Connexin Channels,Connexin Mimetic Peptides and ATP Release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP₃elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Connexin channels, connexin mimetic peptides and ATP release

Connexin hemichannels, that is, half gap junction channels (not connecting cells), have been implicated in the release of various messengers such as ATP and glutamate. We used connexin mimetic peptides, which are, small peptides mimicking a sequence on the connexin subunit, to investigate hemichannel functioning in endothelial cell lines. Short exposure (30 min) to synthetic peptides mimicking a sequence on the first or second extracellular loop of the connexin subunit strongly supressed ATP release and dye uptake triggered by either intracellular InsP(3) elevation or exposure to zero extracellular calcium, while gap junctional coupling was not affected under these conditions. The effect was dependent on the expression of connexin-43 in the cells. Connexin mimetic peptides thus appear to be interesting tools to distinguish connexin hemichannel from gap junction channel functioning. In addition, they are well suited to further explore the role of connexins in cellular release or uptake processes, to investigate hemichannel gating and to reveal new unknown functions of the large conductance hemichannel pathway between the cell and its environment. Work performed up to now with these peptides should be re-interpreted in terms of these new findings.     

Intracellular calcium changes trigger connexin 32 hemichannel opening

Connexin hemichannels have been proposed as a diffusion pathway for the release of extracellular messengers like ATP and others, based on connexin expression models and inhibition by gap junction blockers. Hemichannels are opened by various experimental stimuli, but the physiological intracellular triggers are currently not known. We investigated the hypothesis that an increase of cytoplasmic calcium concentration ([Ca2+]i) triggers hemichannel opening, making use of peptides that are identical to a short amino-acid sequence on the connexin subunit to specifically block hemichannels, but not gap junction channels. Our work performed on connexin 32 (Cx32)-expressing cells showed that an increase in [Ca2+]i triggers ATP release and dye uptake that is dependent on Cx32 expression, blocked by Cx32 (but not Cx43) mimetic peptides and a calmodulin antagonist, and critically dependent on [Ca2+]i elevation within a window situated around 500 nM. Our results indicate that [Ca2+]i elevation triggers hemichannel opening, and suggest that these channels are under physiological control.     

Connexin hemichannel and pannexin channel electrophysiology: How do they differ?

Connexin hemichannels are postulated to form a cell permeabilization pore for the uptake of fluorescent dyes and release of cellular ATP. Connexin hemichannel activity is enhanced by low external [Ca²⁺]_o, membrane depolarization, metabolic inhibition, and some disease-causing gain-of-function connexin mutations. This paper briefly reviews the electrophysiological channel conductance, permeability, and pharmacology properties of connexin hemichannels, pannexin 1 channels, and purinergic P2X₇ receptor channels as studied in exogenous expression systems including Xenopus oocytes and mammalian cell lines such as HEK293 cells. Overlapping pharmacological inhibitory and channel conductance and permeability profiles makes distinguishing between these channel types sometimes difficult. Selective pharmacology for Cx43 hemichannels (Gap19 peptide), probenecid or FD&C Blue #1 (Brilliant Blue FCF, BB FCF) for Panx1, and A740003, A438079, or oxidized ATP (oATP) for P2X7 channels may be the best way to distinguish between these three cell permeabilizing channel types. Endogenous connexin, pannexin, and P2X₇ expression should be considered when performing exogenous cellular expression channel studies. Cell pair electrophysiological assays permit the relative assessment of the connexin hemichannel/gap junction channel ratio not often considered when performing isolated cell hemichannel studies.     

Characterizing the mode of action of extracellular Connexin43 channel blocking mimetic peptides in an in vitro ischemia injury model

BackgroundNon-selective Connexin43 hemichannels contribute to secondary lesion spread. The hemichannel blocking peptidomimetic Peptide5, derived from the second extracellular loop of the human Connexin43 protein, prevents lesion spread and reduces vascular permeability in preclinical models of central nervous system injury. The molecular mode of action of Peptide5, however, was unknown and is described here.MethodsHuman cerebral microvascular endothelial cells and APRE-19 cells were used. Scrape loading was used to assess gap junction function and hypoxic, acidic ion-shifted Ringer solution induced ATP release used to assess hemichannel function. Peptide modifications, including amino acid substitutions and truncations, and competition assays were used to demonstrate Peptide5 functional specificity and site of action respectively.ResultsPeptide5 inhibits Connexin43 hemichannel-mediated ATP release by acting on extracellular loop two of Connexin43, adjacent to its matching sequence within the protein. Precise sequence specificity is important for hemichannel block, but less so for uncoupling of gap junction channels (seen only at high concentrations). The SRPTEKT motif is central to Peptide5 function but on its own is not sufficient to inhibit hemichannels. Both the SRPTEKT motif and Peptide5 reduce gap junction communication, but neither uncoupling below 50%.ConclusionsReduced gap junction coupling at high peptide concentrations appears to be relatively non-specific. However, Peptide5 at low concentrations acts upon extracellular loop two of Connexin43 to block hemichannels in a precise, sequence specific manner.General significanceThe concentration dependent and sequence specific action of Peptide5 supports its development for the treatment of retinal injury and chronic disease, as well as other central nervous system injury and disease conditions.     

Modulation of membrane channel currents by gap junction protein mimetic peptides: size matters

Connexin mimetic peptides are widely used to assess the contribution of nonjunctional connexin channels in several processes, including ATP release. These peptides are derived from various connexin sequences and have been shown to attenuate processes downstream of the putative channel activity. Yet so far, no documentation of effects of peptides on connexin channels has been presented. We tested several connexin and pannexin mimetic peptides and observed attenuation of channel currents that is not compatible with sequence specific actions of the peptides. Connexin mimetic peptides inhibited pannexin channel currents but not the currents of the channel formed by connexins from which the sequence was derived. Pannexin mimetic peptides did inhibit pannexin channel currents but also the channels formed by connexin 46. The same pattern of effects was observed for dye transfer, except that the inhibition levels were more pronounced than for the currents. The channel inhibition by peptides shares commonalities with channel effects of polyethylene glycol (PEG), suggesting a steric block as a mechanism. PEG accessibility is in the size range expected for the pore of innexin gap junction channels, consistent with a functional relatedness of innexin and pannexin channels. mimetic peptide; polyethylene glycol; calcium wave     

Atrial Fibrillation-Linked Germline GJA5/Connexin40 Mutants Showed an Increased Hemichannel Function

Mutations in GJA5 encoding the gap junction protein connexin40 (Cx40) have been linked to lone atrial fibrillation. Some of these mutants result in impaired gap junction function due to either abnormal connexin localization or impaired gap junction channels, which may play a role in promoting atrial fibrillation. However, the effects of the atrial fibrillation-linked Cx40 mutants on hemichannel function have not been studied. Here we investigated two atrial fibrillation-linked germline Cx40 mutants, V85I and L221I. These two mutants formed putative gap junction plaques at cell-cell interfaces, with similar gap junction coupling conductance as that of wild-type Cx40. Connexin deficient HeLa cells expressing either one of these two mutants displayed prominent propidium iodide-uptake distinct from cells expressing wild-type Cx40 or other atrial fibrillation-linked Cx40 mutants, I75F, L229M, and Q49X. Propidium iodide-uptake was sensitive to [Ca²⁺]_o and the hemichannel blockers, carbenoxolone, flufenamic acid and mefloquine, but was not affected by the pannexin 1 channel blocking agent, probenecid, indicating that uptake is most likely mediated via connexin hemichannels. A gain-of-hemichannel function in these two atrial fibrillation-linked Cx40 mutants may provide a novel mechanism underlying the etiology of atrial fibrillation.     

Gap junction-mimetic peptides do work, but in unexpected ways

Gap junction mimetic peptides containing sequences of the extracellular loops of connexins inhibit the de-novo formation of gap junction channels but do not impair the function of existing cell-cell channels. Recently, a flurry of publications appeared showing that such “GAP” peptides attenuate ATP release and/or surrogate measures of it. Although no direct effect on putative connexin “hemichannels” has ever been shown, the peptide effect has been used as diagnostic tool for demonstrating the existence of such channels. However, testing of the peptides on genuine unapposed membrane channels formed by connexins failed to reveal any inhibitory action of the peptides on channel activity. Instead, membrane channels formed by the unrelated pannexin1 were inhibited in the same concentration range as described for the release of ATP. Consequently, rather than indicating connexin involvement in ATP release, the GAP peptide effects represent supporting evidence for a role of pannexin1 in this process.     

Connexin 43 mimetic peptides reduce swelling, astrogliosis, and neuronal cell death after spinal cord injury

Connexin 43 (Cx43) is up-regulated after spinal cord injury (SCI). The authors tested whether mimetic peptides, corresponding to short sequences of rat Cx43, would reduce the severity of damage in a rodent ex vivo model of SCI. Eleven peptides (peptides 1 to 11) corresponding to short amino acid sequences of the extracellular loops of rat Cx43 were tested. Two of these peptides, peptide 4 (corresponding to Gap27) and peptide 5, significantly reduced the degree of swelling after SCI in this model. Peptide 5 produced the more significant reduction in swelling and was analyzed further. Treatment with peptide 5 reduced both the level of Cx43 and the number of glial fibrillary acidic protein (GFAP)-positive astrocytes, and at the same time reduced the loss of NeuN-and SMI-32-positive neurons in a concentration-and time-dependent manner. In cell culture, low concentrations of peptide 5 prevented hemichannel opening, but did not disrupt gap junctional communication. Higher concentrations prevented hemichannel opening, but also uncoupled existing gap junctions. This study supports the idea that regulation of Cx43 hemichannel opening using mimetic peptides may be a useful treatment for reducing the spread of damage after SCI.     

Connexin mimetic peptides reversibly inhibit Ca(2+) signaling through gap junctions in airway cells

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca(2+) signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca(2+) waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20-40 min. The peptides were shown to be more effective in inhibiting Ca(2+) waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca(2+) waves by connexin mimetic peptides did not affect the Ca(2+) response to extracellular ATP. Although the intracellular Ca(2+) response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca(2+) signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca(2+) wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.     

Tumour necrosis factor alpha inhibits purinergic calcium signalling in blood-brain barrier endothelial cells

The breaching of the blood-brain barrier is an essential aspect in the pathogenesis of neuroinflammatory diseases, in which tumour necrosis factor alpha (TNF-alpha) as well as endothelial calcium ions play a key role. We investigated whether TNF-alpha could influence the communication of calcium signals between brain endothelial cells (GP8 and RBE4). Intercellular calcium waves triggered by mechanical stimulation or photoliberation of InsP3 in single cells were significantly reduced in size after TNF-alpha exposure (1000 U/mL, 2 and 24 h). Calcium signals are communicated between cells by means of gap junctional and paracrine purinergic signalling. TNF-alpha significantly inhibited gap junctional coupling, stimulated the basal release of ATP, and dose-dependently blocked the triggered component of ATP release. The cytokine displayed similar effects on the uptake of a fluorescent reporter dye into the cells. Previous work with connexin mimetic peptides demonstrated that the triggered ATP release in these cells is connexin-related; these peptides did, however, not influence the elevated basal ATP release caused by TNF-alpha. We conclude that TNF-alpha depresses calcium signal communication in blood-brain barrier endothelial cells, by reducing gap junctional coupling and by inhibiting triggered ATP release. The cytokine thus inhibits connexin-related communication pathways like gap junctions and connexin hemichannels.     

Connexin mimetic peptides fail to inhibit vascular conducted calcium responses in renal arterioles

Vascular conducted responses are believed to play a central role in controlling the microcirculatory blood flow. The responses most likely spread through gap junctions in the vascular wall. At present, four different connexins (Cx) have been detected in the renal vasculature, but their role in transmission of conducted vasoconstrictor signals in the preglomerular arterioles is unknown. Connexin mimetic peptides were previously reported to target and inhibit specific connexins. We, therefore, investigated whether conducted vasoconstriction in isolated renal arterioles could be blocked by the use of mimetic peptides directed against one or more connexins. Preglomerular resistance vessels were microdissected from kidneys of Sprague-Dawley rats and loaded with fura 2. The vessels were stimulated locally by applying electrical current through a micropipette, and the conducted calcium response was measured 500 mum from the site of stimulation. Application of connexin mimetic peptides directed against Cx40, 37/43, 45, or a cocktail with equimolar amounts of each, did not inhibit the propagated response, whereas the nonselective gap junction uncoupler carbenoxolone completely abolished the propagated response. However, the connexin mimetic peptides were able to reduce dye coupling between rat aorta endothelial cells shown to express primarily Cx40. In conclusion, we did not observe any attenuating effects on conducted calcium responses in isolated rat interlobular arteries when exposed to connexin mimetic peptides directed against Cx40, 37/43, or 45. Further studies are needed to determine whether conducted vasoconstriction is mediated via previously undescribed pathways.     

Air-Stimulated ATP Release from Keratinocytes Occurs through Connexin Hemichannels

Cutaneous ATP release plays an important role in both epidermal stratification and chronic pain, but little is known about ATP release mechanisms in keratinocytes that comprise the epidermis. In this study, we analyzed ATP release from cultured human neonatal keratinocytes briefly exposed to air, a process previously demonstrated to trigger ATP release from these cells. We show that exposing keratinocytes to air by removing media for 15 seconds causes a robust, long-lasting ATP release. This air-stimulated ATP release was increased in calcium differentiated cultures which showed a corresponding increase in connexin 43 mRNA, a major component of keratinocyte hemichannels. The known connexin hemichannel inhibitors 1-octanol and carbenoxolone both significantly reduced air-stimulated ATP release, as did two drugs traditionally used as ABC transporter inhibitors (glibenclamide and verapamil). These same 4 inhibitors also prevented an increase in the uptake of a connexin permeable dye induced by air exposure, confirming that connexin hemichannels are open during air-stimulated ATP release. In contrast, activity of the MDR1 ABC transporter was reduced by air exposure and the drugs that inhibited air-stimulated ATP release had differential effects on this transporter. These results indicate that air exposure elicits non-vesicular release of ATP from keratinocytes through connexin hemichannels and that drugs used to target connexin hemichannels and ABC transporters may cross-inhibit. Connexins represent a novel, peripheral target for the treatment of chronic pain and dermatological disease.     

Pharmacological sensitivity of ATP release triggered by photoliberation of inositol-1,4,5-trisphosphate and zero extracellular calcium in brain endothelial cells

Recently, ATP has gained much interest as an extracellular messenger involved in the communication of calcium signals between cells. The mechanism of ATP release is, however, still a matter of debate. In the present study we investigated the possible contribution of connexin hemichannels or ion channels in the release of ATP in GP8, a rat brain endothelial cell line. Release of ATP was triggered by photoactivation of InsP(3) or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP(3)-triggered ATP release was completely blocked by alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptides gap 26 and 27, and the trivalent ions gadolinium and lanthanum. ATP release triggered by zero calcium was, in addition to these substances, also blocked by flufenamic acid (FFA), niflumic acid, and NPPB. Gap 27 selectively blocked zero calcium-triggered ATP release in connexin-43 transfected HeLa cells, while having no effect in wild-type and connexin-32 transfected cells. Of all the agents used, only alpha-GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP(3) and zero calcium-triggered ATP release show major similarities but also some differences in their sensitivity to the agents applied. It is suggested that both stimuli trigger ATP release through the same mechanism, which is connexin-dependent, permeable in both directions, potently blocked by connexin mimetic peptides, and consistent with the opening of connexin hemichannels.     

Gap junction intercellular communication during lymphocyte transendothelial migration

Migration of lymphocytes across the endothelium of central or peripheral tissues, a process occurring following activation or differentiation, involves cell to cell interactions featuring adhesion and heterotypic signalling 'cross-talk'. Since lymphocytes and endothelial cells express connexins, the subunit proteins of gap junction intercellular channels, we investigated whether these channels feature in heterotypic signalling during transendothelial migration of lymphocytes. We show, using FACS analysis, that calcein, a gap junction permeant fluorescent dye, was transferred from endothelial cell layers to lymphocytes. The gap junction involvement in intercellular dye transfer was reinforced by studies showing that the process was inhibited by connexin mimetic peptides, a new class of reagents shown to block gap junction communication. Further evidence for the involvement of lymphocyte gap junctions in intercellular communication during transendothelial migration was obtained by two-photon laser scanning microscopy. Although gap junctional communication was inhibited by connexin mimetic peptides, they had little influence on the transmigration process.     

Photoliberating inositol-1,4,5-trisphosphate triggers ATP release that is blocked by the connexin mimetic peptide gap 26

Calcium signals can be communicated between cells by the diffusion of a second messenger through gap junction channels or by the release of an extracellular purinergic messenger. We investigated the contribution of these two pathways in endothelial cell lines by photoliberating InsP(3) or calcium from intracellular caged precursors, and recording either the resulting intercellular calcium wave or else the released ATP with a luciferin/luciferase assay. Photoliberating InsP(3) in a single cell within a confluent culture triggered an intercellular calcium wave, which was inhibited by the gap junction blocker alpha-glycyrrhetinic acid (alpha-GA), the connexin mimetic peptide gap 26, the purinergic inhibitors suramin, PPADS and apyrase and by purinergic receptor desensitisation. InsP(3)-triggered calcium waves were able to cross 20 microm wide cell-free zones. Photoliberating InsP(3) triggered ATP release that was blocked by buffering intracellular calcium with BAPTA and by applying gap 26. Gap 26, however, did not inhibit the gap junctional coupling between the cells as measured by fluorescence recovery after photobleaching. Photoliberating calcium did not trigger intercellular calcium waves or ATP release. We conclude that InsP(3)-triggered ATP release through connexin hemichannels contributes to the intercellular propagation of calcium signals.     

Involvement of Connexin43 in the Infrasonic Noise-Induced Glutamate Release by Cultured Astrocytes

Infrasonic noise/infrasound is a type of environmental noise that threatens public health as a nonspecific biological stressor. Glutamate-related excitotoxicity is thought to be responsible for infrasound-induced impairment of learning and memory. In addition to neurons, astrocytes are also capable of releasing glutamate. In the present study, to identify the effect of infrasound on astroglial glutamate release, cultured astrocytes were exposed to infrasound at 16 Hz, 130 dB for different times. We found that infrasound exposure caused a significant increase in glutamate levels in the extracellular fluid. Moreover, blocking the connexin43 (Cx43) hemichannel or gap junction, decreasing the probability of Cx43 being open or inhibiting of Cx43 expression blocked this increase. The results suggest that glutamate release by Cx43 hemichannels/gap junctions is involved in the response of cultured astrocytes to infrasound.     

Changes in Connexin43 Expression and Localization During Pancreatic Cancer Progression

Gap junctions and gap junction communication have long been recognized to play roles in tissue organization and remodeling through both cell autonomous and intercellular means. We hypothesized that these processes become dysregulated during pancreas cancer progression. Molecular and histological characterization of the gap junction protein, connexin43, during progression of pancreatic ductal adenocarcinoma could yield insight into how these events may contribute to or be modulated during carcinogenesis. In a mouse model of pancreatic ductal adenocarcinoma generated through targeted endogenous expression of Kras(G12D) in the murine pancreas, we examined the evolving expression and localization of connexin43. Overall, connexin43 expression increased over time, and its localization became more widespread. At early stages, connexin43 is found almost exclusively in association with the basolateral membrane of duct cells found in invasive lesions. Connexin43 became increasingly associated with the surrounding stroma over time. Connexin43 phosphorylation was also altered during tumorigenesis, as assessed by migrational changes of the protein in immunoblots. These data suggest a potential role for gap junctions and connexin43 in mediating interactions between and amongst the stromal and epithelial cells in pancreatic ductal adenocarcinoma.     

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.     

Analysis of connexin phosphorylation sites

Most connexins, the proteins that form gap junction channels, are phosphoproteins. Connexin phosphorylation has been thought to regulate gap junctional protein trafficking, gap junction assembly, channel gating, and turnover. Connexin phosphorylation has been investigated in a variety of ways. Some connexins show mobility shifts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis on phosphorylation. Kinase modulators can change the level of connexin phosphorylation and affect gap junctional communication levels. Metabolic labeling of cultured cells has allowed both phosphoamino acid identification and generation of phosphotryptic peptide maps. However, identification of the location of phosphorylated residues within the connexin sequence has required either targeted peptide synthesis, in vitro phosphorylation of known sites, and two-dimensional comigration studies or liquid chromatographic separation and N-terminal sequencing of peptides. In addition to these conventional methods, we discuss new applications of mass spectrometry to the identification of phosphorylated peptides and the specific residues phosphorylated within the connexin-derived peptide.