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.

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.

Autocrine activation of P2X7 receptors mediates catecholamine secretion in chromaffin cells.

BACKGROUND AND PURPOSE: ATP is highly accumulated in secretory vesicles and secreted upon exocytosis from neurons and endocrine cells. In adrenal chromaffin granules, intraluminal ATP reaches concentrations over 100 mM. However, how these large amounts of ATP contribute to exocytosis has not been investigated. EXPERIMENTAL APPROACH: Exocytotic events in bovine and mouse adrenal chromaffin cells were measured with single cell amperometry. Cytosolic Ca2+ measurements were carried out in Fluo-4 loaded cells. Submembrane Ca2+ was examined in PC12 cells transfected with a membrane-tethered Ca2+ indicator Lck-GCaMP3. ATP release was measured using the luciferin/luciferase assay. Knockdown of P2X7 receptors was induced with short interfering RNA (siRNA). Direct Ca2+ influx through this receptor was measured using a P2X7 receptor-GCamp6 construct. KEY RESULTS: ATP induced exocytosis in chromaffin cells, whereas the ectonucleotidase apyrase reduced the release events induced by the nicotinic agonist dimethylphenylpiperazinium (DMPP), high KCl, or ionomycin. The purinergic agonist BzATP also promoted a secretory response that was dependent on extracellular Ca2+. A740003, a P2X7 receptor antagonist, abolished secretory responses of these secretagogues. Exocytosis was also diminished in chromaffin cells when P2X7 receptors were silenced using siRNAs and in cells of P2X7 receptor knockout mice. In PC12 cells, DMPP induced ATP release, triggering Ca2+ influx through P2X7 receptors. Furthermore, BzATP, DMPP, and KCl allowed the formation of submembrane Ca2+ microdomains inhibited by A740003. CONCLUSION AND IMPLICATIONS: Autocrine activation of P2X7 receptors constitutes a crucial feedback system that amplifies the secretion of catecholamines in chromaffin cells by favouring submembrane Ca2+ microdomains.

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.

Pannexin-1 Modulates Inhibitory Transmission and Hippocampal Synaptic Plasticity.

Pannexin-1 (Panx1) hemichannel is a non-selective transmembrane channel that may play important roles in intercellular signaling by allowing the permeation of ions and metabolites, such as ATP. Although recent evidence shows that the Panx1 hemichannel is involved in controlling excitatory synaptic transmission, the role of Panx1 in inhibitory transmission remains unknown. Here, we studied the contribution of Panx1 to the GABAergic synaptic efficacy onto CA1 pyramidal neurons (PyNs) by using patch-clamp recordings and pharmacological approaches in wild-type and Panx1 knock-out (Panx1-KO) mice. We reported that blockage of the Panx1 hemichannel with the mimetic peptide 10Panx1 increases the synaptic level of endocannabinoids (eCB) and the activation of cannabinoid receptors type 1 (CB1Rs), which results in a decrease in hippocampal GABAergic efficacy, shifting excitation/inhibition (E/I) balance toward excitation and facilitating the induction of long-term potentiation. Our finding provides important insight unveiling that Panx1 can strongly influence the overall neuronal excitability and play a key role in shaping synaptic changes affecting the amplitude and direction of plasticity, as well as learning and memory processes.

MATE1 expression in the cochlea and its potential involvement in cisplatin cellular uptake and ototoxicity.

BACKGROUND: Cisplatin appears to enter the cochlear cells through the organic cation transporter 2 (OCT2). There is recent evidence that multidrug and toxin extrusion protein 1 (MATE1) is involved in cisplatin-induced nephrotoxicity. Its presence and role in the ear are unknown. AIMS/OBJECTIVES: Evaluate the presence and localization of MATE1, and determine the localization of OCT2, in the cochlea. Evaluate cisplatin uptake with regard to MATE1 and OCT2 expression. MATERIAL AND METHODS: Murine cochlear explants and paraffin-embedded cochleae were evaluated with immunohistochemistry for OCT2 and MATE1. Explant cultures were also treated with Texas Red cisplatin to determine their cellular uptake. RESULTS: MATE1 is present in the cochlea. Most intense labeling of MATE1 and OCT2 was seen in the outer hair cells (OHCs) and pillar cells, respectively. Both transporters were observed in the spiral ganglion neurons and stria vascularis. Expression levels of OCT2 and MATE1 decreased following cisplatin exposure. Texas Red cisplatin staining was strong in OHCs and pillar cells. CONCLUSIONS AND SIGNIFICANCE: To the best of our knowledge, this is the first study demonstrating the presence and localization of MATE1 in the cochlea. OCT2 labeling was seen in pillar cells. Consistently, OHCs and pillar cells uptake Texas Red cisplatin.

The Long-Term Pannexin 1 Ablation Produces Structural and Functional Modifications in Hippocampal Neurons.

Enhanced activity and overexpression of Pannexin 1 (Panx1) channels contribute to neuronal pathologies such as epilepsy and Alzheimer's disease (AD). The Panx1 channel ablation alters the hippocampus's glutamatergic neurotransmission, synaptic plasticity, and memory flexibility. Nevertheless, Panx1-knockout (Panx1-KO) mice still retain the ability to learn, suggesting that compensatory mechanisms stabilize their neuronal activity. Here, we show that the absence of Panx1 in the adult brain promotes a series of structural and functional modifications in the Panx1-KO hippocampal synapses, preserving spontaneous activity. Compared to the wild-type (WT) condition, the adult hippocampal neurons of Panx1-KO mice exhibit enhanced excitability, a more complex dendritic branching, enhanced spine maturation, and an increased proportion of multiple synaptic contacts. These modifications seem to rely on the actin-cytoskeleton dynamics as an increase in the actin polymerization and an imbalance between the Rac1 and the RhoA GTPase activities were observed in Panx1-KO brain tissues. Our findings highlight a novel interaction between Panx1 channels, actin, and Rho GTPases, which appear to be relevant for synapse stability.

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.

Supporting Cells and Their Potential Roles in Cisplatin-Induced Ototoxicity.

Cisplatin is a known ototoxic chemotherapy drug, causing irreversible hearing loss. Evidence has shown that cisplatin causes inner ear damage as a result of adduct formation, a proinflammatory environment and the generation of reactive oxygen species within the inner ear. The main cochlear targets for cisplatin are commonly known to be the outer hair cells, the stria vascularis and the spiral ganglion neurons. Further evidence has shown that certain transporters can mediate cisplatin influx into the inner ear cells including organic cation transporter 2 (OCT2) and the copper transporter Ctr1. However, the expression profiles for these transporters within inner ear cells are not consistent in the literature, and expression of OCT2 and Ctr1 has also been observed in supporting cells. Organ of Corti supporting cells are essential for hair cell activity and survival. Special interest has been devoted to gap junction expression by these cells as certain mutations have been linked to hearing loss. Interestingly, cisplatin appears to affect connexin expression in the inner ear. While investigations regarding cisplatin-induced hearing loss have been focused mainly on the known targets previously mentioned, the role of supporting cells for cisplatin-induced ototoxicity has been overlooked. In this mini review, we discuss the implications of supporting cells expressing OCT2 and Ctr1 as well as the potential role of gap junctions in cisplatin-induced cytotoxicity.

Expression of KID syndromic mutation Cx26S17F produces hyperactive hemichannels in supporting cells of the organ of Corti.

Some mutations in gap junction protein Connexin 26 (Cx26) lead to syndromic deafness, where hearing impairment is associated with skin disease, like in Keratitis Ichthyosis Deafness (KID) syndrome. This condition has been linked to hyperactivity of connexin hemichannels but this has never been demonstrated in cochlear tissue. Moreover, some KID mutants, like Cx26S17F, form hyperactive HCs only when co-expressed with other wild-type connexins. In this work, we evaluated the functional consequences of expressing a KID syndromic mutation, Cx26S17F, in the transgenic mouse cochlea and whether co-expression of Cx26S17F and Cx30 leads to the formation of hyperactive HCs. Indeed, we found that cochlear explants from a constitutive knock-in Cx26S17F mouse or conditional in vitro cochlear expression of Cx26S17F produces hyperactive HCs in supporting cells of the organ of Corti. These conditions also produce loss of hair cells stereocilia. In supporting cells, we found high co-localization between Cx26S17F and Cx30. The functional properties of HCs formed in cells co-expressing Cx26S17F and Cx30 were also studied in oocytes and HeLa cells. Under the recording conditions used in this study Cx26S17F did not form functional HCs and GJCs, but cells co-expressing Cx26S17F and Cx30 present hyperactive HCs insensitive to HCs blockers, Ca2+ and La3+, resulting in more Ca2+ influx and cellular damage. Molecular dynamic analysis of putative heteromeric HC formed by Cx26S17F and Cx30 presents alterations in extracellular Ca2+ binding sites. These results support that in KID syndrome, hyperactive HCs are formed by the interaction between Cx26S17F and Cx30 in supporting cells probably causing damage to hair cells associated to deafness.

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.

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.

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.

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.

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.].

Pannexin-1 Channels Are Essential for Mast Cell Degranulation Triggered During Type I Hypersensitivity Reactions.

Mast cells (MCs) release pro-inflammatory mediators through a process called degranulation response. The latter may be induced by several conditions, including antigen recognition through immunoglobulin E (IgE) or "cross-linking," classically associated with Type I hypersensitivity reactions. Early in this reaction, Ca2+ influx and subsequent increase of intracellular free Ca2+ concentration are essential for MC degranulation. Several membrane channels that mediate Ca2+ influx have been proposed, but their role remains elusive. Here, we evaluated the possible contribution of pannexin-1 channels (Panx1 Chs), well-known as ATP-releasing channels, in the increase of intracellular Ca2+ triggered during cross-linking reaction of MCs. The contribution of Panx1 Chs in the degranulation response was evaluated in MCs from wild type (WT) and Panx1 knock out (Panx1-/-) mice after anti-ovalbumin (OVA) IgE sensitization. Notably, the degranulation response (toluidine blue and histamine release) was absent in Panx1-/- MCs. Moreover, WT MCs showed a rapid and transient increase in Ca2+ signal followed by a sustained increase after antigen stimulation. However, the sustained increase in Ca2+ signal triggered by OVA was absent in Panx1-/- MCs. Furthermore, OVA stimulation increased the membrane permeability assessed by dye uptake, a prevented response by Panx1 Ch but not by connexin hemichannel blockers and without effect on Panx1-/- MCs. Interestingly, the increase in membrane permeability of WT MCs was also prevented by suramin, a P2 purinergic inhibitor, suggesting that Panx1 Chs act as ATP-releasing channels impermeable to Ca2+. Accordingly, stimulation with exogenous ATP restored the degranulation response and sustained increase in Ca2+ signal of OVA stimulated Panx1-/- MCs. Moreover, opening of Panx1 Chs in Panx1 transfected HeLa cells increased dye uptake and ATP release but did not promote Ca2+ influx, confirming that Panx1 Chs permeable to ATP are not permeable to Ca2+. These data strongly suggest that during antigen recognition, Panx1 Chs contribute to the sustained Ca2+ signal increase via release of ATP that activates P2 receptors, playing a critical role in the sequential events that leads to degranulation response during Type I hypersensitivity reactions.

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.

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.

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.