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-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.     

Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism

Linoleic acid (LA) is known to activate G-protein coupled receptors and connexin hemichannels (Cx HCs) but possible interlinks between these two responses remain unexplored. Here, we evaluated the mechanism of action of LA on the membrane permeability mediated by Cx HCs in MKN28 cells. These cells were found to express connexins, GPR40, GPR120, and CD36 receptors. The Cx HC activity of these cells increased after 5 min of treatment with LA or GW9508, an agonist of GPR40/GPR120; or exposure to extracellular divalent cation-free solution (DCFS), known to increase the open probability of Cx HCs, yields an immediate increase in Cx HC activity of similar intensity and additive with LA-induced change. Treatment with a CD36 blocker or transfection with siRNA-GPR120 maintains the LA-induced Cx HC activity. However, cells transfected with siRNA-GPR40 did not show LA-induced Cx HC activity but activity was increased upon exposure to DCFS, confirming the presence of activatable Cx HCs in the cell membrane. Treatment with AKTi (Akt inhibitor) abrogated the LA-induced Cx HC activity. In HeLa cells transfected with Cx43 (HeLa-Cx43), LA induced phosphorylation of surface Cx43 at serine 373 (S373), site for Akt phosphorylation. HeLa-Cx43 but not HeLa-Cx43 cells with a S373A mutation showed a LA-induced Cx HC activity directly related to an increase in cell surface Cx43 levels. Thus, the increase in membrane permeability induced by LA is mediated by an intracellular signaling pathway activated by GPR40 that leads to an increase in membrane levels of Cx43 phosphorylated at serine 373 via Akt.     

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.     

Distinct permeation profiles of the connexin 30 and 43 hemichannels

Connexin 43 (Cx43) hemichannels may form open channels in the plasma membrane when exposed to specific stimuli, e.g. reduced extracellular concentration of divalent cations, and allow passage of fluorescent molecules and presumably a range of smaller physiologically relevant molecules. However, the permeability profile of Cx43 hemichannels remains unresolved. Exposure of Cx43-expressing Xenopus laevis oocytes to divalent cation free solution induced a gadolinium-sensitive uptake of the fluorescent dye ethidium. In spite thereof, a range of biological molecules smaller than ethidium, such as glutamate, lactate, and glucose, did not permeate the pore whereas ATP did. In contrast, permeability of glutamate, glucose and ATP was observed in oocytes expressing Cx30. Exposure to divalent cation free solutions induced a robust membrane conductance in Cx30-expressing oocytes but none in Cx43-expressing oocytes. C-terminally truncated Cx43 (M257) displayed increased dye uptake and, unlike wild type Cx43 channels, conducted current. Neither Cx30 nor Cx43 acted as water channels in their hemichannel configuration. Our results demonstrate that connexin hemichannels have isoform-specific permeability profiles and that dye uptake cannot be equaled to permeability of smaller physiologically relevant molecules in given settings.     

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.     

Mechanism of regulation of the gap junction protein connexin 43 by protein kinase C-mediated phosphorylation

Phosphorylation of the gap junction protein connexin 43 (Cx43) by protein kinase C (PKC) decreases dye coupling in many cell types. We report an investigation of the regulation by PKC of Cx43 gap junctional hemichannels (GJH) expressed in Xenopus laevis oocytes. The activity of GJH was assessed from the uptake of hydrophilic fluorescent probes. PKC inhibitors increased probe uptake in isolated oocytes expressing recombinant Cx43, indicating that the regulatory effect occurs at the hemichannel level. We identified by mutational analysis the carboxy-terminal (CT) domain sequences involved in this response. We found that 1) Ser368 is responsible for the regulation of Cx43 GJH solute permeability by PKC-mediated phosphorylation, 2) CT domain residues 253-270 and 288-359 are not necessary for the effect of PKC, and 3) the prolinerich CT region is not involved in the effect of phosphorylation by PKC. Our results demonstrate that Ser368 (but not Ser372) is involved in the regulation of Cx43 solute permeability by PKC-mediated phosphorylation, and we conclude that different molecular mechanisms underlie the regulation of Cx43 by intracellular pH and PKC-mediated phosphorylation. protein kinase C blocker; dye loading; hemichannel     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

Connexin hemichannels and gap junction channels are differentially influenced by lipopolysaccharide and basic fibroblast growth factor

Gap junction (GJ) channels are formed by two hemichannels (connexons), each contributed by the cells taking part in this direct cell-cell communication conduit. Hemichannels that do not interact with their counterparts on neighboring cells feature as a release pathway for small paracrine messengers such as nucleotides, glutamate, and prostaglandins. Connexins are phosphorylated by various kinases, and we compared the effect of various kinase-activating stimuli on GJ channels and hemichannels. Using peptides identical to a short connexin (Cx) amino acid sequence to specifically block hemichannels, we found that protein kinase C, Src, and lysophosphatidic acid (LPA) inhibited GJs and hemichannel-mediated ATP release in Cx43-expressing C6 glioma cells (C6-Cx43). Lipopolysaccharide (LPS) and basic fibroblast growth factor (bFGF) inhibited GJs, but they stimulated ATP release via hemichannels in C6-Cx43. LPS and bFGF inhibited hemichannel-mediated ATP release in HeLa-Cx43 cells, but they stimulated it in HeLa-Cx43 with a truncated carboxy-terminal (CT) domain or in HeLa-Cx26, which has a very short CT. Hemichannel potentiation by LPS was inhibited by blockers of the arachidonic acid metabolism, and arachidonic acid had a potentiating effect like LPS and bFGF. We conclude that GJ channels and hemichannels display similar or oppositely directed responses to modulatory influences, depending on the balance between kinase activity and the activity of the arachidonic acid pathway. Distinctive hemichannel responses to pathological stimulation with LPS or bFGF may serve to optimize the cell response, directed at strictly controlling cellular ATP release, switching from direct GJ communication to indirect paracrine signaling, or maximizing cell-protective strategies.     

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 43 hemichannels contribute to the propagation of apoptotic cell death in a rat C6 glioma cell model

Gap junctions (GJs) have been demonstrated to communicate cell death signals from apoptotic to healthy cells, thereby spatially extending apoptosis. Before being incorporated into GJs, hemichannels (hemi-GJs) are normally closed but recent evidence suggests that they can be opened by various messengers and conditions, thereby forming a pore through which molecules can enter or leave the cell potentially leading to cell death. The aim of this study was to determine the contribution of GJs and hemichannels in the communication of apoptosis toward surrounding cells. We induced apoptosis in C6 glioma cells stably transfected with connexin (Cx)43, with cytochrome C (cytC) using in situ electroporation and found that healthy surrounding cells underwent apoptotic transformation. Work with various cell death markers, wild-type (WT) and Cx43-expressing cells, inhibitors of GJs and/or hemichannels, and Cx43 gene silencing showed that GJs contribute to the spread of apoptosis in a zone next to where apoptosis was triggered whereas hemichannels also promoted cell death beyond this area. Buffering cytoplasmic Ca(2+) changes inhibited the spread of apoptosis in both cases. We conclude that Cx43 hemichannels, in concert with their GJ counterparts, play a role in communicating cytC-induced apoptotic cell death messages.     

Functioning of cx43 hemichannels demonstrated by single channel properties

It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was approximately 220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of approximately 75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.     

Functioning of Cx43 Hemichannels Demonstrated by Single Channel Properties

It has been suggested that the opening of non-junctional connexin 43 (Cx43) hemichannels may play a role in cell physiology, but some workers doubt the reality of hemichannel openings. Here we show data on unitary conductance and voltage gating properties demonstrating that Cx43 hemichannels can open. Membrane depolarization > +60 mV induced single hemichannel currents in HeLa cells expressing Cx43 or Cx43 with enhanced green fluorescent protein attached to the carboxy terminal (Cx43-EGFP). The conductance of single hemichannels was ～220 pS, about twice that of the cell-cell channels. Cx43 and Cx43-EGFP hemichannels exhibited slow transitions (>5 ms) between closed and fully open states. Cx43 hemichannels also exhibited fast transitions (<1 ms) between the fully open state and a substate of ～75 pS. Similar gating was described for their respective cell-cell channels. No comparable single channel activity was detected in the parental (nontransfected cells) or HeLa cells expressing Cx43 fused at the amino terminal with EGFP (EGFP-Cx43). The latter chimera was inserted into the surface and formed plaques, but did not express functional hemichannels or cell-cell channels. These data convincingly demonstrate the opening of Cx43 hemichannels.     

Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.     

Activation,Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

Astrocytes and neurons express several large pore (hemi)channels that may open in response to various stimuli, allowing fluorescent dyes, ions, and cytoplasmic molecules such as ATP and glutamate to permeate. Several of these large pore (hemi)channels have similar characteristics with regard to activation, permeability, and inhibitor sensitivity. Consequently, their behaviors and roles in astrocytic and neuronal (patho)physiology remain undefined. We took advantage of the Xenopus laevis expression system to determine the individual characteristics of several large pore channels in isolation. Expression of connexins Cx26, Cx30, Cx36, or Cx43, the pannexins Px1 or Px2, or the purinergic receptor P2X₇ yielded functional (hemi)channels with isoform-specific characteristics. Connexin hemichannels had distinct sensitivity to alterations of extracellular Ca²⁺ and their permeability to dyes and small atomic ions (conductance) were not proportional. Px1 and Px2 exhibited conductance at positive membrane potentials, but only Px1 displayed detectable fluorescent dye uptake. P2X₇, in the absence of Px1, was permeable to fluorescent dyes in an agonist-dependent manner. The large pore channels displayed overlapping sensitivity to the inhibitors Brilliant Blue, gadolinium, and carbenoxolone. These results demonstrated isoform-specific characteristics among the large pore membrane channels; an open (hemi)channel is not a nonselective channel. With these isoform-specific properties in mind, we characterized the divalent cation-sensitive permeation pathway in primary cultured astrocytes. We observed no activation of membrane conductance or Cx43-mediated dye uptake in astrocytes nor in Cx43-expressing C6 cells. Our data underscore that although Cx43-mediated transport is observed in overexpressing cell systems, such transport may not be detectable in native cells under comparable experimental conditions.     

Role of connexin 43 in the mechanism of action of alendronate: dissociation of anti-apoptotic and proliferative signaling pathways

Bisphosphonates (BPs) inhibit osteocyte and osteoblast apoptosis via opening of connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. Previously, we hypothesized that intracellular survival signaling is initiated by interaction of BPs with Cx43. However, using whole cell binding assays with [(3)H]-alendronate, herein we demonstrated the presence of saturable, specific and high affinity binding sites in the Cx43-expressing ROS 17/2.8 osteoblastic cells, authentic osteoblasts and MLO-Y4 cells expressing Cx43 or not, as well as in HeLa cells lacking Cx43 expression and ROS 17/2.8 cells pretreated with agents that disassemble Cx channels. In addition, both BPs and the PTP inhibitor Na(3)VO(4) increased proliferation of cells expressing Cx43 or not. Furthermore, although BPs are internalized and inhibit intracellular enzymes in osteoclasts, whether the drugs penetrate non-resorptive bone cells is not known. To clarify this, we evaluated the osteoblastic uptake of AF-ALN, a fluorescently labeled analog of alendronate. AF-ALN was rapidly internalized in cells expressing Cx43 or not indicating that this process is not mediated via Cx43 hemichannels. Altogether, these findings suggest that although required for triggering intracellular survival signaling by BPs, Cx43 is dispensable for cellular BP binding, its uptake, as well as the proliferative effects of these agents.     

Green fluorescent protein changes the conductance of connexin 43 (Cx43) hemichannels reconstituted in planar lipid bilayers

In mammalian tissues, connexin 43 (Cx43) is the most prominent member of the connexin family. In a single lipid bilayer, six connexin subunits assemble into a hemichannel (connexon). Direct communication of apposing cells is realized by two adjacent hemichannels, which can form gap junction channels. Here, we established an expression system in Pichia pastoris to recombinantly produce and purify Cx43 as well as Cx43 fused to green fluorescent protein (GFP). Proteins were isolated from crude cell membrane fractions via affinity chromatography. Cx43 and Cx43-GFP hemichannels were reconstituted in giant unilamellar vesicles as proven by fluorescence microscopy, and their electrophysiological behavior was analyzed on the single channel level by planar patch clamping. Cx43 and Cx43-GFP both showed an ohmic behavior and a voltage-dependent open probability. Cx43 hemichannels exhibited one major mean conductance of 224 ± 26 picosiemens (pS). In addition, a subconductance state at 124 ± 5 pS was identified. In contrast, the analysis of Cx43-GFP single channels revealed 10 distinct conductance states in the range of 15 to 250 pS, with a larger open probability at 0 mV as compared with Cx43, which suggests that intermolecular interactions between the GFP molecules alter the electrophysiology of the protein.     

Asparagine 175 of connexin32 is a critical residue for docking and forming functional heterotypic gap junction channels with connexin26

The gap junction channel is formed by proper docking of two hemichannels. Depending on the connexin(s) in the hemichannels, homotypic and heterotypic gap junction channels can be formed. Previous studies suggest that the extracellular loop 2 (E2) is an important molecular domain for heterotypic compatibility. Based on the crystal structure of the Cx26 gap junction channel and homology models of heterotypic channels, we analyzed docking selectivity for several hemichannel pairs and found that the hydrogen bonds between E2 domains are conserved in a group of heterotypically compatible hemichannels, including Cx26 and Cx32 hemichannels. According to our model analysis, Cx32N175Y mutant destroys three hydrogen bonds in the E2-E2 interactions due to steric hindrance at the heterotypic docking interface, which makes it unlikely to dock with the Cx26 hemichannel properly. Our experimental data showed that Cx26-red fluorescent protein (RFP) and Cx32-GFP were able to traffic to cell-cell interfaces forming gap junction plaques and functional channels in transfected HeLa/N2A cells. However, Cx32N175Y-GFP exhibited mostly intracellular distribution and was occasionally observed in cell-cell junctions. Double patch clamp analysis demonstrated that Cx32N175Y did not form functional homotypic channels, and dye uptake assay indicated that Cx32N175Y could form hemichannels on the cell surface similar to wild-type Cx32. When Cx32N175Y-GFP- and Cx26-RFP-transfected cells were co-cultured, no colocalization was found at the cell-cell junctions between Cx32N175Y-GFP- and Cx26-RFP-expressing cells; also, no functional Cx32N175Y-GFP/Cx26-RFP heterotypic channels were identified. Both our modeling and experimental data suggest that Asn(175) of Cx32 is a critical residue for heterotypic docking and functional gap junction channel formation between the Cx32 and Cx26 hemichannels.     

Connexin43 in MDCK cells: regulation by a tumor-promoting phorbol ester and Ca2+.

Prior to confluence, cultures of Madin Darby canine kidney (MDCK) cells expressed gap junctional communication, as assessed by fluorescent dye transfer, as well as relatively high levels of an anti-connexin43 immunoreactive component referred to as connexin43 (Cx43). After confluence, dye coupling and levels of Cx43 were dramatically reduced. Immunofluorescence analysis of the distribution of Cx43 in subconfluent cultures showed punctate labeling on the plasma membrane at regions of cell apposition and a more diffuse labeling in perinuclear regions. Western blots of total cell homogenates showed that the dephosphorylated form of Cx43 was more abundant than the phosphorylated forms. Phosphorylation of Cx43 was not significantly affected by 8-Bromo-cAMP or 8-Bromo-cGMP. However, 12-O-tetradecanoylphorbol-13-acetate (TPA) inhibited dye coupling and induced an increase in the amount of phosphorylated forms of Cx43 at the expense of the dephosphorylated form. This effect occurred as rapidly as 5 min after TPA treatment without apparent changes in distribution of Cx43 or cell morphology. These results suggest that second messenger pathways involving protein kinase C, but not cAMP- or cGMP-dependent protein kinase, led to changes in electrophoretic mobility of Cx43, revealed by Western blot, consistent with an alteration in the state of phosphorylation of the gap junction protein. Treatments with staurosporine, a protein kinase inhibitor, or okadaic acid, a protein phosphatase inhibitor, either alone or in combination with TPA, indicated that the abundance of the dephosphorylated form of Cx43 in MDCK cells was due to low kinase activity. It was also found that lowering the concentration of extracellular Ca2+, which reduced cell contact, did not affect the abundance, the state of phosphorylation, or the TPA-induced phosphorylation of Cx43. These results suggest that neither extracellular Ca2+ nor cell contact is required for basal or TPA-induced phosphorylation of Cx43.     

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.