Site-directed mutagenesis reveals putative regions of protein interaction within the transmembrane domains of connexins.

Through cysteine-scanning mutagenesis, the authors have compared sites within the transmembrane domains of two connexins, one from the alpha-class (Cx50) and one from the beta-class (Cx32), where amino acid substitution disrupts the function of gap junction channels. In Cx32, 11 sites resulted in no channel function, or an aberrant voltage gating phenotype referred to as "reverse gating," whereas in Cx50, 7 such sites were identified. In both connexins, the sites lie along specific faces of transmembrane helices, suggesting that these may be sites of transmembrane domain interactions. In Cx32, one broad face of the M1 transmembrane domain and a narrower, polar face of M3 were identified, including one site that was shown to come into close apposition with M4 in the closed state. In Cx50, the same face of M3 was identified, but sensitive sites in M1 differed from Cx32. Many fewer sites in M1 disrupted channel function in Cx50, and those that did were on a different helical face to the sensitive sites in Cx32. A more in depth study of two sites in M1 and M2 of Cx32 showed that side-chain length or branching are important for maintenance of normal channel behavior, consistent with this being a site of transmembrane domain interaction.

Aberrant gating, but a normal expression pattern, underlies the recessive phenotype of the deafness mutant Connexin26M34T.

Mutations in the gene GJB2, encoding the gap junction protein Connexin26 (Cx26), are the most prevalent cause of inherited hearing loss, and Cx26M34T was one of the first mutations linked to deafness (Kelsell et al., 1997; Nature 387, 80-83). We report the first characterization of the gating properties of M34T, which had previously been reported to be nonfunctional. Although homotypic mutant channels did not produce detectable currents, heterotypic pairings with wtCx26 confirmed that M34T formed intercellular channels, although the gating properties were altered. Cx26M34T displayed an inverted response to transjunctional voltage (Vj), mediating currents that activate in a time- and Vj-dependent manner. These characteristics suggest that the channel population is only partially open at rest, consistent with previous reports that dye transfer in M34T-expressing cells is reduced or abolished (e.g., Thonnissen et al., Human Genet. 111, 190-197). To investigate the controversial recessive/dominant behavior of this mutant, we coexpressed M34T with wtCx26 RNA at equimolar levels, mimicking the situation in heterozygotic individuals. Under these conditions, M34T did not significantly reduce Cx26/Cx26 coupling, or alter the electrophysiological properties of the wt channels, consistent with the recessive nature of the allele. Overexpression of the mutant did have some inhibitory effects on conductance, possibly explaining some of the previous reports in exogenous expression systems and some patients. Consistent with its electrophysiological behavior, we also show that M34T localizes to cell junctions in both transfected HeLa cells and patient-derived tissue.

Connexin expression and cell coupling fail to reverse the v-src transformed growth characteristics of a Cx43-/- cell line.

Gap junctions, composed of connexins, have been shown to suppress transformation in a variety of malignancies and transformed cell types. In addition, transforming factors such as the src oncogene have been shown to directly phosphorylate some connexins (e.g., Cx43) and inhibit coupling. To investigate the role of gap junctions in cell transformsation by v-src, we utilized a clonal cell line derived from Cx43 knockout mice (KoA) that was immortalized, but not transformed. Transfection by v-src induced a marked transformed phenotype characterized by growth in low serum and anchorage-independent conditions. Subsequent transfections by Cx43, Cx32 or vector alone were then tested for their effects on growth. Activity of pp60v-src was confirmed in all transfectants as well as the ability of pp60v-src to phosphorylate Cx43 in several clones. Despite the documented effect of pp60v-src on Cx43 channel closure, modest coupling was still retained in many of the Cx43 and Cx32 transfectants. However, none of the four Cx43 transfected clones showed significant inhibitory effects on proliferation in either anchorage-independent or low serum growth conditions. Of the Cx32 clones, only one in five showed effects on growth in both assays, which was the same ratio observed for the control transfectants. Thus, based on the levels of expression achieved, which were comparable to endogenous levels in established cell lines, neither Cx43 nor Cx32 serve as effective suppressors of the transformed growth phenotype of this v-src expressing cell line.

Mutagenic approaches to modifying gap junction phenotype.

Intercellular communication through gap junctions is essential for the regulation of normal cellular processes. In the diseased state, however, gap junctions may be decreased, inappropriately expressed, or constitutively expressed in either the open or closed state. Thus, it may prove important to develop therapeutic agents to either induce or prevent channel closure. To address this dilemma, the mechanisms that cause channel gating as well as the structure-function and permeability determinants of connexins provide useful information. Residues in the C-terminal tail of Connexin 43 are implicated as sites for phosphorylation by kinases that directly mediate channel gating as well as binding sites that influence gating properties. Gating of gap junctions by pH, insulin, and other growth factors has also been associated with the C-terminal domain. The rational design of inhibitors to channel gating may prove useful for the development of therapeutic agents to maintain Connexin 43 in the open state, with potential benefits in diseases such as cancer, arrhythmias, and the diabetic lens. Alternatively, modeling approaches to obtain gap junctions that are constitutively closed might be targeted to designing compounds that could potentially occlude the pore. In this case, knowledge of the pore-lining residues, as well as permeability determinants, would be useful for developing connexin-specific inhibitors that may have future therapeutic potential for tumor invasiveness and stroke treatment. Thus, information from existing and future studies may lead to the development of site-directed, specific modulators of gap junction communication with potential implications in the therapeutic treatment of disease.

Identification of amino acid residues lining the pore of a gap junction channel.

Gap junctions represent a ubiquitous and integral part of multicellular organisms, providing the only conduit for direct exchange of nutrients, messengers and ions between neighboring cells. However, at the molecular level we have limited knowledge of their endogenous permeants and selectivity features. By probing the accessibility of systematically substituted cysteine residues to thiol blockers (a technique called SCAM), we have identified the pore-lining residues of a gap junction channel composed of Cx32. Analysis of 45 sites in perfused Xenopus oocyte pairs defined M3 as the major pore-lining helix, with M2 (open state) or M1 (closed state) also contributing to the wider cytoplasmic opening of the channel. Additional mapping of a close association between M3 and M4 allowed the helices of the low resolution map (Unger et al., 1999. Science. 283:1176-1180) to be tentatively assigned to the connexin transmembrane domains. Contrary to previous conceptions of the gap junction channel, the residues lining the pore are largely hydrophobic. This indicates that the selective permeabilities of this unique channel class may result from novel mechanisms, including complex van der Waals interactions of permeants with the pore wall, rather than mechanisms involving fixed charges or chelation chemistry as reported for other ion channels.

Application of SCAM (substituted cysteine accessibility method) to gap junction intercellular channels.

The pore-lining residues of gap junction channels determine their permeability to ions and small cellular metabolites. These residues can be identified through systematic cysteine substitution and accessibility analysis, commonly known as SCAM (Substituted Cysteine Accessibility Method). However, application of this technique to intercellular channels is more complicated than for their transmembrane counterparts. We have utilized a novel dual-oocyte perfusion device to apply cysteine reagents to the cytoplasmic face of paired, voltage-clamped Xenopus oocytes. In this configuration, a large and irreversible cysteine reagent MBB (maliemidobutyryl biocytin, mw 537) was shown to readily traverse the gap junction pore and induce conductance changes upon reaction of accessible sites. Of the 11 reactive sites identified, 6 were located in M3, where they span the bilayer. They display a periodicity characteristic of the tilted helix that lines the pore in the gap junction structure of Unger et al. (1999). Access to several of the other sites was attributed to aqueous crevices between transmembrane helices. Reactive sites were slightly different than those identified for gap junction hemichannels (Zhou et al. 1997), suggesting that conformational changes occur upon docking.

Size selectivity between gap junction channels composed of different connexins.

Gap junction channels are traditionally viewed as large, nonspecific pores connecting cells. Recently the diversity in the connexin family has drawn more attention to their permeability characteristics. Several studies have shown that both size and charge contribute to the permeability of gap junctional channels. We have used a graded series of neutral polyethylene glycol probes (PEGs), which eliminate charge contribution completely, to specifically assess the physical exclusion limits of gap junction channels formed by different connexins. Cx 26, 32 and 37 were expressed in paired Xenopus oocytes to form homotypic gap junctional channels. PEG probes were perfused intracellularly into one side of the oocyte pair. A reversible drop in conductance of the gap juctional channels indicated that the probe was small enough to enter the pore and hinder ion flux. Our data suggest that Cx32 channels have a size cut-off between PEG 400 (11.2 A) and PEG 300 (9.6 A) despite their relatively small single channel conductance (approximately 55 pS). Cx26 channels (approximately 130 pS single channel conductance) have a size exclusion limit around PEG 200 (8.0 A), while Cx37 channels show the most restricted size cut-off between PEG 200 (8.0 A) and TriEG (6.8 A), despite having the largest unitary conductance (approximately 300 pS).

Molecular and functional diversity of neural connexins in the retina.

Electrical synapses (gap junctions) in neuronal circuits have become a major focus in the study of network properties such as synchronization and oscillation (Galarreta and Hestrin, 1999; Gibson et al., 1999). Despite the recent progress made in unraveling the contribution of gap junctions to network behavior, little is known about the molecular composition of the junctional constituents. By cloning gap junction proteins [connexins (Cxs)] from zebrafish retina and through functional expression, we demonstrate that the retina possesses a high degree of connexin diversity, which may account for differential functional properties of electrical synapses. Three new Cxs, designated as zebrafish Cx27.5 (zfCx27.5), zfCx44.1, and zfCx55.5, and the carp ortholog of mammalian Cx43 were cloned. By in situ hybridization and in situ RT-PCR, we demonstrate that the four fish connexin mRNAs show differential localization in the retina. Transient functional expression in paired Xenopus oocytes and in the neuroblastoma N2A cell line indicate an extreme range of electrophysiological properties of these connexins in terms of voltage dependence and unitary conductance. For instance, the new zfCx44.1 exhibited high sensitivity to voltage-induced closure with currents decaying rapidly for transjunctional potentials >10 mV, whereas zfCx55.5 channels showed an opposite voltage dependence in response to voltage steps of either polarity. Moreover, although zfCx44.1 channels showed unitary conductance as high as any previously reported for junctional channels (nearly 300 pS), zfCx55. 5 and zfCx27.5 exhibited much lower unitary conductances (<60 pS).

Connexin43 suppresses MFG-E8 while inducing contact growth inhibition of glioma cells.

Gap junction expression has been reported to control the growth of a variety of transformed cells. We undertook parallel analysis of connexins Cx32 and Cx43 in glioma cells, which revealed potential mechanisms underlying this phenomenon and led to several novel findings. Cx43, but not Cx32, suppressed C6 glioma cell growth. Paradoxically, Cx32 transfection resulted in severalfold more dye transfer than Cx43. However, Cx43 transfectants shared endogenous metabolites more efficiently than Cx32 transfectants. Interestingly, a significant portion of Cx43 permeants were incorporated into macromolecules more readily than those that transferred via Cx32. Cx43 induced contact inhibition of cell growth but in contrast to other reports, did not affect log phase growth rates. Cell death, senescence, or suppression of growth factor signaling was not involved because no significant alterations were seen in cell viability, telomerase, or mitogen-activated protein kinase activity. However, suppression of cell growth by Cx43 entailed the secretion of growth-regulatory factors. Most notably, a major component of conditioned medium that was affected by Cx43 was found to be MFG-E8 (milk fat globule epidermal growth factor 8), which is involved in cell anchorage and integrin signaling. These results indicate that Cx43 regulates cell growth by the modulation of extracellular growth factors including MFG-E8. Furthermore, the ability of a Cx to regulate cell growth may rely on its ability to mediate the intercellular transfer of endogenous metabolites but not artificial dyes.

The molecular basis of selective permeability of connexins is complex and includes both size and charge.

Although gap junction channels are still widely viewed as large, non-specific pores connecting cells, the diversity in the connexin family has led more attention to be focused on their permeability characteristics. We summarize here the current status of these investigations, both published and on-going, that reveal both charge and size selectivity between gap junction channels composed of different connexins. In particular, this review will focus on quantitative approaches that monitor the expression level of the connexins, so that it is clear that differences that are seen can be attributed to channel properties. The degree of selectivity that is observed is modest compared to other channels, but is likely to be significant for biological molecules that are labile within the cell. Of particular relevance to the in vivo function of gap junctions, recent studies are summarized that demonstrate that the connexin phenotype can control the nature of the endogenous traffic between cells, with consequent effects on biological effects of gap junctions such as tumor suppression.

Different ionic selectivities for connexins 26 and 32 produce rectifying gap junction channels.

The functional diversity of gap junction intercellular channels arising from the large number of connexin isoforms is significantly increased by heterotypic interactions between members of this family. This is particularly evident in the rectifying behavior of Cx26/Cx32 heterotypic channels (. Proc. Natl. Acad. Sci. USA. 88:8410-8414). The channel properties responsible for producing the rectifying current observed for Cx26/Cx32 heterotypic gap junction channels were determined in transfected mouse neuroblastoma 2A (N2A) cells. Transfectants revealed maximum unitary conductances (gamma(j)) of 135 pS for Cx26 and 53 pS for Cx32 homotypic channels in 120 mM KCl. Anionic substitution of glutamate for Cl indicated that Cx26 channels favored cations by 2.6:1, whereas Cx32 channels were relatively nonselective with respect to charge. In Cx26/Cx32 heterotypic cell pairs, the macroscopic fast rectification of the current-voltage relationship was fully explained at the single-channel level by a rectifying gamma(j) that increased by a factor of 2.9 as the transjunctional voltage (V(j)) changed from -100 to +100 mV with the Cx26 cell as the positive pole. A model of electrodiffusion of ions through the gap junction pore based on Nernst-Planck equations for ion concentrations and the Poisson equation for the electrical potential within the junction is developed. Selectivity characteristics are ascribed to each hemichannel based on either pore features (treated as uniform along the length of the hemichannel) or entrance effects unique to each connexin. Both analytical GHK approximations and full numerical solutions predict rectifying characteristics for Cx32/Cx26 heterotypic channels, although not to the full extent seen empirically. The model predicts that asymmetries in the conductance/permeability properties of the hemichannels (also cast as Donnan potentials) will produce either an accumulation or a depletion of ions within the channel, depending on voltage polarity, that will result in rectification.

Dissection of the molecular basis of pp60(v-src) induced gating of connexin 43 gap junction channels.

Suppression of gap-junctional communication by various protein kinases, growth factors, and oncogenes frequently correlates with enhanced mitogenesis. The oncogene v-src appears to cause acute closure of gap junction channels. Tyr265 in the COOH-terminal tail of connexin 43 (Cx43) has been implicated as a potential target of v-src, although v-src action has also been associated with changes in serine phosphorylation. We have investigated the mechanism of this acute regulation through mutagenesis of Cx43 expressed in Xenopus laevis oocyte pairs. Truncations of the COOH-terminal domain led to an almost complete loss of response of Cx43 to v-src, but this was restored by coexpression of the independent COOH-terminal polypeptide. This suggests a ball and chain gating mechanism, similar to the mechanism proposed for pH gating of Cx43, and K+ channel inactivation. Surprisingly, we found that v-src mediated gating of Cx43 did not require the tyrosine site, but did seem to depend on the presence of two potential SH3 binding domains and the mitogen-activated protein (MAP) kinase phosphorylation sites within them. Further point mutagenesis and pharmacological studies in normal rat kidney (NRK) cells implicated MAP kinase in the gating response to v-src, while the stable binding of v-src to Cx43 (in part mediated by SH3 domains) did not correlate with its ability to mediate channel closure. This suggests a common link between closure of gap junctions by v-src and other mitogens, such as EGF and lysophosphatidic acid (LPA).

The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for the docking interface of gap junctions.

Connexins, like true cell adhesion molecules, have extracellular domains that provide strong and specific homophilic, and in some cases, heterophilic interactions between cells. Though the structure of the binding domains of adhesion proteins have been determined, the extracellular domains of connexins, consisting of two loops of approximately 34-37 amino acids each, are not easily studied in isolation from the rest of the molecule. As an alternative, we used a novel application of site-directed mutagenesis in which four of the six conserved cysteines in the extracellular loops of connexin 32 were moved individually and in all possible pairwise and some quadruple combinations. This mapping allowed us to deduce that all disulfides form between the two loops of a single connexin, with the first cysteine in one loop connected to the third of the other. Furthermore, the periodicity of movements that produced functional channels indicated that these loops are likely to form antiparallel beta sheets. A possible model that could explain how these domains from apposed connexins interact to form a complete channel is discussed.

Direct isolation and analysis of endogenous transjunctional ADP from Cx43 transfected C6 glioma cells.

Gap junctional communication has been implicated in numerous cellular processes. However, the repertoire of specific transjunctional substances which mediate these processes remains relatively unexplored. A few selected secondary messengers have been identified, at least indirectly (e.g., cAMP and IP3) and phenotypic complementation experiments have indicated that gap junctions enable communicating cells to distribute nucleotide pools as a shared resource. The latter would include high energy compounds such as ADP and ATP, allowing cells to share energy resources. We have utilized a nonbiased process to directly capture, identify, and quantify transjunctional compounds from C6 glioma cells, the transformed phenotype of which has been ameliorated by transfection with connexin43 (Cx43). This technique involves the direct isolation, identification, and quantitation of radioactive transjunctional molecules that travel from metabolically labeled "donor" cells to "receiver" cells. This report demonstrates that ADP and/or ATP represents over 6% of the transjunctional material derived from glucose in Cx43-transfected C6 glioma cells. Furthermore, equilibration of these high energy metabolites among first order neighbors is shown to occur in less than 20 min of communication.

A quantitative analysis of connexin-specific permeability differences of gap junctions expressed in HeLa transfectants and Xenopus oocytes.

Gap junctions provide direct intercellular communication by linking adjacent cells with aqueous pores permeable to molecules up to 1 kDa in molecular mass and 8-14 A in diameter. The identification of over a dozen connexins in the mammalian gap junction family has stimulated interest in the functional significance of this diversity, including the possibility of selectivity for permeants as seen in other channel classes. Here we present a quantitative comparison of channel permeabilities of different connexins expressed in both HeLa transfectants (rat Cx26, rat Cx32 and mouse Cx45) and Xenopus oocytes (rat Cx26 and rat Cx32). In HeLa cells, we examined permeability to two fluorescent molecules: Lucifer Yellow (LY: anionic, MW 457) and 4',6-diamidino-2-phenylindole, dihydrochloride (DAPI, cationic, MW 350). A comparison of the kinetics of fluorescent dye transfer showed Cx32, Cx26 and Cx45 to have progressively decreasing permeabilities to LY, but increasing permeabilities to DAPI. This pattern was inconsistent with selection based on physical size of the probe, nor could it be accounted for by the differences between clones in the electrical conductance of the monolayers. In Xenopus oocytes, where electrical and dye coupling could be assessed in the same cells, Cx32 coupled oocytes showed an estimated 6-fold greater permeability to LY than those coupled by Cx26, a comparable result to that seen in HeLa cells, where an approximately 9-fold difference was seen. The oocyte system also allowed an examination of Cx32/Cx26 heterotypic gap junction that proved to have a permeability intermediate between the two homotypic forms. Thus, independent of the expression system, it appears that connexins show differential permeabilities that cannot be predicted based on size considerations, but must depend on other features of the probe, such as charge.

Molecular cloning and functional expression of mouse connexin-30,a gap junction gene highly expressed in adult brain and skin.

A new gap junction gene isolated from the mouse genome codes for a connexin protein of 261 amino acids. Because of its theoretical molecular mass of 30.366 kDa, it is named connexin-30. Within the connexin gene family, this protein is most closely related to connexin-26 (77% amino acid sequence identity). The coding region of mouse connexin-30 is uninterrupted by introns and is detected in the mouse genome as a single copy gene that is assigned to mouse chromosome 14 by analysis of mouse x hamster somatic cell hybrids. Abundant amounts of connexin-30 mRNA (two transcripts of 2.0 and 2.3 kilobase pairs) were found after 4 weeks of postnatal development in mouse brain and skin. Microinjection of connexin-30 cRNA into Xenopus oocytes induced formation of functional gap junction channels that gated somewhat asymmetrically in response to transjunctional voltage and at significantly lower voltage (Vo = +38 and -46 mV) than the closely homologous connexin-26 channels (Vo = 89 mV). Heterotypic pairings of connexin-30 with connexin-26 and connexin-32 produced channels with highly asymmetric and rectifying voltage gating, respectively. This suggests that the polarity of voltage gating and the cationic selectivity of connexin-30 are similar to those of its closest homologue, connexin-26.

Structure of gap junction intercellular channels.

Gap junctions are formed by a multigene family of polytopic membrane channel proteins, connexins, that have four hydrophobic transmembrane domains and their N and C termini located on the cytoplasmic membrane face. The C-terminal tail plays important roles in channel regulation by pH and phosphorylation. Conserved cysteine residues stabilize the conformation of the extracellular loops that mediate the 'docking' between connexons in the intercellular channel. Over the past year, electron cryocrystallography of two-dimensional crystals of a truncated recombinant alpha 1 (Cx43) has revealed that the transmembrane boundary of the intercellular channel is lined with alpha helices. Furthermore, a ring of alpha helices resides at the interface with the membrane lipids. A three-dimensional analysis based on images recorded from tilted crystals should reveal the location and secondary structure of additional transmembrane domains, as well as provide important structural details about the interactions between connexins within a hemi-channel and connexon-connexon interactions in the extracellular gap.

Membrane integration of in vitro-translated gap junctional proteins: co- and post-translational mechanisms.

Connexins (Cx) are protein components of gap junction channels that permit the passage of small molecules between neighboring cells. cDNAs of a large family of connexins have been isolated and sequenced. A gap junction channel consists of two connexons, one from each cell in contact, composed of six connexin subunits. It has been suggested by Musil and coworkers that the oligomerization of formation of a connexon occurs at the level of the trans-Golgi network. In the present study, we initiated an analysis of the early stages of protein synthesis and membrane insertion of Cx32 and Cx26, two connexins that we have demonstrated are co-expressed in the same junctions in hepatocytes. Using an in vitro transcription and a coupled cell-free translation and translocation system, we observed that both Cx32 and Cx26 could insert into microsome membranes co-translationally, producing a topological structure indistinguishable from that in isolated gap junctions. To our surprise, Cx26 could also insert into membranes post-translationally with a native orientation. This post-translational membrane insertion process is dependent on nucleotides but not their hydrolysis. Cx32, on the other hand, could not insert into membranes post-translationally. These disparate properties of Cx32 and Cx26 are not due to the significant difference in the lengths of their C-terminal domains, but rather to their internal amino acid sequences. These observations raise the possibility that there may be another pathway for Cx26 to insert into membranes in cells and this feature may be important for the regulation of its functions. These findings may also lead us to a new approach to reconstitution without detergent extraction.