesis we generated rPanx1S394A-EGFP (enhanced green fluorescent protein), which lost its sensitivity to MS, and rPanx1S394D-EGFP, mimicking phosphorylation, which shows high DAPI uptake rate without MS stimulation or cleavage of the C terminus. Using whole-cell patch-clamp and outside-out excised patch configurations, we found that rPanx1-EGFP and rPanx1S394D-EGFP channels showed current at all voltages between ±100 mV, similar single channel currents with outward rectification, and unitary conductance (∼30 to 70 pS). However, using cell-attached configuration we found that rPanx1S394D-EGFP channels show increased spontaneous unitary events independent of MS stimulation. In silico studies revealed that phosphorylation of S394 caused conformational changes in the selectivity filter and increased the average volume of lateral tunnels, allowing ATP to be released via these conduits and DAPI uptake directly from the channel mouth to the cytoplasmic space. These results could explain one possible mechanism for activation of rPanx1 upon increase in cytoplasmic Ca2+ signal elicited by diverse physiological conditions in which the C-terminal domain is not cleaved.

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.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

The interplay between α7 nicotinic acetylcholine receptors, pannexin-1 channels and P2X7 receptors elicit exocytosis in chromaffin cells.

Pannexin-1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1-mediated dye uptake and ATP release in the neuroblastoma cell line SH-SY5Y-α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH-SY5Y-α7 cells expressing a membrane-tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR-Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

An Update on Connexin Gap Junction and Hemichannels in Diabetic Retinopathy.

Diabetic retinopathy (DR) is one of the main causes of vision loss in the working age population. It is characterized by a progressive deterioration of the retinal microvasculature, caused by long-term metabolic alterations inherent to diabetes, leading to a progressive loss of retinal integrity and function. The mammalian retina presents an orderly layered structure that executes initial but complex visual processing and analysis. Gap junction channels (GJC) forming electrical synapses are present in each retinal layer and contribute to the communication between different cell types. In addition, connexin hemichannels (HCs) have emerged as relevant players that influence diverse physiological and pathological processes in the retina. This article highlights the impact of diabetic conditions on GJC and HCs physiology and their involvement in DR pathogenesis. Microvascular damage and concomitant loss of endothelial cells and pericytes are related to alterations in gap junction intercellular communication (GJIC) and decreased connexin 43 (Cx43) expression. On the other hand, it has been shown that the expression and activity of HCs are upregulated in DR, becoming a key element in the establishment of proinflammatory conditions that emerge during hyperglycemia. Hence, novel connexin HCs blockers or drugs to enhance GJIC are promising tools for the development of pharmacological interventions for diabetic retinopathy, and initial in vitro and in vivo studies have shown favorable results in this regard.

Restraint of Human Skin Fibroblast Motility, Migration, and Cell Surface Actin Dynamics, by Pannexin 1 and P2X7 Receptor Signaling.

Wound healing is a dynamic process required to maintain skin integrity and which relies on the precise migration of different cell types. A key molecule that regulates this process is ATP. However, the mechanisms involved in extracellular ATP management are poorly understood, particularly in the human dermis. Here, we explore the role, in human fibroblast migration during wound healing, of Pannexin 1 channels and their relationship with purinergic signals and in vivo cell surface filamentous actin dynamics. Using siRNA against Panx isoforms and different Panx1 channel inhibitors, we demonstrate in cultured human dermal fibroblasts that the absence or inhibition of Panx1 channels accelerates cell migration, increases single-cell motility, and promotes actin redistribution. These changes occur through a mechanism that involves the release of ATP to the extracellular space through a Panx1-dependent mechanism and the activation of the purinergic receptor P2X7. Together, these findings point to a pivotal role of Panx1 channels in skin fibroblast migration and suggest that these channels could be a useful pharmacological target to promote damaged skin healing.

Stretch-Induced Activation of Pannexin 1 Channels Can Be Prevented by PKA-Dependent Phosphorylation.

Pannexin 1 channels located in the cell membrane are permeable to ions, metabolites, and signaling molecules. While the activity of these channels is known to be modulated by phosphorylation on T198, T308, and S206, the possible involvement of other putative phosphorylation sites remains unknown. Here, we describe that the activity of Panx1 channels induced by mechanical stretch is reduced by adenosine via a PKA-dependent pathway. The mechanical stretch-induced activity-measured by changes in DAPI uptake-of Panx1 channels expressed in HeLa cell transfectants was inhibited by adenosine or cAMP analogs that permeate the cell membrane. Moreover, inhibition of PKA but not PKC, p38 MAPK, Akt, or PKG prevented the effects of cAMP analogs, suggesting the involvement of Panx1 phosphorylation by PKA. Accordingly, alanine substitution of T302 or S328, two putative PKA phosphorylation sites, prevented the inhibitory effect of cAMP analogs. Moreover, phosphomimetic mutation of either T302 or S328 to aspartate prevented the mechanical stretch-induced activation of Panx1 channels. A molecular dynamics simulation revealed that T302 and S328 are located in the water-lipid interphase near the lateral tunnel of the intracellular region, suggesting that their phosphorylation could promote conformational changes in lateral tunnels. Thus, Panx1 phosphorylation via PKA could be modulated by G protein-coupled receptors associated with the Gs subunit.

Gap junctions coordinate the propagation of glycogenolysis induced by norepinephrine in the pineal gland.

Chemical and electrical synapses are the two major communication systems that permit cell-to-cell communication within the nervous system. Although most studies are focused on chemical synapses (glutamate, γ-aminobutyric acid, and other neurotransmitters), clearly both types of synapses interact and cooperate to allow the coordination of several cell functions within the nervous system. The pineal gland has limited independent axonal innervation and not every cell has access to nerve terminals. Thus, additional communication systems, such as gap junctions, have been postulated to coordinate metabolism and signaling. Using acutely isolated glands and dissociated cells, we found that gap junctions spread glycogenolytic signals from cells containing adrenoreceptors to the entire gland lacking these receptors. Our data using glycogen and lactate quantification, electrical stimulation, and high-performance liquid chromatography with electrochemical detection, demonstrate that gap junctional communication between cells of the rat pineal gland allows cell-to-cell propagation of norepinephrine-induced signal that promotes glycogenolysis throughout the entire gland. Thus, the interplay of both synapses is essential for coordinating glycogen metabolism and lactate production in the pineal gland.

The Ca2+ channel subunit CaV ß2a-subunit down-regulates voltage-activated ion current densities by disrupting actin-dependent traffic in chromaffin cells.

ß-Subunits of the Ca2+ channel have been conventionally regarded as auxiliary subunits that regulate the expression and activity of the pore-forming α1 subunit. However, they comprise protein-protein interaction domains, such as a SRC homology 3 domain (SH3) domain, which make them potential signaling molecules. Here we evaluated the role of the ß2a subunit of the Ca2+ channels (CaV ß2a) and its SH3 domain (ß2a-SH3) in late stages of channel trafficking in bovine adrenal chromaffin cells. Cultured bovine adrenal chromaffin cells were injected with CaV ß2a or ß2a-SH3 under different conditions, in order to acutely interfere with endogenous associations of these proteins. As assayed by whole-cell patch clamp recordings, Ca2+ currents were reduced by CaV ß2a in the presence of exogenous α1-interaction domain. ß2a-SH3, but not its dimerization-deficient mutant, also reduced Ca2+ currents. Na+ currents were also diminished following ß2a-SH3 injection. Furthermore, ß2a-SH3 was still able to reduce Ca2+ currents when dynamin-2 function was disrupted, but not when SNARE-dependent exocytosis or actin polymerization was inhibited. Together with the additional finding that both CaV ß2a and ß2a-SH3 diminished the incorporation of new actin monomers to cortical actin filaments, ß2a-SH3 emerges as a signaling module that might down-regulate forward trafficking of ion channels by modulating actin dynamics.

ß-catenin aggregation in models of ALS motor neurons: GSK3ß inhibition effect and neuronal differentiation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron death. A 20% of familial ALS cases are associated with mutations in the gene coding for superoxide dismutase 1 (SOD1). The accumulation of abnormal aggregates of different proteins is a common feature in motor neurons of patients and transgenic ALS mice models, which are thought to contribute to disease pathogenesis. Developmental morphogens, such as the Wnt family, regulate numerous features of neuronal physiology in the adult brain and have been implicated in neurodegeneration. ß-catenin is a central mediator of both, Wnt signaling activity and cell-cell interactions. We previously reported that the expression of mutant SOD1 in the NSC34 motor neuron cell line decreases basal Wnt pathway activity, which correlates with cytosolic ß-catenin accumulation and impaired neuronal differentiation. In this work, we aimed a deeper characterization of ß-catenin distribution in models of ALS motor neurons. We observed extensive accumulation of ß-catenin supramolecular structures in motor neuron somas of pre-symptomatic mutant SOD1 mice. In cell-cell appositional zones of NSC34 cells expressing mutant SOD1, ß-catenin displays a reduced co-distribution with E-cadherin accompanied by an increased association with the gap junction protein Connexin-43; these findings correlate with impaired intercellular adhesion and exacerbated cell coupling. Remarkably, pharmacological inhibition of the glycogen synthase kinase-3ß (GSK3ß) in both NSC34 cell lines reverted both, ß-catenin aggregation and the adverse effects of mutant SOD1 expression on neuronal differentiation. Our findings suggest that early defects in ß-catenin distribution could be an underlying factor affecting the onset of neurodegeneration in familial ALS.

Redox-mediated regulation of connexin proteins; focus on nitric oxide.

Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels.

Charged Residues at the First Transmembrane Region Contribute to the Voltage Dependence of the Slow Gate of Connexins.

Connexins (Cxs) are a family of membrane-spanning proteins that form gap junction channels and hemichannels. Connexin-based channels exhibit two distinct voltage-dependent gating mechanisms termed slow and fast gating. Residues located at the C terminus of the first transmembrane segment (TM-1) are important structural components of the slow gate. Here, we determined the role of the charged residues at the end of TM-1 in voltage sensing in Cx26, Cx46, and Cx50. Conductance/voltage curves obtained from tail currents together with kinetics analysis reveal that the fast and slow gates of Cx26 involves the movement of two and four charges across the electric field, respectively. Primary sequence alignment of different Cxs shows the presence of well conserved glutamate residues in the C terminus of TM-1; only Cx26 contains a lysine in that position (lysine 41). Neutralization of lysine 41 in Cx26 increases the voltage dependence of the slow gate. Swapping of lysine 41 with glutamate 42 maintains the voltage dependence. In Cx46, neutralization of negative charges or addition of a positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50, the addition of a glutamate in the same region decreased the voltage dependence, and the neutralization of a negative charge increased it. These results indicate that the charges at the end of TM-1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage-dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity.

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.

Carbon monoxide: A new player in the redox regulation of connexin hemichannels.

Carbon monoxide (CO) is a gaseous transmitter that is known to be involved in several physiological processes, but surprisingly it is also becoming a promising molecule to treat several pathologies including stroke and cancer. CO can cross the plasma membrane and activate guanylate cyclase, increasing the cGMP concentration and activating some kinases, including PKG. The other mechanism of action involves induction of protein carbonylation. CO is known to directly and indirectly modulate the function of ion channels at the plasma membrane, which in turn have important repercussions in the cellular behavior. One group of these channels is hemichannels, which are formed by proteins known as connexins (Cxs). Hemichannel allows not only the flow of ions through their pore but also the release of molecules such as ATP and glutamate. Therefore, their modulation not only impacts cellular function but also cellular communication, having the capability to affect tissular behavior. Here, we review the most recent results regarding the effect of CO on Cx hemichannels and their possible repercussions on pathologies.

Characterization of a novel water pocket inside the human Cx26 hemichannel structure.

Connexins (Cxs) are a family of vertebrate proteins constituents of gap junction channels (GJCs) that connect the cytoplasm of adjacent cells by the end-to-end docking of two Cx hemichannels. The intercellular transfer through GJCs occurs by passive diffusion allowing the exchange of water, ions, and small molecules. Despite the broad interest to understand, at the molecular level, the functional state of Cx-based channels, there are still many unanswered questions regarding structure-function relationships, perm-selectivity, and gating mechanisms. In particular, the ordering, structure, and dynamics of water inside Cx GJCs and hemichannels remains largely unexplored. In this work, we describe the identification and characterization of a believed novel water pocket-termed the IC pocket-located in-between the four transmembrane helices of each human Cx26 (hCx26) monomer at the intracellular (IC) side. Using molecular dynamics (MD) simulations to characterize hCx26 internal water structure and dynamics, six IC pockets were identified per hemichannel. A detailed characterization of the dynamics and ordering of water including conformational variability of residues forming the IC pockets, together with multiple sequence alignments, allowed us to propose a functional role for this cavity. An in vitro assessment of tracer uptake suggests that the IC pocket residue Arg-143 plays an essential role on the modulation of the hCx26 hemichannel permeability.

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.

Cx40 Levels Regulate Hypoxia-Induced Changes in the Migration, Proliferation, and Formation of Gap Junction Plaques in an Extravillous Trophoblast Cell Model.

BACKGROUND: Extravillous trophoblasts (EVTs) form stratified columns at the placenta-uterus interface. In the closest part to fetal structures, EVTs have a proliferative phenotype, whereas in the closest part to maternal structures, they present a migratory phenotype. During the placentation process, Connexin 40 (Cx40) participates in both the proliferation and migration of EVTs, which occurs under hypoxia. However, a possible interaction between hypoxia and Cx40 has not yet been established. METHODS: We developed two cellular models, one with "low Cx40" (Jeg-3), which reflected the expression of this protein found in migratory EVTs, and one with "high Cx40" (Jeg-3/hCx40), which reflected the expression of this protein in proliferative cells. We analyzed the migration and proliferation of these cells under normoxic and hypoxic conditions for 24 h. Jeg-3 cells under hypoxia increased their migratory capacity over their proliferative capacity. However, in Jeg-3/hCx40, the opposite effect was induced. On the other hand, hypoxia promoted gap junction (GJ) plaque formation between neighboring Jeg-3 cells. Similarly, the activation of a nitro oxide (NO)/cGMP/PKG-dependent pathway induced an increase in GJ-plaque formation in Jeg-3 cells. CONCLUSIONS: The expression patterns of Cx40 play a crucial role in shaping the responses of EVTs to hypoxia, thereby influencing their migratory or proliferative phenotype. Simultaneously, hypoxia triggers an increase in Cx40 gap junction (GJ) plaque formation through a pathway dependent on NO.

Correlative light and X-ray tomography jointly unveil the critical role of connexin43 channels on inflammation-induced cellular ultrastructural alterations.

Non-junctional connexin43 (Cx43) plasma membrane hemichannels have been implicated in several inflammatory diseases, particularly playing a role in ATP release that triggers activation of the inflammasome. Therapies targeting the blocking of the hemichannels to prevent the pathological release or uptake of ions and signalling molecules through its pores are of therapeutic interest. To date, there is no close-to-native, high-definition documentation of the impact of Cx43 hemichannel-mediated inflammation on cellular ultrastructure, neither is there a robust account of the ultrastructural changes that occur following treatment with selective Cx43 hemichannel blockers such as Xentry-Gap19 (XG19). A combination of same-sample correlative high-resolution three-dimensional fluorescence microscopy and soft X-ray tomography at cryogenic temperatures, enabled in the identification of novel 3D molecular interactions within the cellular milieu when comparing behaviour in healthy states and during the early onset or late stages under inflammatory conditions. Notably, our findings suggest that XG19 blockage of connexin hemichannels under pro-inflammatory conditions may be crucial in preventing the direct degradation of connexosomes by lysosomes, without affecting connexin protein translation and trafficking. We also delineated fine and gross cellular phenotypes, characteristic of inflammatory insult or road-to-recovery from inflammation, where XG19 could indirectly prevent and reverse inflammatory cytokine-induced mitochondrial swelling and cellular hypertrophy through its action on Cx43 hemichannels. Our findings suggest that XG19 might have prophylactic and therapeutic effects on the inflammatory response, in line with functional studies.