Patricia E. M. Martin

Gap junctions: structure and function (Review)

Gap junctions are plasma membrane spatial microdomains constructed of assemblies of channel proteins called connexins in vertebrates and innexins in invertebrates. The channels provide direct intercellular communication pathways allowing rapid exchange of ions and metabolites up to ~1 kD in size. Approximately 20 connexins are identified in the human or mouse genome, and orthologues are increasingly characterized in other vertebrates. Most cell types express multiple connexin isoforms, making likely the construction of a spectrum of heteromeric hemichannels and heterotypic gap junctions that could provide a structural basis for the charge and size selectivity of these intercellular channels. The precise nature of the potential signalling information traversing junctions in physiologically defined situations remains elusive, but extensive progress has been made in elucidating how connexins are assembled into gap junctions. Also, participation of gap junction hemichannels in the propagation of calcium waves via an extracellular purinergic pathway is emerging. Connexin mutations have been identified in a number of genetically inherited channel communication-opathies. These are detected in connexin 32 in Charcot Marie Tooth-X linked disease, in connexins 26 and 30 in deafness and skin diseases, and in connexins 46 and 50 in hereditary cataracts. Biochemical approaches indicate that many of the mutated connexins are mistargeted to gap junctions and/or fail to oligomerize correctly into hemichannels. Genetic ablation approaches are helping to map out a connexin code and point to specific connexins being required for cell growth and differentiation as well as underwriting basic intercellular communication.

The endothelial component of cannabinoid-induced relaxation in rabbit mesenteric artery depends on gap junctional communication

1 We have shown that the endocannabinoid anandamide and its stable analogue methanandamide relax rings of rabbit superior mesenteric artery through endothelium‐dependent and ‐independent mechanisms that are unaffected by blockade of NO synthase and cyclooxygenase. 2 The endothelium‐dependent component of the responses was attenuated by the gap junction inhibitor 18α‐glycyrrhetinic acid (18α‐GA; 50 μm), and a synthetic connexin‐mimetic peptide homologous to the extracellular Gap 27 sequence of connexin 43 (43Gap 27, SRPTEKTIFII; 300 μm). By contrast, the corresponding connexin 40 peptide (40Gap 27, SRPTEKNVFIV) was inactive. 3 The cannabinoid CB1 receptor antagonist SR141716A (10 μm) also attenuated endothelium‐dependent relaxations but this inhibition was not observed with the CB1 receptor antagonist LY320135 (10 μm). Furthermore, SR141716A mimicked the effects of 43Gap 27 peptide in blocking Lucifer Yellow dye transfer between coupled COS‐7 cells (a monkey fibroblast cell line), whereas LY320135 was without effect, thus suggesting that the action of SR141716A was directly attributable to effects on gap junctions. 4 The endothelium‐dependent component of cannabinoid‐induced relaxation was also attenuated by AM404 (10 μm), an inhibitor of the high‐affinity anandamide transporter, which was without effect on dye transfer. 5 Taken together, the findings suggest that cannabinoids derived from arachidonic acid gain access to the endothelial cytosol via a transporter mechanism and subsequently stimulate relaxation by promoting diffusion of an to adjacent smooth muscle cells via gap junctions. 6 Relaxations of endothelium‐denuded preparations to anandamide and methanandamide were unaffected by 43Gap 27 peptide, 18α‐GA, SR141716A, AM404 and indomethacin and their genesis remains to be established.

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.

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.

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 InsP3 or by reducing the extracellular calcium concentration. Both trigger protocols induced ATP release significantly above baseline. InsP3‐triggered ATP release was completely blocked by α‐glycyrrhetinic acid (α‐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 α‐GA, FFA and NPPB significantly reduced gap junctional coupling. In conclusion, InsP3 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. J. Cell. Physiol. 197: 205–213, 2003© 2003 Wiley‐Liss, Inc.

Assembly of Chimeric Connexin-Aequorin Proteins into Functional Gap Junction Channels REPORTING INTRACELLULAR AND PLASMA MEMBRANE CALCIUM ENVIRONMENTS

Chimeric proteins comprising connexins 26, 32, and 43 and aequorin, a chemiluminescent calcium indicator, were made by fusing the amino terminus of aequorin to the carboxyl terminus of connexins. The retention of function by the chimeric partners was investigated. Connexin 32-aequorin and connexin 43-aequorin retained chemiluminescent activity whereas that of connexin 26-aequorin was negligible. Immunofluorescent staining of COS-7 cells expressing the chimerae showed they were targeted to the plasma membrane. Gap junction intercellular channel formation by the chimerae alone and in combination with wild-type connexins was investigated. Stable HeLa cells expressing connexin 43-aequorin were functional, as demonstrated by Lucifer yellow transfer. Pairs of Xenopus oocytes expressing connexin 43-aequorin were electrophysiologically coupled, but those expressing chimeric connexin 26 or 32 showed no detectable levels of coupling. The formation of heteromeric channels constructed of chimeric connexin 32 or connexin 43 and the respective wild-type connexins was inferred from the novel voltage gating properties of the junctional conductance. The results show that the preservation of function by each partner of the chimeric protein is dictated mainly by the nature of the connexin, especially the length of the cytoplasmic carboxyl-terminal domain. The aequorin partner of the connexin 43 chimera reported calcium levels in COS-7 cells in at least two different calcium environments.

Role of gap junctions in endothelium-derived hyperpolarizing factor responses and mechanisms of K+-relaxation

We have examined the effects of ouabain (1 mM), the gap junction inhibitors, 18 alpha-glycyrrhetinic acid (100 microM), N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2, 4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide hydrochloride (SR141716A; 10 microM) and palmitoleic acid (50 microM), and clotrimazole (10 microM) against endothelium-derived hyperpolarizing factor (EDHF)-mediated and K(+)-induced vasorelaxations in the rat mesentery. In the presence of indomethacin (10 microM) and 300-microM N(G)nitro-L-arginine methyl ester (L-NAME), carbachol caused EDHF-mediated relaxations (R(max)=85.3+/-4.0%). In the presence of ouabain, these responses were substantially reduced (R(max)=11.0+/-2.3%). 18 alpha-glycyrrhetinic acid, SR141716A, palmitoleic acid and clotrimazole also significantly inhibited these EDHF-mediated responses. K(+) caused vasorelaxation of preparations perfused with K(+)-free buffer (R(max)=73.7+/-2.4%), which were reduced by 10-microM indomethacin (R(max)=56.4+/-6.2%). K(+) vasorelaxation was essentially abolished by endothelial denudation. Both ouabain and 18 alpha-glycyrrhetinic acid opposed K(+) relaxations, however, neither SR141716A, clotrimazole nor palmitoleic acid had any effect. Direct cell-cell coupling via gap junctions was attenuated by ouabain, clotrimazole and palmitoleic acid. We conclude that: (i) that gap junctional communication plays a major role in EDHF-mediated relaxations, (ii) that K(+)-vasorelaxation is endothelium-dependent (thus, K(+) is unlikely to represent an EDHF), and (iii) that the inhibitory actions of ouabain and clotrimazole on gap junctions might contribute towards their effects against EDHF.

Analysis of gap junction assembly using mutated connexins detected in Charcot-Marie-Tooth X-linked disease

Abstract: The assembly of gap junction intercellular communication channels was studied by analysis of the molecular basis of the dysfunction of connexin 32 mutations associated with the X‐linked form of Charcot—Marie—Tooth disease in which peripheral nervous transmission is impaired. A cell‐free translation system showed that six recombinant connexin 32 mutated proteins—four point mutations at the cytoplasmic amino terminus, one at the membrane aspect of the cytoplasmic carboxyl terminus, and a deletion in the intracellular loop—were inserted into microsomal membranes and oligomerised into connexon hemichannels with varying efficiencies. The functionality of the connexons was determined by the ability of HeLa cells expressing the respective connexin cDNAs to transfer Lucifer yellow. The intracellular trafficking properties of the mutated connexins were determined by immunocytochemistry. The results show a relationship between intracellular interruption of connexin trafficking, the efficiency of intercellular communication, and the severity of the disease phenotype. Intracellular retention was explained either by deficiencies in the ability of connexins to oligomerise or by mutational changes at two targeting motifs. The results point to dominance of two specific targeting motifs: one at the amino terminus and one at the membrane aspect of the cytoplasmically located carboxyl tail. An intracellular loop deletion of six amino acids, associated with a mild phenotype, showed partial oligomerisation and low intercellular dye transfer compared with wild‐type connexin 32. The results show that modifications in trafficking and assembly of gap junction channels emerge as a major feature of Charcot—Marie—Tooth X‐linked disease.

Elevated Endothelial Nitric Oxide Bioactivity and Resistance to Angiotensin-Dependent Hypertension in 12/15-Lipoxygenase Knockout Mice

12/15-Lipoxygenase (12/15-LOX) plays a pathogenic role in atherosclerosis. To characterize whether 12/15-LOX also contributes to endothelial dysfunction and hypertension, regulation of vessel tone and angiotensin II (ang II) responses were characterized in mice deficient in 12/15-LOX. There was a twofold increase in the magnitude of l-nitroarginine-methyl ester-inhibitable, acetylcholine-dependent relaxation or phenylephrine-dependent constriction in aortic rings isolated from 12/15-LOX(-/-) mice. Plasma NO metabolites and aortic endothelial NO synthase (eNOS) expression were also elevated twofold. Angiotensin II failed to vasoconstrict 12/15-LOX(-/-) aortic rings in the absence of L-nitroarginine-methyl ester, and ang II impaired acetylcholine-induced relaxation in wild-type, but not 12/15-LOX(-/-) rings. In vivo, 12/15-LOX(-/-) mice had similar basal systolic blood pressure measurements to wild type, however, blood pressure elevations in response to ang II infusion (1.1 mg/kg/day) were significantly attenuated (maximal pressure, 143.4 +/- 4 mmHg versus 122.1 +/- 5.3 mmHg for wild type and 12/15-LOX(-/-), respectively). In contrast, vascular hypertrophic responses to ang II, and ang II type 1 receptor (AT1-R) expression were similar in both strains. This study shows that 12/15-LOX(-/-) mice have increased NO biosynthesis and impaired ang II-dependent vascular responses in vitro and in vivo, suggesting that 12/15-LOX signaling contributes to impaired NO bioactivity in vascular disease in vivo.

Ouabain exerts biphasic effects on connexin functionality and expression in vascular smooth muscle cells.

We have compared the effects of ouabain on the maintenance of gap junctional communication in rat aortic A7r5 smooth muscle cells, monkey COS‐1 fibroblasts and human HeLa epithelial cells. Ouabain (1 mM) interrupted dye coupling between confluent A7r5 cells within ∼1 h, and high concentrations of ouabain were similarly required to reduce coupling between COS‐1 cells selected to express the rat α1 Na+/K+‐ATPase subunit, which is ouabain resistant. By contrast, low concentrations of ouabain (1–10 μM) attenuated dye transfer in wild‐type COS‐1 and HeLa cells, whose endogenous α1 subunits possess relatively high affinity for the glycoside (Ki∼0.3 vs ∼100 μM) Ouabain‐induced reductions in dye transfer therefore correlated with the ability of the glycoside to bind to the Na+/K+‐ATPase isoenzymes expressed in these different cell lines. No consistent relationship between inhibition of intercellular dye transfer and secondary changes in [Ca2+]i or pHi could be identified following incubation with ouabain. In separate experiments, the effects of ouabain on real‐time trafficking of connexin protein were monitored by time‐lapse microscopy of A7r5 cells transfected to express a fluorescent Cx43‐green fluorescent protein (GFP) and the ability of the glycoside to modulate endogenous expression of connexins (Cx) 40 and 43 evaluated in A7r5 cells by immunochemical and Western blot analysis. Ouabain (1 mM) depressed vesicular trafficking of Cx43‐GFP after ∼1 h, and caused a time‐dependent loss of endogenous Cx40 and Cx43 protein that was first evident at 2 h and almost complete after 4 h. These effects of ouabain on Cx expression were reversed ∼90 min following washout of the glycoside. We conclude that ouabain exerts biphasic effects on the intercellular communication that involve an initial decrease in gap junctional permeability followed by a global reduction in the expression of Cx protein. Further studies are necessary to establish to what extent these actions of ouabain reflect inversion of the normal [Na+]i/[K+]i ratio and/or conversion of the Na+/K+‐ATPase into a general signal transducer that regulates downstream protein synthesis.