Intracellular calcium regulation of connexin43

The mechanism by which intracellular Ca(2+) concentration ([Ca(2+)](i)) regulates the permeability of gap junctions composed of connexin43 (Cx43) was investigated in HeLa cells stably transfected with this connexin. Extracellular addition of Ca(2+) in the presence of the Ca(2+) ionophore ionomycin produced a sustained elevation in [Ca(2+)](i) that resulted in an inhibition of the cell-to-cell transfer of the fluorescent dye Alexa fluor 594 (IC(50) of 360 nM Ca(2+)). The Ca(2+) dependency of this inhibition of Cx43 gap junctional permeability is very similar to that described in sheep lens epithelial cell cultures that express the three sheep lens connexins (Cx43, Cx44, and Cx49). The intracellular Ca(2+)-mediated decrease in cell-to-cell dye transfer was prevented by an inhibitor of calmodulin action but not by inhibitors of Ca(2+)/calmodulin-dependent protein kinase II or protein kinase C. In experiments that used HeLa cells transfected with a Cx43 COOH-terminus truncation mutant (Cx43(Delta257)), cell-to-cell coupling was similarly decreased by an elevation of [Ca(2+)](i) (IC(50) of 310 nM Ca(2+)) and similarly prevented by the addition of an inhibitor of calmodulin. These data indicate that physiological concentrations of [Ca(2+)](i) regulate the permeability of Cx43 in a calmodulin-dependent manner that does not require the major portion of the COOH terminus of Cx43.     

Osteocytic connexin 43 channels affect fracture healing

The cross-talk between cells is very critical for moving forward fracture healing in an orderly manner. Connexin (Cx) 43-formed gap junctions and hemichannels mediate the communication between adjacent cells and cells and extracellular environment. Loss of Cx43 in osteoblasts/osteocytes results in delayed fracture healing. For investigating the role of two channels in osteocytes in bone repair, two transgenic mouse models with Cx43 dominant negative mutants driven by a 10 kb-DMP1 promoter were generated: R76W (gap junctions are blocked, whereas hemichannels are promoted) and Δ130–136 (both gap junctions and hemichannels are blocked). R76W mice (promotion of hemichannels) showed a significant increase of new bone formation, whereas delayed osteoclastogenesis and healing was observed in Δ130–136 (impairment of gap junctions), but not in R76W mice (hemichannel promotion may recover the delay). These results suggest that gap junctions and hemichannels play some similar and cooperative roles in bone repair.     

Gap junction channels formed by coexpressed connexin40 and connexin43

Many cardiovascular cells coexpress multiple connexins (Cx), leading to the potential formation of mixed (heteromeric) gap junction hemichannels whose biophysical properties may differ from homomeric channels containing only one connexin type. We examined the potential interaction of connexin Cx43 and Cx40 in HeLa cells sequentially stably transfected with these two connexins. Immunoblots verified the production of comparable amounts of both connexins, cross-linking showed that both connexins formed oligomers, and immunofluorescence showed extensive colocalization. Moreover, Cx40 copurified with (His)(6)-tagged Cx43 by affinity chromatography of detergent-solubilized connexons, demonstrating the presence of both connexins in some hemichannels. The dual whole cell patch-clamp method was used to compare the gating properties of gap junctions in HeLa Cx43/Cx40 cells with homotypic (Cx40-Cx40 and Cx43-Cx43) and heterotypic (Cx40-Cx43) gap junctions. Many of the observed single channel conductances resembled those of homotypic or heterotypic channels. The steady-state junctional conductance (g(j,ss)) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells (V(j)) compared with homotypic gap junctions and/or an asymmetrical V(j) dependence reminiscent of heterotypic gap junctions. These gating properties could be fit using a combination of homotypic and heterotypic channel properties. Thus, whereas our biochemical evidence suggests that Cx40 and Cx43 form heteromeric connexons, we conclude that they are functionally insignificant with regard to voltage-dependent gating.     

Gating properties of gap junction channels assembled from connexin43 and connexin43 fused with green fluorescent protein.

We used cell lines expressing wild-type connexin43 (Cx43) and Cx43 fused with enhanced green fluorescent protein (Cx43-EGFP) to examine mechanisms of gap junction channel gating. Previously it was suggested that each hemichannel in a cell-cell channel possesses two gates, a fast gate that closes channels to a nonzero conductance or residual state via fast (< approximately 2 ms) transitions and a slow gate that fully closes channels via slow transitions (> approximately 10 ms). Here we demonstrate that transjunctional voltage (V(j)) regulates both gates and that they are operating in series and in a contingent manner in which the state of one gate affects gating of the other. Cx43-EGFP channels lack fast V(j) gating to a residual state but show slow V(j) gating. Both Cx43 and Cx43-EGFP channels exhibit slow gating by chemical uncouplers such as CO(2) and alkanols. Chemical uncouplers do not induce obvious changes in Cx43-EGFP junctional plaques, indicating that uncoupling is not caused by dispersion or internalization of junctional plaques. Similarity of gating transitions during chemical gating and slow V(j) gating suggests that both gating mechanisms share common structural elements. Cx43/Cx43-EGFP heterotypic channels showed asymmetrical V(j) gating with fast transitions between open and residual states only when the Cx43 side was relatively negative. This result indicates that the fast V(j) gate of Cx43 hemichannels closes for relative negativity at its cytoplasmic end.     

Selective effect of PDGF on connexin43 versus connexin40 comprised gap junction channels

The goals of the current study were to determine whether the conductance of Cx40 and Cx40-Cx43 mixed composition junctions was regulated by platelet-derived growth factor (PDGF)-activated signaling cascades, to ascertain the minimum number of Cx43 subunits/connexon required to confer PDGF sensitivity, and to identify specific residues in Cx43 required for this regulation. Junctional and channel conductances (g(j) and gamma(j), respectively) were determined for Cx40/Cx40, Cx43/Cx43, Cx40/Cx43, and Cx40-Cx43/Cx40-Cx43 mixed composition channels. PDGF had no effect on g(j) in Cx40/Cx40 pairs, but decreased g(j) in the remaining combinations by 53% (Cx43/Cx43), 48% (Cx40/Cx43), 41% (4:1 Cx40:Cx43 expression ratio) and 24% (10:1 Cx40:Cx43 expression ratio). Based on the predicted connexin composition of channels in cells expressing Cx40 and Cx43 at either 4:1 or 10:1 ratios, these decreases in g(j) suggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g(j) involved a decrease in both gamma(j) and Po and required serine 368 in the C-terminus. These data implicate protein kinase C as the mediator of the PDGF effect and strongly suggest that acute regulation of gap junction function by PDGF-activated signaling cascades is conferred by low levels of expression of a sensitive connexin in cells that otherwise express insensitive connexins.     

Evidence for heteromeric gap junction channels formed from rat connexin43 and human connexin37

Homomeric gap junction channels are composed solely of oneconnexin type, whereas heterotypic forms contain two homomeric hemichannels but the six identical connexins of each are different fromeach other. A heteromeric gap junction channel is one that containsdifferent connexins within either or both hemichannels. The existenceof heteromeric forms has been suggested, and many cell types are knownto coexpress connexins. To determine if coexpressed connexins wouldform heteromers, we cotransfected rat connexin43 (rCx43) and humanconnexin37 (hCx37) into a cell line normally devoid of any connexinexpression and used dual whole cell patch clamp to compare the observedgap junction channel activity with that seen in cells transfected onlywith rCx43 or hCx37. We also cocultured cells transfected with hCx37 orrCx43, in which one population was tagged with a fluorescent marker tomonitor heterotypic channel activity. The cotransfected cells possessedchannel types unlike the homotypic forms of rCx43 or hCx37 or theheterotypic forms. In addition, the noninstantaneous transjunctionalconductance-transjunctional voltage(G_j/V_j)relationship for cotransfected cell pairs showed a large range ofvariability that was unlike that of the homotypic or heterotypic form.The heterotypic cell pairs displayed asymmetric voltage dependence. Theresults from the heteromeric cell pairs are inconsistent with summedbehavior of two independent homotypic populations or mixed populationsof homotypic and heterotypic channels types. TheG_j/V_jdata imply that the connexin-to-connexin interactions are significantlyaltered in cotransfected cell pairs relative to the homotypic andheterotypic forms. Heteromeric channels are a population of channelswhose characteristics could well impact differently from theirhomotypic counterparts with regard to multicellular coordinatedresponses.

     

PH regulation of connexin43: molecular analysis of the gating particle.

Gap junction channels allow for the passage of ions and small molecules between neighboring cells. These channels are formed by multimers of an integral membrane protein named connexin. In the heart and other tissues, the most abundant connexin is a 43-kDa, 382-amino acid protein termed connexin43 (Cx43). A characteristic property of connexin channels is that they close upon acidification of the intracellular space. Previous studies have shown that truncation of the carboxyl terminal of Cx43 impairs pH sensitivity. In the present study, we have used a combination of optical, electrophysiological, and molecular biological techniques and the oocyte expression system to further localize the regions of the carboxyl terminal that are involved in pH regulation of Cx43 channels. Our results show that regions 261-300 and 374-382 are essential components of a pH-dependent "gating particle," which is responsible for acidification-induced uncoupling of Cx43-expressing cells. Regions 261-300 and 374-382 seem to be interdependent. The function of region 261-300 may be related to the presence of a poly-proline repeat between amino acids 274 and 285. Furthermore, site-directed mutagenesis studies show that the function of region 374-382 is not directly related to its net balance of charges, although mutation of only one amino acid (aspartate 379) for asparagine impairs pH sensitivity to the same extent as truncation of the carboxyl terminal domain (from amino acid 257). The mutation in which serine 364 is substituted for proline, which has been associated with some cases of cardiac congenital malformations in humans, also disrupts the pH gating of Cx43, although deletion of amino acids 364-373 has no effect on acidification-induced uncoupling. These results provide new insight into the molecular mechanisms responsible for acidification-induced uncoupling of gap junction channels in the heart and in other Cx43-expressing structures.     

Spatio-temporal regulation of connexin43 phosphorylation and gap junction dynamics

Gap junctions are specialized membrane domains containing tens to thousands of intercellular channels. These channels permit exchange of small molecules (< 1000 Da) including ions, amino acids, nucleotides, metabolites and secondary messengers (e.g., calcium, glucose, cAMP, cGMP, IP₃) between cells. The common reductionist view of these structures is that they are composed entirely of integral membrane proteins encoded by the 21 member connexin human gene family. However, it is clear that the normal physiological function of this structure requires interaction and regulation by a variety of proteins, especially kinases. Phosphorylation is capable of directly modulating connexin channel function but the most dramatic effects on gap junction activity occur via the organization of the gap junction structures themselves. This is a direct result of the short half-life of the primary gap junction protein, connexin, which requires them to be constantly assembled, remodeled and turned over. The biological consequences of this remodeling are well illustrated during cardiac ischemia, a process wherein gap junctions are disassembled and remodeled resulting in arrhythmia and ultimately heart failure. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.     

Pharmacological modulation and differential regulation of the cardiac gap junction proteins connexin 43 and connexin 40

Gap junction channels provide the basis for the electrical syncytial properties of the heart as a communicating electrical network. Cardiac gap junction channels are predominantly composed of connexin 40 or connexin 43. The conductance of these channels (g(j)) can be regulated pharmacologically: substances which activate protein kinase C, protein kinase A or protein kinase G may alter Cx43 gap junction conductance. However, for PKC, this seems to be subtype specific. Thus, antiarrhythmic peptides can enhance g(j) via activation of PKCepsilon, while FGF-2 reduces g(j) via PKCepsilon. Lipophilic drugs can uncouple the channels. Besides an acute regulation of g(j), the expression of the cardiac connexins can also be regulated. A decrease in Cx43 with a concomitant increase in Cx40 has been found in end-stage failing hearts, while in renovascular hypertension, an increase in Cx43 has been described. Mediators like endothelin-1, angiotensin-II, TGF-beta, VEGF, and cAMP have been shown to increase Cx43. Interestingly, endothelin-1 and angiotensin-II increased Cx43 but did not affect Cx40 expression. In contrast, in humans suffering from atrial fibrillation (AF), the content in Cx40 can be enhanced while Cx43 was unaltered, although in several other studies, other changes of the cardiac connexins were found, which might be related to the type of AF. Regarding the role of calcium, the content in both Cx40 and Cx43 was decreased in cultured neonatal rat cardiomyocytes after 24 h administration of 100 nM verapamil. Thus, gap junctional channels can be affected pharmacologically either acutely by modulating gap junction conductance or chronically by altering gap junction protein expression. Interestingly, it appears that the expression of Cx43 and Cx40 can be differentially regulated.     

Opposite regulation of connexin33 and connexin43 by LPS and IL-1alpha in spermatogenesis

The gap junction proteins, connexins (Cxs), are present in the testis, and among them, Cx43 play an essential role in spermatogenesis. In the present study, we investigated the testicular expression and regulation of another Cx, Cx33, previously described as a negative regulator of gap junction communication. Cx33 mRNA was present in testis and undetectable in heart, liver, ovary, and uterus. In the mature testis, Cx33 was specifically immunolocalized in the basal compartment of the seminiferous tubules, whereas Cx43 was present in both seminiferous tubule and interstitial compartments. During stages IX and X of spermatogenesis, characterized by Sertoli cell phagocytosis of residual bodies, Cx43 was poorly expressed within seminiferous tubules, while Cx33 signal was strong. To evaluate the role of phagocytosis in the control of Cx33 and Cx43 expression, the effect of LPS was analyzed in the Sertoli cell line 42GPA9. We show herein that phagocytosis activation by LPS concomitantly stimulated Cx33 and inhibited Cx43 mRNA levels. These effects appear to have been mediated through IL-1, because the exposure of Sertoli cells to the IL-1 receptor antagonist partly reversed these effects. IL-1 enhanced and reduced, respectively, the levels of Cx33 and Cx43 mRNA in a time- and dose-dependent manner. These data reveal that Cx33 and Cx43 genes are controlled differently within the testis and suggest that these two Cxs may exert opposite and complementary effects on spermatogenesis. Sertoli cell; germ cell proliferation     

Conductance and permeability of the residual state of connexin43 gap junction channels

We used cell lines expressing wild-type connexin43 and connexin43 fused with the enhanced green fluorescent protein (Cx43-EGFP) to examine conductance and perm-selectivity of the residual state of Cx43 homotypic and Cx43/Cx43-EGFP heterotypic gap junction channels. Each hemichannel in Cx43 cell-cell channel possesses two gates: a fast gate that closes channels to the residual state and a slow gate that fully closes channels; the transjunctional voltage (V(j)) closes the fast gate in the hemichannel that is on the relatively negative side. Here, we demonstrate macroscopically and at the single-channel level that the I-V relationship of the residual state rectifies, exhibiting higher conductance at higher V(j)s that are negative on the side of gated hemichannel. The degree of rectification increases when Cl(-) is replaced by Asp(-) and decreases when K(+) is replaced by TEA(+). These data are consistent with an increased anionic selectivity of the residual state. The V(j)-gated channel is not permeable to monovalent positively and negatively charged dyes, which are readily permeable through the fully open channel. These data indicate that a narrowing of the channel pore accompanies gating to the residual state. We suggest that the fast gate operates through a conformational change that introduces positive charge at the cytoplasmic vestibule of the gated hemichannel, thereby producing current rectification, increased anionic selectivity, and a narrowing of channel pore that is largely responsible for reducing channel conductance and restricting dye transfer. Consequently, the fast V(j)-sensitive gating mechanism can serve as a selectivity filter, which allows electrical coupling but limits metabolic communication.     

Enantioselective protein-sterol interactions mediate regulation of both prokaryotic and eukaryotic inward rectifier K+ channels by cholesterol

Cholesterol is the major sterol component of all mammalian cell plasma membranes and plays a critical role in cell function and growth. Previous studies have shown that cholesterol inhibits inward rectifier K(+) (Kir) channels, but have not distinguished whether this is due directly to protein-sterol interactions or indirectly to changes in the physical properties of the lipid bilayer. Using purified bacterial and eukaryotic Kir channels reconstituted into liposomes of controlled lipid composition, we demonstrate by (86)Rb(+) influx assays that bacterial Kir channels (KirBac1.1 and KirBac3.1) and human Kir2.1 are all inhibited by cholesterol, most likely by locking the channels into prolonged closed states, whereas the enantiomer, ent-cholesterol, does not inhibit these channels. These data indicate that cholesterol regulates Kir channels through direct protein-sterol interactions likely taking advantage of an evolutionarily conserved binding pocket.     

LacSwitch II regulation of connexin43 cDNA expression enables gap-junction single-channel analysis

Metabolic and electrical coupling through gap junction channels is implicated in cell differentiation, tissue homeostasis, and electrotonic propagation of signals in excitable tissues. The characterization of gating properties of these channels requires electrophysiological recordings at both single- and multiple-channel levels. Hence, a system that is able to control connexin expression by external means would provide a useful tool. To regulate the expression of connexins in cells, plasmids encoding a transactivator and/or a lac-operon IPTG response-dependent Cx43 target gene were transfected into communication-deficient N2a neuroblastoma cells. Immunoblotting, dye coupling, and electrophysiological methods revealed that expression of Cx43 in selected clones could be tightly regulated. After 15-20 h of acute induction with IPTG, cell-to-cell communication reached its peak with junctional conductances of 15-30 nS. Chronic induction at specific doses of IPTG produced constant, controlled levels of Cx43 expression, which were reflected by predictable junctional coupling levels. These conditions allowed prolonged recordings from either lowly or highly coupled cells, making lac operon an ideal regulatory system for channel gating studies at a single-channel level.     

Cannabinoids prevent the opposite regulation of astroglial connexin43 hemichannels and gap junction channels induced by pro‐inflammatory treatments

Brain injuries as well as neurodegenerative diseases, are associated with neuro‐inflammation characterized by astroglial and microglial activation and/or proliferation. Recently, we reported that lipopolysaccharide (LPS)‐activation of microglia inhibits junctional channels and promotes hemichannels, two connexin43 functions in astrocytes. This opposite regulation is mediated by two pro‐inflammatory cytokines, interleukin‐1 beta and tumor necrosis factor‐alpha, released from activated microglia. Because cannabinoids (CBs) have anti‐inflammatory properties and their receptors are expressed by glial cells, we investigated on primary cortical cultures the effects of CB agonists, methanandamide and synthetic CBs on (i) cytokines released from LPS‐activated microglia and (ii) connexin43 functions in astrocytes subjected to pro‐inflammatory treatments. We observed that CBs inhibited the LPS‐induced release of interleukin‐1 beta and tumor necrosis factor‐alpha from microglia. Moreover, the connexin43 dual regulation evoked by the pro‐inflammatory treatments, was prevented by CB treatments. Pharmacological characterizations of CB actions on astrocytic connexin43 channels revealed that these effects were mainly mediated through CB1 receptors activation, although non‐CB1/CB2 receptors seemed to mediate the action of the methanandamide. Altogether these data demonstrate that in inflammatory situations CBs exert, through the activation of different sub‐types of glial CB receptors, a regulation on two functions of connexin43 channels in astrocytes known to be involved in neuron survival.     

Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation

The regulation of gap junctional permeability by phosphorylation was examined in a model system in which connexin 43 (Cx43) gap junction hemichannels were reconstituted in lipid vesicles. Cx43 was immunoaffinity-purified from rat brain, and Cx43 channels were reconstituted into unilamellar phospholipid liposomes. The activities of the reconstituted channels were measured by monitoring liposome permeability. Liposomes containing the Cx43 protein were fractionated on the basis of permeability to sucrose using sedimentation in an iso-osmolar density gradient. The gradient allowed separation of the sucrose-permeable and -impermeable liposomes. Liposomes that were permeable to sucrose were also permeable to the communicating dye molecule lucifer yellow. Permeability, and therefore activity of the reconstituted Cx43 channels, were directly dependent on the state of Cx43 phosphorylation. The permeability of liposomes containing Cx43 channels was increased by treatment of liposomes with calf intestinal phosphatase. Moreover, liposomes formed with Cx43 that had been dephosphorylated by calf intestinal phosphatase treatment showed increased permeability to sucrose. The role of phosphorylation in the gating mechanism of Cx43 channels was supported further by the observation that phosphorylation of Cx43 by mitogen-activated protein kinase reversibly reduced the permeability of liposomes containing dephosphorylated Cx43. Our results show a direct correlation between gap junctional permeability and the phosphorylation state of Cx43.     

The highly conserved Gln49 and Ser50 of mammalian connexin43 are present in chick connexin43 and essential for functional gap junction channels

In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 ±5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 ±8 pS) compared to wild-type controls. Low or high pH of the bathing solution had no effect on the Q49H-Cx43 channels. We conclude that glutamine49 is important for channel function, and replacement of this residue with histidine most likely distorts secondary structure of the first extracellular loop, possibly by changing the orientation of conserved cysteines, and this inhibits channel function. The S50V substitution may also cause similar but less severe structural changes.     

New protein–protein interactions of mitochondrial connexin 43 in mouse heart

Connexin 43 (Cx43), the gap junction protein involved in cell‐to‐cell coupling in the heart, is also present in the subsarcolemmal fraction of cardiomyocyte mitochondria. It has been described to regulate mitochondrial potassium influx and respiration and to be important for ischaemic preconditioning protection, although the molecular effectors involved are not fully characterized. In this study, we looked for potential partners of mitochondrial Cx43 in an attempt to identify new molecular pathways for cardioprotection. Mass spectrometry analysis of native immunoprecipitated mitochondrial extracts showed that Cx43 interacts with several proteins related with mitochondrial function and metabolism. Among them, we selected for further analysis only those present in the subsarcolemmal mitochondrial fraction and known to be related with the respiratory chain. Apoptosis‐inducing factor (AIF) and the beta‐subunit of the electron‐transfer protein (ETFB), two proteins unrelated to date with Cx43, fulfilled these conditions, and their interaction with Cx43 was proven by direct and reverse co‐immunoprecipitation. Furthermore, a previously unknown molecular interaction between AIF and ETFB was established, and protein content and sub‐cellular localization appeared to be independent from the presence of Cx43. Our results identify new protein–protein interactions between AIF‐Cx43, ETFB‐Cx43 and AIF‐ETFB as possible players in the regulation of the mitochondrial redox state.