RhoA GTPase Switch Controls Cx43-Hemichannel Activity through the Contractile System

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 Ca²⁺ exerts a bell-shaped-dependent effect on Cx43-hemichannel opening. While low-intracellular [Ca²⁺] (<500 nM) provokes opening of the channel, high-intracellular [Ca²⁺] (> 500 nM) favours closing of the channel. The mechanism underlying this negative regulation of Cx43-hemichannel activity by high-intracellular [Ca²⁺] 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 [Ca²⁺] rise or external [Ca²⁺] 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.

Connexin43 (Cx43) hemichannels form a pathway for cellular communication between the cell and its extracellular environment. Under pathological conditions, Cx43 hemichannels release adenosine triphosphate (ATP), which triggers inflammation. Over the past two years, azithromycin, chloroquine, dexamethasone, favipiravir, hydroxychloroquine, lopinavir, remdesivir, ribavirin, and ritonavir have been proposed as drugs for the treatment of the coronavirus disease 2019 (COVID-19), which is associated with prominent systemic inflammation. The current study aimed to investigate if Cx43 hemichannels, being key players in inflammation, could be affected by these drugs which were formerly designated as COVID-19 drugs. For this purpose, Cx43-transduced cells were exposed to these drugs. The effects on Cx43 hemichannel activity were assessed by measuring extracellular ATP release, while the effects at the transcriptional and translational levels were monitored by means of real-time quantitative reverse transcriptase polymerase chain reaction analysis and immunoblot analysis, respectively. Exposure to lopinavir and ritonavir combined (4:1 ratio), as well as to remdesivir, reduced Cx43 mRNA levels. None of the tested drugs affected Cx43 protein expression.

β(2)-Adrenergic receptors (β(2)ARs) regulate cellular functions through G protein-transduced and βArrestin-transduced signals. β(2)ARs have been shown to regulate cancer cell migration, but the underlying mechanisms are not well understood. Here, we report that β(2)AR regulates formation of focal adhesions, whose dynamic remodeling is critical for directed cell migration. β(2)ARs induce activation of RhoA, which is dependent on βArrestin2 but not G(s). βArrestin2 forms a complex with p115RhoGEF, a guanine nucleotide exchange factor for RhoA that is well known to be activated by G(12/13)-coupled receptors. Our results show that βArrestin2 forms a complex with p115RhoGEF in the cytosol in resting cells. Upon β(2)AR activation, both βArrestin2 and p115RhoGEF translocate to the plasma membrane, with concomitant activation of RhoA and formation of focal adhesions and stress fibers. Activation of RhoA and focal adhesion remodeling may explain, at least in part, the role of β(2)ARs in cell migration. These results suggest that βArrestin2 may serve as a convergence point for non-G(12/13) and non-G(q) protein-coupled receptors to activate RhoA.

Astrocytes are electrically nonexcitable cells that communicate by means of Ca(2+) signaling. Long-distance intercellular Ca(2+) waves are initiated by release of ATP and activation of purinergic receptors on nearby cells. Previous studies have implicated connexin 43 (Cx43) in ATP release, but definitive proof that ATP exits through Cx43 hemichannels does not exist. Here, through several alternative approaches, we show that ATP anions can permeate through Cx43 hemichannels. First, openings of Cx43 hemichannels were detected in both cell-attached and inside-out patch recordings in C6 cells expressing Cx43, but not in C6 cells expressing Cx43-eGFP (enhanced green fluorescent protein) or a C-terminus truncation mutant of Cx43. Second, Cx43 hemichannel openings were inhibited by three structurally different gap-junction channel blockers, but not by the P2X(7) blocker Brilliant blue G. Third, bioluminescence imaging of ATP combined with single-channel recording in the inside-out patch configuration showed that ATP efflux coincided with channel openings and was absent when the Cx43 hemichannel was closed. Fourth, ion replacement experiments confirmed that Cx43 hemichannels are permeable to ATP. In summary, these observations provide the first direct evidence for efflux of ATP through Cx43 hemichannels. Furthermore, a putative Cx43 hemichannel with characteristics identical to the Cx43 hemichannel in C6 cells was identified in the membrane of hippocampal astrocytes in acutely prepared slices.

The tumor suppressor in lung cancer 1 (TSLC1/IGSF4) encodes an immunoglobulin-superfamily cell adhesion molecule whose cytoplasmic domain contains a protein 4.1-binding motif (protein 4.1-BM) and a PDZ-binding motif (PDZ-BM). Loss of TSLC1 expression is frequently observed in advanced cancers implying its involvement in tumor invasion and/or metastasis. Using Madin-Darby canine kidney cells expressing a full-length TSLC1 or various cytoplasmic deletion mutants of TSLC1, we examined the role of TSLC1 in epithelial mesenchymal transitions during the hepatocyte growth factor (HGF)-induced tubulogenesis and cell scattering. In a three-dimensional culture, the full-length TSLC1, which was localized to the lateral membrane of Madin-Darby canine kidney cysts, inhibited HGF-induced tubulogenesis. In contrast, the mutants lacking either the protein 4.1-BM or the PDZ-BM abolished the inhibitory effect on tubulogenesis. In addition, these mutants showed aberrant subcellular localization indicating that lateral localization is correlated with the effect of TSLC1. In a two-dimensional culture, the full-length TSLC1, but not the mutants lacking the protein 4.1-BM or the PDZ-BM, suppressed HGF-induced cell scattering. Furthermore, the cells expressing full-length TSLC1 retained E-cadherin-based cell-cell adhesion even after being treated with HGF. These cells showed prolonged activation of Rac and low activity of Rho, whereas the HGF-treated parental cells induced transient activation of Rac and sustained activation of Rho. Prolonged Rac activation caused by the expression of TSLC1 required its cytoplasmic tail. These findings, taken together, suggest that TSLC1 plays a role in suppressing induction of epithelial mesenchymal transitions by regulating the activation of small Rho GTPases.

To determine the presence of Kir2.1 channels in native bovine corneal endothelial (BCE) cells and assess their contribution to the resting membrane potential. RT-PCR and Western blot analysis were used to detect the expression of Kir2.1 mRNA and protein in native BCE cells. Whole-cell patch-clamp recording was used to characterize Kir2.1 currents in freshly isolated, single BCE cells, as well as in BCE cell clusters. The contribution of Kir2.1 channels to the membrane potential (V(m)) was assessed by whole-cell recording in the zero-current clamp mode in the absence and presence of Ba(2+). RT-PCR analysis confirmed that Kir2.1 was expressed in the native BCE cells. Western blot analysis with native BCE cell protein and a polyclonal anti-Kir2.1 antibody revealed a approximately 60-kDa band that was blocked by the corresponding synthetic Kir2.1 peptide. Both single BCE cells and BCE cell clusters exhibited an inwardly rectifying K(+) (Kir) current that was dependent on the extracellular K(+) concentration. The Kir current was blocked by external Ba(2+) or Cs(+) in a voltage- and concentration-dependent manner. In 5 mM K(+) Ringer's, the V(m) of cell clusters averaged -40.0 +/- 4.1 mV (n = 14) and in 140 mM K(+) Ringer's it depolarized to -7.4 +/- 1.8 mV. Application of Ba(2+) in 5 mM K(+) Ringer's produced a concentration-dependent depolarization of V(m), with 10 mM Ba(2+) depolarizing V(m) from -53.4 +/- 4.8 mV to -27.8 +/- 6.3 mV (n = 6). Native BCE cells express functional Kir2.1 channels that help determine the membrane potential and probably also play a role in transendothelial transport.

Connexins can act either as hemichannels to facilitate ion and small-molecule movement from the cytosol to the extracellular space or as gap junction channels to provide a pathway for solute exchange between cells. Connexins are ubiquitously expressed throughout the body and are implicated in a wide range of processes. The permselectivity of connexin hemichannels for small neurochemicals remains poorly understood. By coexpressing genetically encoded fluorescent sensors for ATP, glutamate, and lactate with a range of connexins, we examined the ability of different hemichannels to permit the release of these compounds under physiological conditions and in response to physiological stimuli (small changes in partial pressure of CO2 and transmembrane depolarization). We found that some connexin hemichannels were relatively nonselective (Cx26, Cx32, Cx43, and Cx31.1) allowing passage of ATP, glutamate, and lactate. By contrast, other connexin hemichannels (Cx36, Cx46, and Cx50) were highly selective. Cx36 and Cx46 hemichannels allowed the release of ATP but not glutamate or lactate. The size of the permeating molecule cannot be the sole determinant of permselectivity. By contrast, Cx50 hemichannels permitted the release of lactate and glutamate but not ATP. We also found that the nature of the opening stimulus could alter the permselectivity of the hemichannel—for some of the relatively nonselective connexins, hemichannel opening via depolarization was ineffective at allowing the release of lactate. By performing a mutational analysis, informed by the differential selectivity of the closely related Cx46 and Cx50 hemichannels, we found that the charge on the N terminus and N terminus–transmembrane 2 interactions are key contributors to permselectivity for ATP.

See related article, pages 50–58 Vascular leakage is a hallmark of many, often life-threatening, inflammatory diseases, and contributes to disease severity in disorders such as sepsis, cancer, diabetes, and atherosclerosis.1 Despite the tremendous medical importance of vascular leakage, few specific therapies are available today to counteract it, and current therapies often fail to do so.2 This is in part because the in vivo molecular targets are incompletely identified, although a wealth of data obtained from in vitro studies is available on signal transduction pathways that regulate vascular permeability.3,4 Interestingly, among various new agents that potentially reduce endothelial hyperpermeability, the cholesterol-lowering statin drugs have been proposed to reduce vascular leakage by means of their inhibitory effects on RhoA proteins.5,6 In the current issue of Circulation Research Gorovoy et al provide an elegant proof-of-principle that increased RhoA activity results in vascular hyperpermeability in vivo.7 They show that elevation of RhoA activity by deletion of one of its inhibitory proteins, RhoGDI, causes a loss of endothelial junctional integrity and decrease in vascular barrier function. Rho GTPases, particularly RhoA, Rac-1 and cdc42 have received much attention as key regulators of cell shape, movement and proliferation. In vitro studies have shown that the balance of activities of these small G proteins determines the status of endothelial barrier8: Cdc42 enhancing recovery of a disturbed barrier,9 Rac-1 being required for establishing a tight barrier,8 and RhoA being involved in inducing endothelial hyperpermeability by various stimuli, such as thrombin, VEGF, angiopoietin-2 and LPA (see Figure).10–12 Similarly, these Rho GTPases are acting …

Infection with the Ad5-SVR4 virus was used to introduce the large T antigen encoding region of the SV40 virus into bovine and human corneal endothelial cells. Expression of large T antigen occurred in 40% of bovine corneal endothelial cells after a 24-h incubation time versus 12% after 8 h of incubation. By 48 h after infection, almost all (92.8%) bovine corneal endothelial cells expressed large T antigen. Bovine and human corneal endothelial cells which expressed large T antigen proliferated and the characteristic morphologic features of corneal endothelium were maintained. This method may enable growth of enough corneal endothelium to perform studies to elucidate the biochemical mechanisms involved in regulating endothelial cell function.

The role of ventral hippocampus (vHipp) astroglial gliotransmission in depression was studied using chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) rodent models. CRS increased Cx43 hemichannel activity and extracellular glutamate levels in the vHipp and blocking astroglial Cx43 hemichannel-dependent gliotransmission during CRS prevented the development of depression and glutamate buildup. Moreover, the acute blockade of Cx43 hemichannels induced antidepressant effects in rats previously subjected to CRS or CUMS. This antidepressant effect was prevented by coinjection of glutamate and D-serine. Furthermore, Cx43 hemichannel blockade decreased postsynaptic NMDAR currents in vHipp slices in a glutamate and D-serine-dependent manner. Notably, chronic microinfusion of glutamate and D-serine, L-serine, or the NMDAR agonist NMDA, into the vHipp induced depressive-like symptoms in nonstressed rats. We also identified a small molecule, cacotheline, which blocks Cx43 hemichannels and its systemic administration induced rapid antidepressant effects, preventing stress-induced increases in astroglial Cx43 hemichannel activity and extracellular glutamate in the vHipp, without sedative or locomotor side effects. In conclusion, chronic stress increases Cx43 hemichannel-dependent release of glutamate and D-/L-serine from astrocytes in the vHipp, overactivating postsynaptic NMDARs and triggering depressive-like symptoms. This study highlights the critical role of astroglial gliotransmitter release in chronic stress-induced depression and suggests it can be used as a target for the prevention and treatment of depression.

Connexin and pannexin signaling in gastrointestinal and liver disease

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

Nonjunctional membrane in many cells contains connexin gap junction hemichannels (or connexons) that can open to allow permeation of small molecules. Opening of Cx43 hemichannels is infrequent in normal extracellular Ca(2+) and enhanced by low Ca(2+), positive membrane potentials, and dephosphorylation of critical residues. Here we report that lowering intracellular redox potential increases Cx43 hemichannel open probability under otherwise normal conditions. We studied dye uptake and single-channel activity in HeLa cells transfected with wild-type Cx43, Cx43 with enhanced GFP attached to its C terminus (Cx43-EGFP), and Cx43 with enhanced GFP attached to its N terminus (EGFP-Cx43). Dithiothreitol [(DTT) 10 mM], a membrane permeant-reducing agent, increased the rate of dye uptake by cells expressing Cx43 and Cx43-EGFP, but not by parental cells or cells expressing EGFP-Cx43. Induced dye uptake was blocked by La(3+), by a peptide gap junction and hemichannel blocker (gap 26), and by flufenamic acid. DTT increased Cx43-EGFP hemichannel opening at positive voltages. Bath application of reduced glutathione, a membrane impermeant-reducing agent, did not increase dye uptake, but glutathione in the recording pipette increased hemichannel opening at positive voltages, suggesting that it acted intracellularly. DTT caused little change in levels of surface Cx43 or Cx43-EGFP, or in intracellular pH. These findings suggest that lowering intracellular redox potential increases the opening of Cx43 and Cx43-EGFP hemichannels, possibly by action on cytoplasmic cysteine residues in the connexin C terminus.

A mathematical model of wound healing in bovine corneal endothelium

Introduction: Ischemia-reperfusion (I-R) injury is a significant complication in vascular surgery or organ transplantation. I-R injury involves intracellular Ca2+ overload, nitric oxide (NO), oxidative stress. I-R injury increases endothelial vascular hyperpermeability leading to tissue edema and damage mediated by endothelial Ca2+ increase. The role of Ca2+ permeable channels in I-R is not defined. Connexin (Cx) proteins form hemichannels, allowing the exchange of molecules between intracellular and extracellular spaces. The Cx43 isoform is highly expressed in endothelial cells (EC). Endothelial Ca2+ increase promotes NO-dependent Cx43 S-nitrosylation. However, whether Cx43 hemichannels are involved in I-R-induced hyperpermeability is unknown. Hypothesis: VEGF-induced hyperpermeability causes increased NO-mediated Cx43 S-nitrosylation and opening of endothelial Cx43 hemichannels, leading to Ca2+ entry and exacerbating reperfusion damage. Methods: We used VEGF as I-R related pro-inflammatory agonist. Using primary cultures of mesenteric endothelial cells, we tested Cx43 hemichannel activity by ethidium uptake and Ca2+ influx with Fluo-4 indicator. We evaluated hyperpermeability in vitro and in vivo Results: Results indicate that VEGF stimulation increased hemichannel activity in EC, associated with a rapid rise of Ca2+ influx. Both hemichannel activation and Ca2+ increase were abolished by treatment with specific Cx43 blocker (Gap19) or blocking NO production with NG-monomethyl-L-arginine (LNMMA). Cx43 inhibition blunted VEGF-induced hyperpermeability both in vitro in primary EC culture and in vivo mouse mesenteric vascular bed. To further understand the participation of NO-mediated S-nitrosylation in the VEGF-induced hyperpermeability, we replaced cysteine 271 by serine to create a Cx43C271S mouse that fails to cause S-nitrosylation of Cx43. We demonstrated that this knockin mouse exhibits a decrease in VEGF-stimulated hyperpermeability in vivo. Conclusions: These results indicate that Cx43 hemichannels activation is critical to VEGF (I-R inflammatory environment)-induced hyperpermeability and the S-nitrosylated Cx43 is a key regulator in I-R damage. AHA support: # 932684 and #23DIVSUP1054931