Nutrient Starvation Decreases Cx43 Levels and Limits Intercellular Communication in Primary Bovine Corneal Endothelial Cells.

Connexin (Cx) proteins form large conductance channels which function as regulators of communication between neighboring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signaling, survival and death processes. Connexin 43 (Cx43), a major connexin isoform in brain and heart, is rapidly turned over. Recent studies implicated that autophagy, a lysosomal degradation pathway induced upon nutrient starvation, mediates connexins, including Cx43, degradation. Here, we examined the impact of nutrient starvation on endogenous Cx43-protein levels and endogenous Cx43-driven intercellular communication in primary bovine corneal endothelial cells (BCECs). Hank's Balanced Salt Solution (HBSS) was used as a starvation condition that induces autophagic flux without impacting the survival of the BCECs. Nutrient starvation of BCECs caused a rapid decline in Cx43-protein levels, both as gap junctions and as hemichannels. The time course of the decline in Cx43-protein levels coincided with the time course of the decline in intercellular communication, assessed as intercellular Ca(2+)-wave propagation in BCECs exposed to a single-cell mechanical stimulus. The decline in Cx43-protein levels, both as gap junctions and as hemichannels, could be prevented by the addition of bafilomycin A1, a lysosomal inhibitor, during the complete nutrient starvation period. Consistent with this, bafilomycin A1 significantly alleviated the decrease in intercellular Ca(2+)-wave propagation. This study further underpins the importance of autophagy as an important degradation pathway for Cx43 proteins during periods of nutrient deprivation, thereby impacting the ability of cells to perform intercellular communication.

Regulation of connexin- and pannexin-based channels by post-translational modifications.

Connexin (Cx) and pannexin (Panx) proteins form large conductance channels, which function as regulators of communication between neighbouring cells via gap junctions and/or hemichannels. Intercellular communication is essential to coordinate cellular responses in tissues and organs, thereby fulfilling an essential role in the spreading of signalling, survival and death processes. The functional properties of gap junctions and hemichannels are modulated by different physiological and pathophysiological stimuli. At the molecular level, Cxs and Panxs function as multi-protein channel complexes, regulating their channel localisation and activity. In addition to this, gap junctional channels and hemichannels are modulated by different post-translational modifications (PTMs), including phosphorylation, glycosylation, proteolysis, N-acetylation, S-nitrosylation, ubiquitination, lipidation, hydroxylation, methylation and deamidation. These PTMs influence almost all aspects of communicating junctional channels in normal cell biology and pathophysiology. In this review, we will provide a systematic overview of PTMs of communicating junction proteins and discuss their effects on Cx and Panx-channel activity and localisation.

Non-channel functions of connexins in cell growth and cell death.

Cellular communication mediated by gap junction channels and hemichannels, both composed of connexin proteins, constitutes two acknowledged regulatory platforms in the accomplishment of tissue homeostasis. In recent years, an abundance of reports has been published indicating functions for connexin proteins in the control of the cellular life cycle that occur independently of their channel activities. This has yet been most exemplified in the context of cell growth and cell death, and is therefore as such addressed in the current paper. Specific attention is hereby paid to the molecular mechanisms that underpin the cellular non-channel roles of connexin proteins, namely the alteration of the expression of tissue homeostasis determinants and the physical interaction with cell growth and cell death regulators. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Negatively charged residues (Asp378 and Asp379) in the last ten amino acids of the C-terminal tail of Cx43 hemichannels are essential for loop/tail interactions.

Connexin 43 (Cx43)-hemichannel activity is controlled by intramolecular interactions between cytoplasmic loop and C-terminal tail. We previously identified the last 10 amino acids of the C-terminal tail of Cx43 as essential for Cx43-hemichannel activity. We developed a cell-permeable peptide covering this sequence (TAT-Cx43CT). In this study, we examined the critical molecular determinants in TAT-Cx43CT to restore Cx43-hemichannel activity. Using amino acid substitutions in TAT-Cx43CT, we identified the two aspartate (Asp378 and Asp379) and two proline (Pro375 and Pro377) residues as critical for TAT-Cx43CT activity, since TAT-Cx43CT(DD/AA) and TAT-Cx43CT(PP/GG) did not overcome the inhibition of Cx43-hemichannel activity induced by thrombin, micromolar cytoplasmic Ca(2+) concentration or truncation of Cx43 at M(239). Consistent with this, we found that biotin-Cx43CT(DD/AA) was much less efficient than biotin-Cx43CT to bind the purified CL domain of Cx43 in surface plasmon resonance experiments. In conclusion, we postulate that Asp378 and Asp379 in the C-terminal part of Cx43 are essential for loop/tail interactions in Cx43 hemichannels, while Pro375 and Pro377 may help to properly coordinate the critical Asp residues.

The contractile system as a negative regulator of the connexin 43 hemichannel.

The molecular mechanisms underlying the regulation of gap junction (GJ) channels based on the 43-kDa connexin isoform (Cx43) have been studied extensively. GJ channels are formed by the docking of opposed hemichannels in adjacent cells. Mounting data indicate that unopposed Cx43 hemichannels are also functional in the plasma membrane. However, our understanding of how Cx43-hemichannel opening and closing is regulated at the molecular level is only poorly understood. Recent work elucidated that actomyosin contractility inhibits potently Cx43 hemichannels. It is known that intracellular Ca²âº exerts a bell-shaped-dependent effect on Cx43-hemichannel opening. While low-intracellular [Ca²âº] (<500 nM) provokes opening of the channel, high-intracellular [Ca²âº] (> 500 nM) favours closing of the channel. The mechanism underlying this negative regulation of Cx43-hemichannel activity by high-intracellular [Ca²âº] seems to be dependent on the activation of the actomyosin contractile system. The activity of Cx43 hemichannels is critically controlled by molecular interactions between the intracellular loop and the C-terminal tail. These interactions are essential for Cx43-hemichannel opening in response to triggers such as cytosolic [Ca²âº] rise or external [Ca²âº] lowering. In this review, we present the hypothesis that the actomyosin contractile system can function as an important brake mechanism on Cx43-hemichannel opening. By controlling loop-tail interactions, the contractile system would prevent aberrant or excessive opening of Cx43 hemichannels.

Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity.

Connexin-assembled gap junctions (GJs) and hemichannels coordinate intercellular signaling processes. Although the regulation of connexins in GJs has been well characterized, the molecular determinants controlling connexin-hemichannel activity are unresolved. Here we investigated the regulation of Cx43-hemichannel activity by actomyosin contractility and intracellular [Ca(2+)] ([Ca(2+)](i)) using plasma membrane-permeable TAT peptides (100 µM) designed to interfere with interactions between the cytoplasmic loop (CL) and carboxy-terminal (CT) in primary bovine corneal endothelial cells and HeLa, C6 glioma, and Xenopus oocytes ectopically expressing Cx43. Peptides corresponding to the last 10 CT aa (TAT-Cx43CT) prevented the inhibition of Cx43-hemichannel activity by contractility/high [Ca(2+)](i), whereas a reverse peptide (TAT-Cx43CTrev) did not. These effects were independent of zonula occludens-1, a cytoskeletal-associated Cx43-binding protein. In contrast, peptides corresponding to CL (TAT-L2) inhibited Cx43-hemichannel responses, whereas a mutant peptide (TAT-L2(H126K/I130N)) did not inhibit. In these assays, TAT-Cx43CT acted as a scaffold for TAT-L2 and vice versa, a finding supported by surface plasmon resonance measurements. Loop/tail interactions appeared essential for Cx43-hemichannel activity, because TAT-Cx43CT restored the activity of nonfunctional hemichannels, consisting of either Cx43 lacking the C-terminal tail (Cx43(M239)) or intact Cx43 ectopically expressed in Xenopus oocytes. We conclude that intramolecular loop/tail interactions control Cx43-hemichannel activity, laying the basis for developing hemichannel-specific blockers.

Pannexins, distant relatives of the connexin family with specific cellular functions?

Intercellular communication (IC) is mediated by gap junctions (GJs) and hemichannels, which consist of proteins. This has been particularly well documented for the connexin (Cx) family. Initially, Cxs were thought to be the only proteins capable of GJ formation in vertebrates. About 10 years ago, however, a new GJ-forming protein family related to invertebrate innexins (Inxs) was discovered in vertebrates, and named the pannexin (Panx) family. Panxs, which are structurally similar to Cxs, but evolutionarily distinct, have been shown to be co-expressed with Cxs in vertebrates. Both protein families show distinct properties and have their own particular function. Identification of the mechanisms that control Panx channel gating is a major challenge for future work. In this review, we focus on the specific properties and role of Panxs in normal and pathological conditions.

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases.

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.

Thrombin inhibits intercellular calcium wave propagation in corneal endothelial cells by modulation of hemichannels and gap junctions.

PURPOSE: Thrombin, a serine protease, breaks down the barrier integrity of corneal endothelial cells by phosphorylation of the regulatory light chain of myosin II (myosin light chain; MLC), which induces contractility of the actin cytoskeleton. This study was undertaken to investigate the effect of thrombin on gap junctional (GJIC) and paracrine (PIC) intercellular communication in cultured bovine corneal endothelial cells (BCECs). METHODS: An intercellular Ca(2+) wave, a form of cell-cell communication, was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. Changes in [Ca(2+)](i) were imaged by fluorescence microscopy with a fluorescent calcium indicator, and the images were used to calculate the area reached by the Ca(2+) wave (active area). GJIC was assessed by fluorescence recovery after photobleaching (FRAP). Activity of hemichannels was assayed by lucifer yellow (LY) uptake and also by adenosine triphosphate (ATP) release by using the luciferin-luciferase technique. RESULTS: RT-PCR showed transcripts for PAR-1 and -2 receptors, but not for PAR-4 receptors. Immunocytochemistry showed thrombin-sensitive PAR receptors as well as trypsin-sensitive PAR-2 receptors. Both thrombin and the selective PAR-1 agonist TRAP-6 reduced the active area of the Ca(2+) wave. These agents also reduced the fluorescence recovery in FRAP experiments. The effect of thrombin on the Ca(2+) wave was inhibited by a peptide antagonist of PAR-1, but not by a PAR-4 antagonist. Pretreatment with ML-7 (an MLCK inhibitor), Y-27632 (a Rho kinase inhibitor) or chelerythrine (a PKC inhibitor) prevented the effect of thrombin on the Ca(2+) wave. Activation of PAR-1 did not affect the Ca(2+) wave propagation in cells pretreated with Gap26, which blocks hemichannels. However, PAR-1 activation decreased the active area in cells pretreated with Gap27, which inhibits gap junctions. Thrombin abolished enhancement of the Ca(2+) wave propagation by ARL-67156 (inhibitor of ecto-ATPases). The effect of the PAR-1 agonists on the Ca(2+) wave was not detectable in cells pretreated with exogenous apyrases. CONCLUSIONS: Thrombin inhibits intercellular Ca(2+) wave propagation in BCECs. This effect is due to activation of PAR-1 receptors and involves MLC phosphorylation by MLCK-, PKC- and Rho kinase-sensitive pathways. Thrombin mainly inhibits the ATP-mediated PIC pathway, and also reduces GJIC to a lesser extent.

Adenosine opposes thrombin-induced inhibition of intercellular calcium wave in corneal endothelial cells.

PURPOSE: In corneal endothelial cells, intercellular Ca(2+) waves elicited by a mechanical stimulus involve paracrine intercellular communication, mediated by ATP release via connexin hemichannels, as well as gap junctional intercellular communication. Both mechanisms are inhibited by thrombin, which activates RhoA and hence results in myosin light chain phosphorylation. This study was conducted to examine the effects of adenosine, which is known to oppose thrombin-induced RhoA activation, thereby leading to myosin light chain dephosphorylation, on gap junctional intercellular communication and paracrine intercellular communication in cultured bovine corneal endothelial cells. METHODS: An intercellular Ca(2+) wave was elicited by applying a mechanical stimulus to a single cell in a confluent monolayer. The area of Ca(2+) wave propagation was measured by [Ca(2+)](i) imaging using the fluorescent dye Fluo-4. Gap junctional intercellular communication was assessed by fluorescence recovery after photobleaching. Activity of hemichannels was determined by uptake of the hydrophilic dye Lucifer yellow in a Ca(2+)-free medium containing 2 mM EGTA. Adenosine triphosphate (ATP) release in response to mechanical stimulation was measured using the luciferin-luciferase technique. Gap26, a connexin mimetic peptide, was used to block hemichannels. RESULTS: Exposure to thrombin or TRAP-6 (a selective PAR-1 agonist) inhibited the Ca(2+) wave propagation by 70%. Pretreatment with adenosine prevented this inhibitory effect of thrombin. NECA (a potent A2B agonist) and forskolin, agents known to elevate cAMP in bovine corneal endothelial cells, also suppressed the effect of thrombin. The A1 receptor agonist CPA failed to inhibit the effect of thrombin. Similar to the effects on Ca(2+) wave propagation, adenosine prevented the thrombin-induced reduction in the fluorescence recovery during photobleaching experiments. Furthermore, pretreatment with adenosine prevented both thrombin and TRAP-6 from blocking the uptake of Lucifer yellow in a Ca(2+)-free medium. However, adenosine was ineffective in overcoming the Gap26-mediated block of Lucifer yellow uptake. In consistence with Lucifer yellow uptake through hemichannels, the thrombin-induced inhibition of ATP release was overcome by pretreatment with adenosine. CONCLUSIONS: Adenosine prevents thrombin-induced inhibition of hemichannel-mediated paracrine intercellular communication and of gap junctional intercellular communication. The mechanism involves an increase in cAMP, which results in inhibition of RhoA and a subsequent decrease in myosin light chain phosphorylation. Since myosin light chain dephosphorylation causes a decrease in contractility of the actin cytoskeleton, the results suggest possible effects of the actin cytoskeleton on gap junctions and connexin hemichannels.

Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels.

Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells. Intercellular calcium wave propagation evoked by a local mechanical stimulus is one of the gap junction assays to study intercellular communication. In experimental settings, an intercellular calcium wave can be elicited by applying a mechanical stimulus to a single cell. Here, we describe the use of monolayers of primary bovine corneal endothelial cells as a model to study intercellular communication. Calcium wave propagation was assayed by imaging fluorescent calcium in bovine corneal endothelial cells loaded with a fluorescent calcium dye using a confocal microscope. Spatial changes in intercellular calcium concentration following mechanical stimulation were measured in the mechanical stimulated cell and in the neighboring cells. The active area (i.e., total surface area of responsive cells) of a calcium wave can be measured and used for studying the function and regulation of gap junction channels as well as hemichannels in a variety of cell systems.

Determinants of the nucleolar targeting of protein phosphatase-1.

The ubiquitously expressed protein Ser/Thr phosphatase-1 isoforms PP1alpha, PP1beta and PP1gamma1 are dynamically targeted to distinct, but overlapping cellular compartments by associated proteins. Within the nucleus of HeLa cells, EGFP-tagged PP1gamma1 and PP1beta were predominantly targeted to the nucleoli, while PP1alpha showed a more diffuse distribution. Using PP1 chimaeras and point mutants we show here that a single N-terminal residue, i.e., Gln20 for PP1alpha, Arg19 for PP1beta and Arg20 for PP1gamma1 accounts for their distinct subnuclear distribution. Our data also suggest that the N-terminus of PP1beta and PP1gamma1 harbours an interaction site for one or more nucleolar interactors.

ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells.

PURPOSE: Intercellular communication (IC) in nonexcitable cells is mediated through gap junctions and/or through the release of paracrine mediators. This study was conducted to investigate adenosine-5' triphosphate (ATP)-dependent paracrine IC in the propagation of Ca2+ waves in confluent monolayers of cultured bovine corneal endothelial cells (BCECs). METHODS: A Ca2+ wave was induced by point mechanical stimulation (PMS) of a single cell by indentation with a glass micropipette (approximately 1 microm tip) for <1 second. Dynamic changes in [Ca2+]i in the mechanically stimulated (MS) cell and in the neighboring (NB) cells were visualized with a confocal microscope, using a fluorescent dye. Normalized fluorescence (NF), calculated as the ratio of the average fluorescence of a cell to the average under resting conditions, was used as a measure of [Ca2+]i. Expression of P2Y receptors and ecto-adenosine triphosphatases (ATPases) was investigated by RT-PCR. ATP release in response to PMS was measured by luciferin-luciferase (LL) bioluminescence. RESULTS: BCECs subjected to PMS showed a transient [Ca2+]i increase. Under control conditions, the maximum NF in the MS cell occurred within 600 ms, and the fluorescence returned to baseline within 170 seconds. NB cells also presented a [Ca2+]i increase with a transient characterized by decreasing maximum NF and increasing latency as a function of the distance from the MS cell. These transients propagated as an intercellular Ca2+ wave to a distance of five or six NB cells away from the MS cell, covering areas (called active areas, AAs) up to 77,000 +/- 3,200 microm2 (N=21). The percentage of responsive cells (defined as cells showing maximum NF >1.1) decreased with increasing distance from the MS cell. The Ca2+ wave crossed cell-free lanes. Pretreatment of cells with the nonselective purinergic receptor antagonist suramin (200 microM), exogenous apyrases, which break down nucleotides (10 U/mL), or the PLC inhibitor U-73122 (10 microM) reduced the wave propagation, whereas the ecto-ATPase inhibitor ARL-67156 (100 microM) significantly enhanced it. ATP-dependent LL bioluminescence increased after PMS. RT-PCR showed mRNAs for P2Y1 and P2Y2 receptors and ecto-ATPases in BCECs. CONCLUSIONS: PMS of BCECs induces release of ATP and a concomitant intercellular Ca2+ wave, even in the absence of direct cell-cell contacts. The AA of the wave is modulated by agents that affect P2Y receptor activity. Thus, PMS-induced intercellular Ca2+ wave propagation in BCECs involves ATP-dependent paracrine IC.

ATP release through connexin hemichannels in corneal endothelial cells.

PURPOSE: Intercellular Ca(2+) wave propagation is a distinct form of cell-cell communication. In corneal endothelial cells, intercellular Ca(2+) wave propagation evoked by a point mechanical stimulus (PMS) is partially mediated by adenosine triphosphate (ATP) release and subsequent activation of P2Y receptors. This study was conducted to investigate the possibility that extrajunctional connexons (hemichannels) play a role in ATP release during PMS-induced Ca(2+) wave propagation in bovine corneal endothelial cells (BCECs). METHODS: A Ca(2+) wave was evoked by a PMS applied to a single cell in a monolayer of cultured BCECs. Changes in [Ca(2+)](i) in the mechanically stimulated cell (MS cell) and in the neighboring (NB) cells were visualized by fluorescence imaging using the Ca(2+)-sensitive dye Fluo-4. From these images, the maximum normalized fluorescence (NF), the percentage of responsive cells (%RC), and the total area of cells reached by the Ca(2+) wave (active area [AA], in square micrometers) were calculated. Intercellular dye transfer, generally attributed to gap junctional coupling, was assessed by fluorescence recovery after photobleaching (FRAP) using 6-carboxyfluorescein diacetate. Opening of hemichannels was investigated by measuring cellular uptake of the fluorescent dye Lucifer yellow, which is known to permeate hemichannels. ATP release was measured by luciferin-luciferase bioluminescence. RESULTS: Flufenamic acid (FFA; 50 microM) and the connexin mimetic peptide Gap26 (300 microM), known blockers of hemichannels, significantly reduced AA in confluent monolayers as well as in contact-free cells. Neither FFA nor Gap26 affected the FRAP, indicating that reduction in AA of the PMS-induced wave by these agents is not due to a block of gap junction channels. FFA as well as Gap26 inhibited the increase in AA of the wave that was observed when cells were pretreated with the ectonucleotidase inhibitor ARL-67156 (100 microM). These findings suggest that the hemichannel blockers reduce the Ca(2+) wave propagation by inhibiting ATP release. Consistent with this finding, PMS or exposure to Ca(2+)-free solution (a maneuver known to induce the opening of hemichannels) led to ATP release; moreover, the release was inhibited by the hemichannel blockers. The extracellular ATP levels in response to both PMS and extracellular Ca(2+) removal were strongly enhanced by ARL-67156, and this effect was inhibited by FFA as well as by Gap26. Moreover, pretreatment of subconfluent BCEC monolayers with FFA or Gap26 inhibited the uptake of Lucifer yellow induced by removal of extracellular Ca(2+). CONCLUSIONS: Hemichannels contribute to ATP release on mechanical stimulation in BCECs. The released ATP contributes to propagation of the Ca(2+) wave.

Cx43-hemichannel function and regulation in physiology and pathophysiology: insights from the bovine corneal endothelial cell system and beyond.

Intercellular communication in primary bovine corneal endothelial cells (BCECs) is mainly driven by the release of extracellular ATP through Cx43 hemichannels. Studying the characteristics of Ca(2+)-wave propagation in BCECs, an important form of intercellular communication, in response to physiological signaling events has led to the discovery of important insights in the functional properties and regulation of native Cx43 hemichannels. Together with ectopic expression models for Cx43 hemichannels and truncated/mutated Cx43 versions, it became very clear that loop/tail interactions play a key role in controlling the activity of Cx43 hemichannels. Interestingly, the negative regulation of Cx43 hemichannels by enhanced actin/myosin contractility seems to impinge upon loss of these loop/tail interactions essential for opening Cx43 hemichannels. Finally, these molecular insights have spurred the development of novel peptide tools that can selectively inhibit Cx43 hemichannels, but neither Cx43 gap junctions nor hemichannels formed by other Cx isoforms. These tools now set the stage to hunt for novel physiological functions for Cx43 hemichannels in primary cells and tissues and to tackle disease conditions associated with excessive, pathological Cx43-hemichannel openings.

Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells.

Intercellular communication is essential for the coordination of physiological processes between cells in a variety of organs and tissues, including the brain, liver, retina, cochlea and vasculature. In experimental settings, intercellular Ca(2+)-waves can be elicited by applying a mechanical stimulus to a single cell. This leads to the release of the intracellular signaling molecules IP3 and Ca(2+) that initiate the propagation of the Ca(2+)-wave concentrically from the mechanically stimulated cell to the neighboring cells. The main molecular pathways that control intercellular Ca(2+)-wave propagation are provided by gap junction channels through the direct transfer of IP3 and by hemichannels through the release of ATP. Identification and characterization of the properties and regulation of different connexin and pannexin isoforms as gap junction channels and hemichannels are allowed by the quantification of the spread of the intercellular Ca(2+)-wave, siRNA, and the use of inhibitors of gap junction channels and hemichannels. Here, we describe a method to measure intercellular Ca(2+)-wave in monolayers of primary corneal endothelial cells loaded with Fluo4-AM in response to a controlled and localized mechanical stimulus provoked by an acute, short-lasting deformation of the cell as a result of touching the cell membrane with a micromanipulator-controlled glass micropipette with a tip diameter of less than 1 µm. We also describe the isolation of primary bovine corneal endothelial cells and its use as model system to assess Cx43-hemichannel activity as the driven force for intercellular Ca(2+)-waves through the release of ATP. Finally, we discuss the use, advantages, limitations and alternatives of this method in the context of gap junction channel and hemichannel research.

Peptides and peptide-derived molecules targeting the intracellular domains of Cx43: gap junctions versus hemichannels.

About a decade ago, the molecular determinants controlling the opening and closing of Cx43 gap junction channels have been identified. Advanced biophysical approaches revealed a critical role for structural rearrangements in the cytoplasmic loop and dimerization of the C-terminal tail, resulting in binding of the C-terminal tail to the cytoplasmic loop and Cx43 gap junction channel closure during cellular acidosis. This has spurred the development of Cx43-mimetic peptides and peptidomimetics that interfere with these loop/tail interactions, thereby preventing the closure of Cx43 gap junctions, e.g. in the heart upon ischemia. Recently, we found that loop/tail interactions control Cx43-hemichannel activity but with an opposite effect. Binding of the C-terminal tail to the cytoplasmic loop is a requisite for the opening of Cx43 hemichannels in response to different stimuli, like decreased extracellular [Ca2+], increased intracellular [Ca2+], positive membrane potentials or ischemia. Strikingly, peptides that favor the open state of Cx43 gap junctions like the L2 peptide inhibit Cx43-hemichannel opening. These tools now provide unprecedented opportunities to selectively inhibit Cx43 hemichannels while maintaining Cx43 gap junction communication, impossible to achieve with siRNA or knockdown approaches both affecting gap junctions and hemichannels. These tools not only are very helpful to unravel the role of Cx43 hemichannels in complex biological systems, but also hold therapeutic potential to counteract excessive Cx43-hemichannel activity like in ischemia/reperfusion in the brain and the heart or to prevent Cx43 hemichannel-mediated gliotransmitter release in the basal amygdala during memory consolidation in response to emotional events. This article is part of the Special Issue Section entitled 'Current Pharmacology of Gap Junction Channels and Hemichannels'.

RhoA GTPase switch controls Cx43-hemichannel activity through the contractile system.

ATP-dependent paracrine signaling, mediated via the release of ATP through plasma membrane-embedded hemichannels of the connexin family, coordinates a synchronized response between neighboring cells. Connexin 43 (Cx43) hemichannels that are present in the plasma membrane need to be tightly regulated to ensure cell viability. In monolayers of bovine corneal endothelial cells (BCEC),Cx43-mediated ATP release is strongly inhibited when the cells are treated with inflammatory mediators, in particular thrombin and histamine. In this study we investigated the involvement of RhoA activation in the inhibition of hemichannel-mediated ATP release in BCEC. We found that RhoA activation occurs rapidly and transiently upon thrombin treatment of BCEC. The RhoA activity correlated with the onset of actomyosin contractility that is involved in the inhibition of Cx43 hemichannels. RhoA activation and inhibition of Cx43-hemichannel activity were both prevented by pre-treatment of the cells with C3-toxin as well as knock down of RhoA by siRNA. These findings provide evidence that RhoA activation is a key player in thrombin-induced inhibition of Cx43-hemichannel activity. This study demonstrates that RhoA GTPase activity is involved in the acute inhibition of ATP-dependent paracrine signaling, mediated by Cx43 hemichannels, in response to the inflammatory mediator thrombin. Therefore, RhoA appears to be an important molecular switch that controls Cx43 hemichannel openings and hemichannel-mediated ATP-dependent paracrine intercellular communication under (patho)physiological conditions of stress.

Pannexin channels in ATP release and beyond: an unexpected rendezvous at the endoplasmic reticulum.

The pannexin (Panx) family of proteins, which is co-expressed with connexins (Cxs) in vertebrates, was found to be a new GJ-forming protein family related to invertebrate innexins. During the past ten years, different studies showed that Panxs mainly form hemichannels in the plasma membrane and mediate paracrine signalling by providing a flux pathway for ions such as Ca²(+), for ATP and perhaps for other compounds, in response to physiological and pathological stimuli. Although the physiological role of Panxs as a hemichannel was questioned, there is increasing evidence that Panx play a role in vasodilatation, initiation of inflammatory responses, ischemic death of neurons, epilepsy and in tumor suppression. Moreover, it is intriguing that Panxs may also function at the endoplasmic reticulum (ER) as intracellular Ca²(+)-leak channel and may be involved in ER-related functions. Although the physiological significance and meaning of such Panx-regulated intracellular Ca²(+) leak requires further exploration, this functional property places Panx at the centre of many physiological and pathophysiological processes, given the fundamental role of intracellular Ca²(+) homeostasis and dynamics in a plethora of physiological processes. In this review, we therefore want to focus on Panx as channels at the plasma membrane and at the ER membranes with a particular emphasis on the potential implications of the latter in intracellular Ca²(+) signalling.