Hemichannel Opening Research Articles

Modified connexin43 mimetic peptide shows higher efficacy in reducing retinal ganglion cell loss and vessel leak after retinal ischemia

AbstractPurpose Optic neuropathy is associated with retinal ganglion cell (RGC) loss leading to optic nerve damage and visual impairment. Transient block of connexin43 (Cx43) hemichannels by mimetic peptides (MP) after retinal ischemia has been shown to inhibit uncontrolled hemichannel opening and may provide improved RGC survival. However, high hydrophilicity and poor peptide stability can limit efficient delivery in a clinical setting. This study evaluated the efficacy of modified Cx43MP using a retinal ischemia reperfusion rat model.Methods Retinal ischemia was created in the left eye (120 mmHg for 60 min) with the right eye serving as a control and Cx43MP formulations were injected intravitreally. Evan’s blue dye was injected intraperitoneally 4 h after reperfusion to visualize vessel leak using confocal microscopy. Retinal whole mounts were also labeled with Cx43, GFAP and Brn3a antibodies to quantify Cx43 and RGC densities.Results Intravitreal injection of native and modified Cx43MP resulted in significantly reduced Cx43 levels compared to untreated eyes 8 h after ischemia, with Cx43MP containing two C12‐lipoamino acid groups reducing Cx43 counts almost down to baseline level. A similar trend was seen for RCG counts with the modified Cx43MP resulting in >93% RCG survival after 28 days compared to <70% for untreated eyes. Vessel leak was also noticeably reduced in Cx43MP treated eyes.Conclusion C12‐lipoamino acid conjugation to Cx43MP increased peptide stability and may have also resulted in better tissue permeability, improving the overall efficacy of Cx43MP. Modified Cx43MP may therefore offer a clinically relevant treatment option for optic neuropathies.

Connexin hemichannel blockade is neuroprotective after, but not during, global cerebral ischemia in near-term fetal sheep

There is increasing evidence that connexin hemichannels, the half gap junctions that sit unopposed in the cell membrane, can open during ischemia and that blockade of connexin43 hemichannels after cerebral ischemia can improve neural outcomes. However, it is unclear whether connexin blockade during ischemia is protective. In the present study global cerebral ischemia was induced by 30min of bilateral carotid artery occlusion in near-term (128±1day gestation age) fetal sheep. A specific mimetic peptide that blocks connexin43 hemichannels was infused into the lateral ventricle for either 1h before and during ischemia (intra-ischemia group, n=6) or for 25h starting 90min after the end of ischemia (post-ischemia group, n=7). The vehicle was infused in the ischemia-vehicle group (n=6) and sham-controls received sham occlusion plus vehicle (n=10). The post-ischemia group showed enhanced recovery of EEG power from day five until the end of the experiment (−5±1.6dB) compared to ischemia-vehicle (−13±1.9dB, p<0.05) and intra-ischemia infusion (−14.4±3.6dB, p<0.05). Post-ischemic infusion was associated with higher neuronal counts compared to ischemia-vehicle and intra-ischemia in the cortex (p<0.05) but not the CA1 and CA3 regions of the hippocampus. Oligodendrocyte cell counts in the intragyral and periventricular white matter were significantly higher in the post-ischemia group compared to ischemia-vehicle and intra-ischemia infusion (p<0.05). These large animal data support the hypothesis that connexin hemichannel opening after, but not during, ischemia contributes to the spread of white and gray matter injury of the developing brain.

Cytotoxicity and Vitreous Stability of Chemically Modified Connexin43 Mimetic Peptides for the Treatment of Optic Neuropathy

Optic neuropathy is associated with retinal ganglion cell (RGC) loss leading to optic nerve damage and visual impairment. Unregulated connexin (Cx) hemichannel opening plays a role in RGC loss. Thus, inhibition via Cx43-specific mimetic peptides (MP) may prevent further cell death. However, the highly hydrophilic character and poor stability of native peptides prevent their efficient delivery across biological membranes. The present study aimed to improve the stability of Cx43 MP by conjugation to C12-lipoamino acid (C12-Laa) or sugar groups. Unmodified and modified Cx43 MP were synthesized using solid-phase peptide synthesis. Their functionality was assessed by propidium iodide (PI) uptake into NT2 cells, a human testicular carcinoma progenitor cell line able to differentiate into astrocytes, whereas the stability in ocular vitreous was measured by reversed-phase high-performance liquid chromatography. PI uptake studies showed inhibition of hemichannel opening for unmodified and modified Cx43 MP. Stability measurements revealed improved stability of modified Cx43 MP, with two Laa groups increasing the peptide half-life in bovine vitreous more than twofold. Conjugation to C12 -Laa or sugar did not affect the functionality of Cx43 MP, but addition of two C12-Laa groups significantly improved peptide stability. Laa-modifications may therefore offer improved stability and retinal delivery of peptides in vivo.

Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

The D50N mutation and syndromic deafness: Altered Cx26 hemichannel properties caused by effects on the pore and intersubunit interactions

Mutations in the GJB2 gene, which encodes Cx26, are the most common cause of sensorineural deafness. In syndromic cases, such as keratitis-ichthyosis-deafness (KID) syndrome, in which deafness is accompanied by corneal inflammation and hyperkeratotic skin, aberrant hemichannel function has emerged as the leading contributing factor. We found that D50N, the most frequent mutation associated with KID syndrome, produces multiple aberrant hemichannel properties, including loss of inhibition by extracellular Ca2+, decreased unitary conductance, increased open hemichannel current rectification and voltage-shifted activation. We demonstrate that D50 is a pore-lining residue and that negative charge at this position strongly influences open hemichannel properties. Examination of two putative intersubunit interactions involving D50 suggested by the Cx26 crystal structure, K61–D50 and Q48–D50, showed no evidence of a K61–D50 interaction in hemichannels. However, our data suggest that Q48 and D50 interact and disruption of this interaction shifts hemichannel activation positive along the voltage axis. Additional shifts in activation by extracellular Ca2+ remained in the absence of a D50–Q48 interaction but required an Asp or Glu at position 50, suggesting a separate electrostatic mechanism that critically involves this position. In gap junction (GJ) channels, D50 substitutions produced loss of function, whereas K61 substitutions functioned as GJ channels but not as hemichannels. These data demonstrate that D50 exerts effects on Cx26 hemichannel and GJ channel function as a result of its dual role as a pore residue and a component of an intersubunit complex in the extracellular region of the hemichannel. Differences in the effects of substitutions in GJ channels and hemichannels suggest that perturbations in structure occur upon hemichannel docking that significantly impact function. Collectively, these data provide insight into Cx26 structure–function and the underlying bases for the phenotypes associated with KID syndrome patients carrying the D50N mutation.

Abstract 3786: Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration.

Abstract Connexin 43 (Cx43) can form both gap junction channels and halves of gap junctions known as hemichannels. Hemichannels allow communication between cells and the extracellular environment. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation through fluid flow shear stress (FFSS) as well as by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug. The opening of hemichannels is known to release small molecules important for bone formation and remodeling. Here, we explore the role of Cx43 hemichannels in osteocytes and how it affects cancer cell migratory behavior. We confirm the opening of Cx43 hemichannels in the presence of AD by measuring uptake of ethidium bromide (EtBr) in osteocytic MLO-Y4 cells. As expected, an increase in EtBr uptake was observed after AD treatment. To ensure the specificity of Cx43 hemichannels, we treated the MLO-Y4 cells with Cx43 (E2) antibody, an antibody which specifically blocks Cx43 hemichannel activity. This prevented the uptake of EtBr by the MLO-Y4 cells. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell migration, we stimulated the opening of Cx43 hemichannels on MLO-Y4 osteocytes with either AD or FFSS. Conditioned media (CM) was collected after treatment and were used to incubate with metastatic MDA-MB-231 breast cancer cells and PC3 prostate cancer cells. Cell migration was measured using wound healing and transwell migration assays. The CM collected from MLO-Y4 osteocytes treated with AD or by FFSS reduced cancer cell migration in a dose-dependent manner. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody. Treatment with this antibody significantly attenuated the inhibitory effect of the CM on the migration of MDA-MB-231 and PC3 cells. Interestingly, the inhibitory effects on cancer cell migration were not observed when we used CM collected from osteoblasts, whose hemichannels were not shown to open by AD treatment. In order to determine whether ATP released from the Cx43 hemichannels in osteocytes would have an effect on cancer cell migration, we depleted ATP from the CM collected from MLO-Y4 cells using apyrase, an ATP hydrolyzing enzyme. The addition of apyrase increased MDA-MB-231 cell migration. To test the effect of purinergic signaling on breast cancer cell migration, we treated the CM with oxidized ATP (oATP), a potent inhibitor of ATP P2X receptors. The addition of oATP attenuated the inhibitory effect of CM on MDA-MB-231 cell migration. Then we stimulated the purinergic receptors with a nonhydrolyzable ATP analogue, ATPγS, the migration of MDA-MB-231 cells decreased. Together, our results point to the role of adenosine nucleotides released from Cx43 hemichannels in tumor migration and metastasis. Citation Format: Jade Z. Zhou, Jean X. Jiang. Osteocytic connexin 43 hemichannels in the inhibition of cancer cell migration. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3786. doi:10.1158/1538-7445.AM2013-3786

Drowning out communication. Focus on “The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity”

<i>Drowning out communication</i>. Focus on “The human Cx26-D50A and Cx26-A88V mutations causing keratitis-ichthyosis-deafness syndrome display increased hemichannel activity”

Pannexin1 hemichannels are critical for HIV infection of human primary CD4+ T lymphocytes

HIV is a major public health issue, and infection of CD4(+) T lymphocytes is one of its key features. Whereas several cellular proteins have been identified that facilitate viral infection and replication, the role of hemichannels in these processes has not been fully characterized. We now show that the HIV isolates, R5 and X4, induced a transient-early (5-30 min) and a later, persistent (48-120 h) opening of Panx1 hemichannels, which was dependent on the binding of HIV to CD4 and CCR5/CXCR4 receptors. Blocking Panx1 hemichannels by reducing their opening or protein expression inhibited HIV replication in CD4(+) T lymphocytes. Thus, our findings demonstrate that Panx1 hemichannels play an essential role in HIV infection.

Disruption of Salt Bridge Interactions Modifies Gating Kinetics of Connexin Hemichannels

Opening and closing of connexin hemichannels is involved in physiological paracrine signaling via release of ATP and other cytosolic molecules, but exacerbated hemichannel opening caused by mutations or pathological conditions is detrimental. The molecular mechanisms that control opening and closing of connexin hemichannels are poorly understood. The crystal structure of human Cx26 (hCx26) channels seems to represent the channel with open gates. This structure, and molecular dynamics studies based on it, reveal that charged residues (D46, E47, R75, R184) at the extracellular entrance of the aqueous pore - a region thought to be involved in gating rearrangements - form an electrostatic network. We explored the role of these salt bridge interactions in gating using mutagenesis, kinetic analysis and chemical modifications. Substitution of neutral residues for D46 or E47, which would eliminate their participation in salt bridges, accelerate deactivation kinetics and moderately increase the apparent affinity of Ca2+ to induce channel closing. These data support a role of these residues in stabilization of the open state. In addition, when D46 is substituted by a cysteine (D46C), modification by MTSES to add a negative charge increases holding and tail currents. This suggests that a negative charge at this position is involved in stabilizing open hemichannels. In wild-type channels, following depolarizing pulses to 0 mV, peak tail currents increase as a function of pulse duration, reaching maximum with pulses of 40 sec. Strikingly, E47A/Q mutations showed peak tail currents that saturate more rapidly, at 15 sec, suggesting that this position also plays a key role in hemichannel activation. Thus far, our data suggest that intra- and inter-subunit electrostatic networks at the extracellular entrance of the hCx26 pore play critical roles in hemichannel gating reactions. Support: R01GM099490.

ATP release and purinergic signaling in NLRP3 inflammasome activation

The NLRP3 inflammasome is a protein complex involved in IL-1β and IL-18 processing that senses pathogen- and danger-associated molecular patterns (PAMPs and DAMPs). One step- or two step-models have been proposed to explain the tight regulation of IL-1β production during inflammation. Moreover, cellular stimulation triggers adenosine triphosphate (ATP) release and subsequent activation of purinergic receptors at the cell surface. Importantly some studies have reported roles for extracellular ATP, in NLRP3 inflammasome activation in response to PAMPs and DAMPs. In this mini review, we will discuss the link between active ATP release, purinergic signaling and NLRP3 inflammasome activation. We will focus on the role of autocrine or paracrine ATP export in particle-induced NLRP3 inflammasome activation and discuss how particle activators are competent to induce maturation and secretion of IL-1β through a process that involves, as a first event, extracellular release of endogenous ATP through hemichannel opening, and as a second event, signaling through purinergic receptors that trigger NLRP3 inflammasome activation. Finally, we will review the evidence for ATP as a key pro-inflammatory mediator released by dying cells. In particular we will discuss how cancer cells dying via autophagy trigger ATP-dependent NLRP3 inflammasome activation in the macrophages engulfing them, eliciting an immunogenic response against tumors.

Extracellular Divalent Cations Regulation of Cx26 and Cx30 Hemichannels

Due to the large size and modest selectivity of the aqueous pore, exacerbated opening of connexin hemichannels leads to loss of electrochemical gradients and of small cytoplasmic metabolites, causing cell death. Control of hemichannel opening is indispensable, and is achieved by extracellular divalent concentrations, which drastically reduces hemichannel activity. Here, we explore the differences between extracellular Ca2+ and Mg2+ regulation in two relatives connexin, hCx26 and hCx30. Our standard protocol for assessment connexin hemichannel activation and deactivation with the two electro-voltage clamp technique is to examine the peak tail currents and their relaxation kinetics following a depolarizing pulse from −80 mV to 0 mV. using this protocol, the peak tail currents increase with reduction of external divalents. We estimate the extracellular Ca2+ and Mg2+ apparent affinity for hCx26 hemichannels at values of 0.33 mM and 1.8 mM, respectively. hCx30 hemichannels showed slightly higher extracellular Ca2+ and Mg2+ apparent affinity with values of 0.17 mM and 1.0 mM, respectively. At physiological Ca2+ concentration (1.0 - 1.8 mM), both hCx26 and hCx30 hemichannels reach ≤ 15% of the maximal response, but at corresponding Mg2+ concentrations they reach ≥ 50%. In addition, deactivation time constant at the tail currents are accelerated as a function of Ca2+ concentrations in hCx26 and hCx30 hemichannels; however, only high extracellular Mg2+ concentrations (> 2.0 mM) are capable to accelerate deactivation kinetics in both connexin types. The holding currents at steady state are significantly increased at physiological extracellular Mg2+ concentrations (1.0 - 1.2 mM) in both hCx26 and hCx30 suggesting an increase in open hemichannels even at negative potentials. Our data support that, under physiological ionic conditions, Ca2+, but not Mg2+ plays a major role stabilizing and facilitating closing of Cx26 and Cx30 hemichannels. Support: R01GM099490

Connexin in Lens Physiology and Cataract Formation

Connexins are a family of proteins that forms hemichannels that communicate the cytoplasm with the extracellular space. When two hemichannels [each one from two neighboring cells] make contact, they form a gap junction channel, which communicates the cytoplasm of adjacent cells. The molecular mechanisms that control the opening and closing of both functional hemichannels and gap junction channels is still matter of intense scrutiny. The lens is a transparent structure located in the anterior segment of the eye, which is critical for normal vision. Its main function is to refract the light, focusing it on the retina. Given this function, the lens requires great transparency and homogeneity which are attained by being avascular to avoid light scattering. To compensate for the lack of blood vessels, lens cells have intercellular connections formed by gap junction channels, which allow passive flux of nutrients and metabolites throughout the entire lens. Cataracts are produced by opacity of the lens, so less light reaches the retina. Recent evidence suggests that dysfunction of gap junction channels and hemichannels may induce cataract formation. Here we review general properties of gap junction channels and hemichannels. Then, we show the role of these channels in lens physiology and cataract formation with emphasis on rodent models lacking particular connexin genes and single point mutations in humans associated to hemichannel dysfunction. Finally, we raise the question of how environmental factors may affect hemichannel and gap junction activity and in turn induce or accelerate cataract formation by discussing the evidence that link molecular modifications [i.e phosphorylation and oxidation] of gap junction channels and hemichannels with cataract formation.

14-3-3θ Facilitates plasma membrane delivery and function of mechanosensitive connexin 43 hemichannels

Intracellular signaling in osteocytes activated by mechanical loading is important for bone formation and remodeling. These signaling events are mediated by small modulators released from Cx43 hemichannels (HC). We have recently shown that integrin α5 senses the mechanical stimulation and induces the opening of Cx43 HC; however, the underlying mechanism is unknown. Here, we show that both Cx43 and integrin α5 interact with 14-3-3θ, and this interaction is required for the opening of Cx43 HC upon mechanical stress. The absence of 14-3-3θ prevented the interaction between Cx43 and integrin α5, and blocked HC opening. Furthermore, it decreased the transport of Cx43 and integrin α5 from the Golgi apparatus to the plasma membrane. Mechanical loading promoted the movement of Cx43 to the surface which was associated not only with an increase in 14-3-3θ levels but also its interaction with Cx43 and integrin α5. This stimulatory effect on forward transport by mechanical loading was attenuated in the absence of 14-3-3θ and the majority of the Cx43 accumulated in the Golgi. Disruption of the Golgi by brefeldin A reduced the association of Cx43 and integrin α5 with 14-3-3θ, further suggesting that the interaction is likely to occur in the Golgi. Together, these results define a previously unidentified, scaffolding role of 14-3-3θ in assisting the delivery of Cx43 and integrin α5 to the plasma membrane for the formation of mechanosensitive HC in osteocytes.

Linoleic acid induces opening of connexin26 hemichannels through a PI3K/Akt/Ca2 +-dependent pathway

Connexin hemichannel (Cx HC) opening is involved in physiological and pathological processes, allowing the cellular release of autocrine/paracrine signaling molecules. Linoleic acid (LA) is known to modulate the functional state of connexin46 (Cx46) HCs. However, the molecular mechanism involved in this effect, or whether LA affects HCs constituted of other connexins, remains unknown. Here, we report the effects of LA on HCs in HeLa cells that express Cx26, one of the main Cxs in the cochlear sensory epithelium. Cx26 HC activity (dye uptake) was increased in a concentration-dependent manner by bath application of LA and inhibited by HC blockers. Moreover, intracellular BAPTA, a Ca2+ chelator, and PI3K/AKT inhibitors were found to reduce the LA-induced Cx26 HC opening, suggesting that the LA effect is mediated by an increase of free intracellular Ca2+ concentration and activation of the PI3K/Akt-dependent pathway. The LA-induced increase in free intracellular Ca2+ concentration was mainly due to Ca2+ influx through Cx26 HCs. In addition, the involvement of SH groups was ruled out, because dithiothreitol (DTT) did not block the LA-induced dye uptake. LA also increased the membrane current mediated by Cx26 HCs expressed in Xenopus oocytes and the dye uptake in HeLa cells expressing Cxs 32, 43 or 45. Since LA is an essential polyunsaturated fatty acid, its effect on HCs might be relevant to cell growth as well as to cellular functions of differentiated cells such as audition.

Abstract P3-13-06: Osteocytic Connexin 43 Hemichannels in Prevention of Bone Metastasis

Abstract Bone metastases are major, potentially fatal complications in patients with advanced cancers including breast, prostate and lung cancers. The gap junction-forming protein connexin 43 (Cx43) has been known to inhibit primary tumor cell growth. In addition to gap junctions, Cx43 also forms halves of gap junctions known as hemichannels which allow communication between the intracellular and extracellular environments. Cx43 hemichannels in osteocytes have been shown to open when induced by mechanical stimulation as well as by treatment with alendronate (AD), an efficacious and commonly used bisphosphonate drug. The opening of hemichannels is known to release small molecules important for bone formation and remodeling. To confirm the opening of Cx43 hemichannels in the presence of AD, we measured uptake of ethidium bromide (EtBr) in MLO-Y4 cells after the addition of AD. As expected, an increase in Cx43 hemichannels EtBr uptake was observed after AD treatment. To elucidate the effect(s) of osteocyte Cx43 hemichannels on cancer cell growth and migration, we treated MLO-Y4 osteocytes with AD. Conditioned media (CM) was collected after treatment and were used to incubate with metastatic MDA-MB-231 breast cancer cells and PC3 prostate cancer cells. Cell migration was measured using wound healing and transwell migration assays, and cell proliferation was measured using the soft agarose anchorage-independent growth assay. The CM collected from MLO-Y4 osteocytes treated with AD reduced cancer cell migration and proliferation in a dose-dependent manner. However, direct treatment with AD had no effect on the cell migration or proliferation of the MDA-MB-231 cells. These data validated that the decrease in migration and proliferation was not a result of direct action by AD on the cancer cells. In order to elucidate the direct functional involvement of Cx43 hemichannels in cell migration, we treated the MLO-Y4 cells with Cx43 (E2) antibody, a specific antibody which blocks hemichannel activity but not gap junction channels formed by Cx43. The inhibitory effect of the CM on the migration and proliferation of MDA-MB-231 and PC3 cells was diminished. Interestingly, the inhibitory effects of osteocytes on cancer cell migration were not observed when using osteoblasts. Together, these results suggest the functional importance of osteocytic Cx43 hemichannels in attenuating cancer cell migration and growth. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-13-06.

Selective inhibition of Cx43 hemichannels by Gap19 and its impact on myocardial ischemia/reperfusion injury

Connexin-43 (Cx43), a predominant cardiac connexin, forms gap junctions (GJs) that facilitate electrical cell-cell coupling and unapposed/nonjunctional hemichannels that provide a pathway for the exchange of ions and metabolites between cytoplasm and extracellular milieu. Uncontrolled opening of hemichannels in the plasma membrane may be deleterious for the myocardium and blocking hemichannels may confer cardioprotection by preventing ionic imbalance, cell swelling and loss of critical metabolites. Currently, all known hemichannel inhibitors also block GJ channels, thereby disturbing electrical cell-cell communication. Here we aimed to characterize a nonapeptide, called Gap19, derived from the cytoplasmic loop (CL) of Cx43 as a hemichannel blocker and examined its effect on hemichannel currents in cardiomyocytes and its influence in cardiac outcome after ischemia/reperfusion. We report that Gap 19 inhibits Cx43 hemichannels without blocking GJ channels or Cx40/pannexin-1 hemichannels. Hemichannel inhibition is due to the binding of Gap19 to the C-terminus (CT) thereby preventing intramolecular CT-CL interactions. The peptide inhibited Cx43 hemichannel unitary currents in both HeLa cells exogenously expressing Cx43 and acutely isolated pig ventricular cardiomyocytes. Treatment with Gap19 prevented metabolic inhibition-enhanced hemichannel openings, protected cardiomyocytes against volume overload and cell death following ischemia/reperfusion in vitro and modestly decreased the infarct size after myocardial ischemia/reperfusion in mice in vivo. We conclude that preventing Cx43 hemichannel opening with Gap19 confers limited protective effects against myocardial ischemia/reperfusion injury.

Connexin mimetic peptides inhibit Cx43 hemichannel opening triggered by voltage and intracellular Ca2+ elevation

Connexin mimetic peptides (CxMPs), such as Gap26 and Gap27, are known as inhibitors of gap junction channels but evidence is accruing that these peptides also inhibit unapposed/non-junctional hemichannels (HCs) residing in the plasma membrane. We used voltage clamp studies to investigate the effect of Gap26/27 at the single channel level. Such an approach allows unequivocal identification of HC currents by their single channel conductance that is typically ~220 pS for Cx43. In HeLa cells stably transfected with Cx43 (HeLa-Cx43), Gap26/27 peptides inhibited Cx43 HC unitary currents over minutes and increased the voltage threshold for HC opening. By contrast, an elevation of intracellular calcium ([Ca(2+)](i)) to 200-500 nM potentiated the unitary HC current activity and lowered the voltage threshold for HC opening. Interestingly, Gap26/27 inhibited the Ca(2+)-potentiated HC currents and prevented lowering of the voltage threshold for HC opening. Experiments on isolated pig ventricular cardiomyocytes, which display strong endogenous Cx43 expression, demonstrated voltage-activated unitary currents with biophysical properties of Cx43 HCs that were inhibited by small interfering RNA targeting Cx43. As observed in HeLa-Cx43 cells, HC current activity in ventricular cardiomyocytes was potentiated by [Ca(2+)](i) elevation to 500 nM and was inhibited by Gap26/27. Our results indicate that under pathological conditions, when [Ca(2+)](i) is elevated, Cx43 HC opening is promoted in cardiomyocytes and CxMPs counteract this effect.

Connexin and pannexin hemichannels in inflammatory responses of glia and neurons

Mammals express ∼20 different connexins, the main gap junction forming proteins in mammals, and 3 pannexins, homologs of innexins, the main gap junction forming proteins in invertebrates. In both classes of gap junction, each channel is formed by two hemichannels, one contributed by each of the coupled cells. There is now general, if not universal, agreement that hemichannels of both classes can open in response to various physiological and pathological stimuli when they are not apposed to another hemichannels and face the external milieu. Connexin (and likely pannexin) hemichannel permeability is consistent with that of the cell-cell channels and open hemichannels can be a release site for relatively large molecules such as ATP and glutamate, which can serve as transmitters between cells. Here we describe three experimental paradigms in which connexin and pannexin hemichannel signaling occurs. (1) In cultures of spinal astrocytes FGF-1 causes the release of ATP, and ATP causes opening of pannexin hemichannels, which then release further ATP. Subsequently, several hours later, connexin hemichannels are also opened by an unknown mechanism. Release of ATP appears to become self sustaining through action of P2X7 receptors to open pannexin hemichannels and then connexin hemichannels, both of which are ATP permeable. (2) Spinal cord injury by dropping a small weight on the exposed cord is followed by release of ATP in the region surrounding the primary lesion. This release is greatly reduced in a mouse in which Cx43 is knocked down in the astrocytes. Application of FGF-1 causes a similar release of ATP in the uninjured spinal cord, and an inhibitor of the FGF-1 receptor, PD173074, inhibits both FGF-1 and injury-induced release. Reduction in ATP release is associated with reduced inflammation and less secondary expansion of the lesion. (3) Cortical astrocytes in culture are permeabilized by hypoxia, and this effect is increased by high or zero glucose. The mechanism of permeabilization is opening of Cx43 hemichannels, which can lead to cell death. Activated microglia secrete TNF-α and IL-1β, which open connexin hemichannels in astrocytes. Astrocytes release ATP and glutamate which can kill neurons in co-culture through activation of neuronal pannexin hemichannels. These studies implicate two kinds of gap junction hemichannel in inflammatory responses and cell death. This article is part of a Special Issue entitled Electrical Synapses.

“INTEGRINating” the connexin hemichannel function in bone osteocytes through the action of integrin α5

Mechanical loading influences skeletal structural integrity and bone remodeling. Application of a mechanical stimulus such as fluid flow shear stress to the bone osteocytes activates the cascade of mechanotransduction mediated by multiple signaling molecules. Hemichannels formed by connexin molecules are emerging as a candidate mechanosensor. Connexin 43 (Cx43) hemichannels open in response to mechanical stimulation to release bone modulators which influence bone remodeling. Our study identified a direct interaction between integrin α5 and Cx43 which was essential for hemichannels to open. Uncoupling the interaction blocked the hemichannels and shear stress enhanced the interaction between the two proteins to promote channel opening. More importantly, integrin α5, independent of its association with fibronectin, was activated upon shear stress through a PI3K signaling pathway. These results suggest a critical regulatory mechanism for Cx43 hemichannel opening through the association of integrin α5, resulting in release of bone anabolic factors required for bone development.

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.