Connexin 43 hemichannels mediate the Ca2+ influx induced by extracellular alkalinization

Although alkaline pH is known to trigger Ca(2+) influx in diverse cells, no pH-sensitive Ca(2+) channel has been identified. Here, we report that extracellular alkalinization induces opening of connexin 43 hemichannels (Cx43 HCs). Increasing extracellular pH from 7.4 to 8.5, in the presence of physiological Ca(2+)/Mg(2+) concentrations, rapidly increased the ethidium uptake rate and open probability of HCs in Cx43 and Cx43EGFP HeLa transfectants (HeLa-Cx3 and HeLa-Cx43EGFP, respectively) but not in parental HeLa cells (HeLa-parental) lacking Cx43 HCs. The increase in ethidium uptake induced by pH 8.5 was not affected by raising the extracellular Ca(2+) concentration from 1.8 to 10 mM but was inhibited by a connexin HC inhibitor (La(3+)). Probenecid, a pannexin HC blocker, had no effect. Extracellular alkalinization increased the intracellular Ca(2+) levels only in cells expressing HCs. The above changes induced by extracellular alkalinization did not change the cellular distribution of Cx43, suggesting that HC activation occurs through a gating mechanism. Experiments on cells expressing a COOH-terminal truncated Cx43 mutant indicated that the effects of alkalinization on intracellular Ca(2+) and ethidium uptake did not depend on the Cx43 C terminus. Moreover, purified dephosphorylated Cx43 HCs reconstituted in liposomes were Ca(2+) permeable, suggesting that Ca(2+) influx through Cx43 HCs could account for the elevation in intracellular Ca(2+) elicited by extracellular alkalinization. These studies identify a membrane pathway for Ca(2+) influx and provide a potential explanation for the activation of cellular events induced by extracellular alkalinization.     

Connexin hemichannel composition determines the FGF-1-induced membrane permeability and free [Ca2+]i responses

Cell surface hemichannels (HCs) composed of different connexin (Cx) types are present in diverse cells and their possible role on FGF-1-induced cellular responses remains unknown. Here, we show that FGF-1 transiently (4-14 h, maximal at 7 h) increases the membrane permeability through HCs in HeLa cells expressing Cx43 or Cx45 under physiological extracellular Ca(2+)/Mg(2+) concentrations. The effect does not occur in HeLa cells expressing HCs constituted of Cx26 or Cx43 with its C-terminus truncated at aa 257, or in parental nontransfected HeLa cells. The increase in membrane permeability is associated with a rise in HC levels at the cell surface and a proportional increase in HC unitary events. The response requires an early intracellular free Ca(2+) concentration increase, activation of a p38 MAP kinase-dependent pathway, and a regulatory site of Cx subunit C-terminus. The FGF-1-induced rise in membrane permeability is also associated with a late increase in intracellular free Ca(2+) concentration, suggesting that responsive HCs allow Ca(2+) influx. The cell density of Cx26 and Cx43 HeLa transfectants cultured in serum-free medium was differentially affected by FGF-1. Thus, the FGF-1-induced cell permeabilization and derived consequences depend on the Cx composition of HCs.     

TRPM5 is critical for linoleic acid-induced CCK secretion from the enteroendocrine cell line, STC-1

Fatty acid-induced stimulation of enteroendocrine cells leads to release of the hormones such as cholecystokinin (CCK) that contribute to satiety. Recently, the fatty acid activated G protein-coupled receptor GPR120 has been shown to mediate long-chain unsaturated free fatty acid-induced CCK release from the enteroendocrine cell line, STC-1, yet the downstream signaling pathway remains unclear. Here we show that linoleic acid (LA) elicits membrane depolarization and an intracellular calcium rise in STC-1 cells and that these responses are significantly reduced when activity of G proteins or phospholipase C is blocked. LA leads to activation of monovalent cation-specific transient receptor potential channel type M5 (TRPM5) in STC-1 cells. LA-induced TRPM5 currents are significantly reduced when expression of TRPM5 or GPR120 is reduced using RNA interference. Furthermore, the LA-induced rise in intracellular calcium and CCK secretion is greatly diminished when expression of TRPM5 channels is reduced using RNA interference, consistent with a role of TRPM5 in LA-induced CCK secretion in STC-1 cells.     

Protein kinase Calpha mediates the effect of antiarrhythmic peptide on gap junction conductance

We investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH2, 50 nM) on pairs of adult guinea pig cardiomyocytes and on pairs of HeLa-cells transfected with rat connexin43 (Cx43). Using double cell voltage clamp technique in cardiomyocytes under control conditions, gap junction conductance (Gj) steadily decreased (by -0.3 to -0.4 nS/min). In contrast, 50 nM AAP10 significantly enhanced Gj (by +0.22 to +0.29 nS/min). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM), and by the protein kinase C (PKC) subtype-specific inhibitors HBDDE (PKCgamma and -alpha) and CGP 54345 (PKCalpha). In HeLa-Cx43 cells we found similar electrophysiological effects of AAP10. For further analysis, we incubated HeLa-Cx43 cells with [32P]orthophosphate (0.05 mCi/ml) for 4 h at 37 degrees C followed by addition of 50 nM AAP10 for 15 min. We found that incorporation of 32P into Cx43 was significantly enhanced in the presence of AAP10, which was completely inhibited in presence of BIM. PKC enzyme-linked immunosorbent assay (ELISA) revealed significant activation of PKC by AAP10 in HeLa-Cx43 cells, which could be inhibited by HBDDE and CGP 54345. Finally, a binding study using [14C]-AAP10 as radioligand was performed. We found a saturable binding of [14C]-AAP10 with a KD of 0.88 nM to cardiac membrane preparations. For assessment of the antiarrhythmic activity in anesthetized rats, we infused aconitine until the occurrence of ventricular fibrillation (VF). The aconitine dose required for initiation of VF was significantly enhanced in the presence of AAP10. In conclusion; AAP10 increases Gj in both adult cardiomyocytes and transfected HeLa-Cx43 cells. AAP10 leads to enhanced phosphorylation of Cx43 via activation of PKCalpha. A membrane receptor exists for antiarrhythmic peptides.     

De novo expression of functional connexins 43 and 45 hemichannels increases sarcolemmal permeability of skeletal myofibers during endotoxemia

Endotoxemia caused by bacterial lipopolysaccharides (LPSs) leads to severe skeletal muscular deterioration, starting with higher membrane permeability and decline in resting membrane potential (RMP). However, the molecular mechanism of such changes remains unclear. Here, we evaluated the possible involvement of connexin43- and connexin45-based hemichannels (Cx43 and Cx45 HCs, respectively) as putative mediators of sarcolemmal dysfunctions induced by LPS in control (Cx43^fl/flCx45^fl/fl) and Cx43/Cx45 expression-deficient (Cx43^fl/flCx45^fl/fl:Myo-Cre) skeletal mice myofibers. At 5 h of endotoxemia, control myofibers presented Cx43 and Cx45 proteins forming functional HCs. Additionally, myofibers from endotoxic control mice showed dye uptake in vivo, which was inhibited by carbenoxolone, a Cx HC blocker. A similar increase in membrane permeability was observed in myofibers freshly isolated from skeletal muscle of mice treated for 5 h with LPS, which was blocked by the Cx HC blocker and was absent in myofibers from mice simultaneously treated with LPS and boldine, which is a Cx HC blocker. The increase in sarcolemmal permeability was mimicked by isolated myofibers treated with pro-inflammatory cytokines (TNF-α and IL-1β) and occurred at 5 h after treatment. Endotoxemia also induced a significant increase in basal intracellular Ca²⁺ signal and a drop in RMP in control myofibers. These two changes were not elicited by myofibers deficient in Cx43/Cx45 expression. Therefore, sarcolemmal dysfunction characterizing endotoxemia is largely explained by the expression of functional Cx43 and Cx45 HCs. Hence, current therapy options for individuals suffering from endotoxic shock could be greatly improved with selective Cx HC inhibitors avoiding the underlying skeletal muscle dysfunction.     

Carbon Monoxide (CO) Is a Novel Inhibitor of Connexin Hemichannels

Hemichannels (HCs) are hexamers of connexins that can form gap-junction channels at points of cell contacts or “free HCs” at non-contacting regions. HCs are involved in paracrine and autocrine cell signaling, and under pathological conditions may induce and/or accelerate cell death. Therefore, studies of HC regulation are of great significance. Nitric oxide affects the activity of Cx43 and Cx46 HCs, whereas carbon monoxide (CO), another gaseous transmitter, modulates the activity of several ion channels, but its effect on HCs has not been explored. We studied the effect of CO donors (CORMs) on Cx46 HCs expressed in Xenopus laevis oocytes using two-electrode voltage clamp and on Cx43 and Cx46 expressed in HeLa cells using a dye-uptake technique. CORM-2 inhibited Cx46 HC currents in a concentration-dependent manner. The C-terminal domain and intracellular Cys were not necessary for the inhibition. The effect of CORM-2 was not prevented by guanylyl-cyclase, protein kinase G, or thioredoxin inhibitors, and was not due to endocytosis of HCs. However, the effect of CORM-2 was reversed by reducing agents that act extracellularly. Additionally, CO inhibited dye uptake of HeLa cells expressing Cx43 or Cx46, and MCF-7 cells, which endogenously express Cx43 and Cx46. Because CORM-2 carbonylates Cx46 in vitro and induces conformational changes, a direct effect of that CO on Cx46 is possible. The inhibition of HCs could help to understand some of the biological actions of CO in physiological and pathological conditions.     

A Potential Role for Cx43-Hemichannels in Staurosporin-Induced Apoptosis

To address the role of gap junction hemichannels in apoptosis, the cell death induced by staurosporine (ST) was evaluated in wild type HeLa cells (HeLa-WT) and transfectants expressing either full-length connexin43 (HeLa-Cx43) or a C-terminal truncation of Cx43 (HeLa-ΔCT). Cell death was measured with fluorescence-activated cell sorting (FACS), both DNA and nuclear fragmentation methods and assays for PARP and caspase 3. The ST-mediated cell death was accelerated in HeLa-Cx43 cells compared to HeLa-WT and HeLa-ΔCT. To determine why HeLa-Cx43 cells were more susceptible to ST, the phosphorylation state and the localization of Cx43 protein within cells were examined using specific Cx43 antibodies. The phosphorylated forms of Cx43 were sharply reduced in HeLa-Cx43 cells treated with ST. Moreover, in ST-treated HeLa-Cx43 cells, Cx43 was mainly observed at the cell surface. In contrast, the truncated form of Cx43 found in HeLa-ΔCT cells, which lacks many of the normal phosphorylation sites, was observed in the cytosol with ST treatment. To examine the hemichannels in the plasma membranes of ST-treated HeLa-Cx43 cells, several dye uptake methods using carboxyfluorescein and propidium iodide were employed. While the number of fluorescent cells did not change in HeLa-WT and HeLa-ΔCT cells with ST treatment, the number of fluorescent HeLa-Cx43 cells increased more than ten-fold. These results indicate that the increases in cell surface Cx43 seen with immunofluorescence and the elevated hemichannel activities detected with dye uptake could help explain the accelerated cell death observed in ST-treated HeLa-Cx43 cells.     

A potential role for cx43-hemichannels in staurosporin-induced apoptosis

To address the role of gap junction hemichannels in apoptosis, the cell death induced by staurosporine (ST) was evaluated in wild type HeLa cells (HeLa-WT) and transfectants expressing either full-length connexin43 (HeLa-Cx43) or a C-terminal truncation of Cx43 (HeLa-DeltaCT). Cell death was measured with fluorescence-activated cell sorting (FACS), both DNA and nuclear fragmentation methods and assays for PARP and caspase 3. The ST-mediated cell death was accelerated in HeLa-Cx43 cells compared to HeLa-WT and HeLa-DeltaCT. To determine why HeLa-Cx43 cells were more susceptible to ST, the phosphorylation state and the localization of Cx43 protein within cells were examined using specific Cx43 antibodies. The phosphorylated forms of Cx43 were sharply reduced in HeLa-Cx43 cells treated with ST. Moreover, in ST-treated HeLa-Cx43 cells, Cx43 was mainly observed at the cell surface. In contrast, the truncated form of Cx43 found in HeLa-DeltaCT cells, which lacks many of the normal phosphorylation sites, was observed in the cytosol with ST treatment. To examine the hemichannels in the plasma membranes of ST-treated HeLa-Cx43 cells, several dye uptake methods using carboxyfluorescein and propidium iodide were employed. While the number of fluorescent cells did not change in HeLa-WT and HeLa-DeltaCT cells with ST treatment, the number of fluorescent HeLa-Cx43 cells increased more than ten-fold. These results indicate that the increases in cell surface Cx43 seen with immunofluorescence and the elevated hemichannel activities detected with dye uptake could help explain the accelerated cell death observed in ST-treated HeLa-Cx43 cells.     

Protein Kinase Cα Mediates the Effect of Antiarrhythmic Peptide on Gap Junction Conductance

We investigated the effects of the antiarrhythmic peptide AAP10 (GAG-4Hyp-PY-CONH₂, 50 nM) on pairs of adult guinea pig cardiomyocytes and on pairs of HeLa-cells transfected with rat connexin43 (Cx43). Using double cell voltage clamp technique in cardiomyocytes under control conditions, gap junction conductance (G_j) steadily decreased (by -0.3 to -0.4 nS/min). In contrast, 50 nM AAP10 significantly enhanced G_j (by +0.22 to +0.29 nS/min). This effect of AAP10 could be significantly antagonized by bisindolylmaleimide I (BIM), and by the protein kinase C (PKC) subtype-specific inhibitors HBDDE (PKCγ and -α) and CGP 54345 (PKCα). In HeLa-Cx43 cells we found similar electrophysiological effects of AAP 10. For further analysis, we incubated HeLa-Cx43 cells with [³²P]orthophosphate (0.05 mCi/ml) for 4 h at 37°C followed by addition of 50 nM AAP10 for 15 min. We found that incorporation of ³²P into Cx43 was significantly enhanced in the presence of AAP 10, which was completely inhibited in presence of BIM. PKC enzyme-linked immunosorbent assay (ELISA) revealed significant activation of PKC by AAP10 in HeLa-Cx43 cells, which could be inhibited by HBDDE and CGP 54345. Finally, a binding study using [¹⁴C]-AAP10 as radioligand was performed. We found a saturable binding of [¹⁴C]-AAP10 with a K₀ of 0.88 nM to cardiac membrane preparations. For assessment of the antiarrhythmic activity in anesthetized rats, we infused aconitine until the occurrence of ventricular fibrillation (VF). The aconitine dose required for initiation of VF was significantly enhanced in the presence of AAP 10. In conclusion; AAP 10 increases G_j in both adult cardiomyocytes and transfected HeLa-Cx43 cells. AAP 10 leads to enhanced phosphorylation of Cx43 via activation of PKCα. A membrane receptor exists for antiarrhythmic peptides.     

A Potential Role for Cx43-Hemichannels in Staurosporin-Induced Apoptosis

To address the role of gap junction hemichannels in apoptosis, the cell death induced by staurosporine (ST) was evaluated in wild type HeLa cells (HeLa-WT) and transfectants expressing either full-length connexin43 (HeLa-Cx43) or a C-terminal truncation of Cx43 (HeLa-ΔCT). Cell death was measured with fluorescence-activated cell sorting (FACS), both DNA and nuclear fragmentation methods and assays for PARP and caspase 3. The ST-mediated cell death was accelerated in HeLa-Cx43 cells compared to HeLa-WT and HeLa-ΔCT. To determine why HeLa-Cx43 cells were more susceptible to ST, the phosphorylation state and the localization of Cx43 protein within cells were examined using specific Cx43 antibodies. The phosphorylated forms of Cx43 were sharply reduced in HeLa-Cx43 cells treated with ST. Moreover, in ST-treated HeLa-Cx43 cells, Cx43 was mainly observed at the cell surface. In contrast, the truncated form of Cx43 found in HeLa-ΔCT cells, which lacks many of the normal phosphorylation sites, was observed in the cytosol with ST treatment. To examine the hemichannels in the plasma membranes of ST-treated HeLa-Cx43 cells, several dye uptake methods using carboxyfluorescein and propidium iodide were employed. While the number of fluorescent cells did not change in HeLa-WT and HeLa-ΔCT cells with ST treatment, the number of fluorescent HeLa-Cx43 cells increased more than ten-fold. These results indicate that the increases in cell surface Cx43 seen with immunofluorescence and the elevated hemichannel activities detected with dye uptake could help explain the accelerated cell death observed in ST-treated HeLa-Cx43 cells.     

Akt phosphorylates Connexin43 on Ser373, a "mode-1" binding site for 14-3-3

Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques.     

Cell membrane permeabilization via connexin hemichannels in living and dying cells

Vertebrate cells that express connexins likely express connexin hemichannels (Cx HCs) at their surface. In diverse cell types, surface Cx HCs can open to serve as a diffusional exchange pathway for ions and small molecules across the cell membrane. Most cells, if not all, also express pannexins that form hemichannels and increase the cell membrane permeability but are not addressed in this review. To date, most characterizations of Cx HCs have utilized cultured cells under resting conditions have and revealed low open probability and unitary conductance close to double that of the corresponding gap junction channels. In addition, the cell membrane permeability through Cx HCs can be markedly affected within seconds to minutes by various changes in the intra and/or extracellular microenvironment (i.e., pH, pCa, redox state, transmembrane voltage and intracellular regulatory proteins) that affect levels, open probability and/or (single channel) permeability of Cx HC. Net increase or decrease in membrane permeability could result from the simultaneous interaction of different mechanisms that affect hemichannels. The permeability of Cx HCs is controlled by complex signaling cascades showing connexin, cell and cell stage dependency. Changes in membrane permeability via hemichannels can have positive consequences in some cells (mainly in healthy cells), whereas in others (mainly in cells affected by acquired and/or genetic diseases) hemichannel activation can be detrimental.     

Functional hemichannels formed by human connexin 26 expressed in bacteria

Gap-junction channels (GJCs) communicate the cytoplasm of adjacent cells and are formed by head-to-head association of two hemichannels (HCs), one from each of the neighbouring cells. GJCs mediate electrical and chemical communication between cells, whereas undocked HCs participate in paracrine signalling because of their permeability to molecules such as ATP. Sustained opening of HCs under pathological conditions results in water and solute fluxes that cannot be compensated by membrane transport and therefore lead to cell damage. Mutations of Cx26 (connexin 26) are the most frequent cause of genetic deafness and it is therefore important to understand the structure–function relationship of wild-type and deafness-associated mutants. Currently available connexin HC expression systems severely limit the pace of structural studies and there is no simple high-throughput HC functional assay. The Escherichia coli-based expression system presented in the present study yields milligram amounts of purified Cx26 HCs suitable for functional and structural studies. We also show evidence of functional activity of recombinant Cx26 HCs in intact bacteria using a new growth complementation assay. The E. coli-based expression system has high potential for structural studies and high-throughput functional screening of HCs.     

Inhibition of Cx43 mediates protective effects on hypoxic/reoxygenated human neuroblastoma cells

Olfactory ensheathing cells (OECs), a special population of glial cells, are able to synthesise several trophic factors exerting a neuroprotective action and promoting growth and functional recovery in both in vitro and in vivo models. In the present work, we investigated the neuroprotective effects of OEC‐conditioned medium (OEC‐CM) on two different human neuron‐like cell lines, SH‐SY5Y and SK‐N‐SH (neuroblastoma cell lines), under normoxic and hypoxic conditions. In addition, we also focused our attention on the role of connexins (Cxs) in the neuroprotective processes. Our results confirmed OEC‐CM mediated neuroprotection as shown by cell adherence, proliferation and cellular viability analyses. Reduced connexin 43 (Cx43) levels in OEC‐CM compared to unconditioned cells in hypoxic conditions prompted us to investigate the role of Cx43‐Gap junctions (GJs) and Cx43‐hemichannels (HCs) in hypoxic/reoxygenation injury using carbenoxolone (non‐selective GJ inhibitor), ioxynil octanoato (selective Cx43‐GJ inhibitor) and Gap19 (selective Cx43‐HC inhibitor). We found that Cx43‐GJ and Cx43‐HC inhibitors are able to protect SH‐SY5Y and allow to these cultures to overcome the injury. Our findings support the hypothesis that both OEC‐CM and the inhibition of Cx43‐GJs and Cx43‐HCs offer a neuroprotective effect by reducing Cx43‐mediated cell‐to‐cell and cell‐to‐extracellular environment communications.     

Cation permeation through connexin 43 hemichannels is cooperative, competitive and saturable with parameters depending on the permeant species

Kinetics of permeation through connexin 43-EGFP hemichannels (Cx43-EGFP HCs) were evaluated in divalent cation-free solutions, which enhance HC open probability and thus, allow measurements during initial velocity. Three cations that become fluorescent upon binding to intracellular nucleic acids [ethidium (Etd), propidium (Prd) and 4′,6-diamidino-2-phenylindole (DAPI)] and Cx43-EGFP or Cx43 wild type HeLa cell transfectants (Cx43-EGFP- and Cx43-WT-HeLa cells, respectively) were used. Levels of Cx43-EGFP at the cell periphery and rate of dye uptake were directly related. The rate of uptake of each dye reached saturation consistent with a facilitated transport mechanism. Before saturation, the relation between rate of uptake and concentration of each dye was sigmoidal with Hill coefficients >1, indicating positive cooperativity of transport at low concentrations. The maximal rate of Etd uptake was not affected by the presence of DAPI and vice versa, but under each condition the apparent affinity constant of the main permeant molecule increased significantly consistent with competitive inhibition or competition for binding sites within the channel. Moreover, Cx43-EGFP and Cx43-WT HCs had similar permeability properties, indicating that EGFP bound to the C-terminal of Cx43 does not significantly alter the permeability of Cx43 HCs to positively charged molecules. Thus, competitive inhibition of permeation through hemichannels might contribute to cellular retention of essential molecules and/or uptake inhibition of toxic compounds.     

Properties and regulation of gap junctional hemichannels in the plasma membranes of cultured cells

During the assembly of gap junctions, a hemichannel in the plasma membrane of one cell is thought to align and dock with another in an apposed membrane to form a cell-to-cell channel. We report here on the existence and properties of nonjunctional, plasma membrane connexin43 (Cx43) hemichannels. The opening of the hemichannels was demonstrated by the cellular uptake of 5(6)-carboxyfluorescein from the culture medium when extracellular calcium levels were reduced. Dye uptake exhibited properties similar to those of gap junction channels. For example, using different dyes, the levels of uptake were correlated with molecular size: 5(6)-carboxyfluorescein (approximately 32%), 7- hydroxycoumarin-3-carboxylic acid (approximately 24%), fura-2 (approximately 11%), and fluorescein-dextran (approximately 0.4%). Octanol and heptanol also reduced dye uptake by approximately 50%. Detailed analysis of one clone of Novikoff cells transfected with a Cx43 antisense expression vector revealed a reduction in dye uptake levels according to uptake assays and a corresponding decrease in intercellular dye transfer rates in microinjection experiments. In addition, a more limited decrease in membrane resistance upon reduction of extracellular calcium was detected in electrophysiological studies of antisense transfectants, in contrast to control cells. Studies of dye uptake in HeLa cells also demonstrated a large increase following transfection with Cx43. Together these observations indicate that Cx43 is responsible for the hemichannel function in these cultured cells. Similar dye uptake results were obtained with normal rat kidney (NRK) cells, which express Cx43. Dye uptake can be dramatically inhibited by 12-O-tetradeconylphorbol-13-acetate-activated protein kinase C in these cell systems and by a temperature-sensitive tyrosine protein kinase, pp60v-src in LA25-NRK cells. We conclude that Cx43 hemichannels are found in the plasma membrane, where they are regulated by multiple signaling pathways, and likely represent an important stage in gap junction assembly.     

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.     

Essential role of ERK activation in neurite outgrowth induced by α-lipoic acid

Background

Neurite outgrowth is an important aspect of neuronal plasticity and regeneration after neuronal injury. Alpha-lipoic acid (LA) has been used as a therapeutic approach for a variety of neural disorders. We recently reported that LA prevents local anesthetics-induced neurite loss. In this study, we hypothesized that LA administration promotes neurite outgrowth.

Methods

To test our hypothesis, we treated mouse neuroblastoma N2a cells and primary neurons with LA. Neurite outgrowth was evaluated by examination of morphological changes and by immunocytochemistry for β-tubulin-3. ROS production was examined, as were the phosphorylation levels of ERK and Akt. In separate experiments, we determined the effects of the inhibition of ERK or PI3K/Akt as well as ROS production on LA-induced neurite outgrowth.

Results

LA promoted significantly neurite outgrowth in a time- and concentration-dependent manner. LA stimulation significantly increased the phosphorylation levels of both Akt and ERK and transiently induced ROS production. PI3K/Akt inhibition did not affect LA-induced neurite outgrowth. However, the inhibition of ERK activation completely abolished LA-induced neurite outgrowth. Importantly, the prevention of ROS production by antioxidants attenuated LA-stimulated ERK activation and completely abolished LA-promoted neurite outgrowth.

Conclusion

Our data suggest that LA stimulates neurite outgrowth through the activation of ERK signaling, an effect mediated through a ROS-dependent mechanism.     

Structural Determinants and Proliferative Consequences of Connexin 37 Hemichannel Function in Insulinoma Cells

Connexin (Cx) 37 suppresses vascular and cancer cell proliferation. The C terminus and a channel able to function are necessary, and neither by itself is sufficient, for Cx37 to mediate growth suppression. Cx37 supports transmembrane and intercellular signaling by forming functional hemichannels (HCs) and gap junction channels (GJCs), respectively. Here we determined whether Cx37 with HC, but not GJC, functionality would suppress proliferation of rat insulinoma (Rin) cells comparably to wild-type Cx37 (Cx37-WT). We mutated extracellular loop residues hypothesized to compromise HC docking but not HC function (six cysteines mutated to alanine, C54A,C61A,C65A, C187A,C192A,C198A (designated as C₆A); N55I; and Q58L). All three mutants trafficked to the plasma membrane and formed protein plaques comparably to Cx37-WT. None of the mutants formed functional GJCs, and Cx37-C₆A did not form functional HCs. Cx37-N55I and -Q58L formed HCs with behavior and permeation properties similar to Cx37-WT (especially Q58L), but none of the mutants suppressed Rin cell proliferation. The data indicate that determinants of Cx37 HC function differ from other Cxs and that HC functions with associated HC-supported protein-protein interactions are not sufficient for Cx37 to suppress Rin cell proliferation. Together with previously published data, these results suggest that Cx37 suppresses Rin cell proliferation only when in a specific conformation achieved by interaction of the C terminus with a Cx37 pore-forming domain able to open as a GJC.