Connexin43 Hemichannel-Mediated Regulation of Connexin43

Background

Many signaling molecules and pathways that regulate gap junctions (GJs) protein expression and function are, in fact, also controlled by GJs. We, therefore, speculated an existence of the GJ channel-mediated self-regulation of GJs. Using a cell culture model in which nonjunctional connexin43 (Cx43) hemichannels were activated by cadmium (Cd²⁺), we tested this hypothesis.

Principal Findings

Incubation of Cx43-transfected LLC-PK1 cells with Cd²⁺ led to an increased expression of Cx43. This effect of Cd²⁺ was tightly associated with JNK activation. Inhibition of JNK abolished the elevation of Cx43. Further analysis revealed that the changes of JNK and Cx43 were controlled by GSH. Supplement of a membrane-permeable GSH analogue GSH ethyl ester or GSH precursor N-acetyl-cystein abrogated the effects of Cd²⁺ on JNK activation and Cx43 expression. Indeed, Cd²⁺ induced extracellular release of GSH. Blockade of Cx43 hemichannels with heptanol or Cx43 mimetic peptide Gap26 to prevent the efflux of GSH significantly attenuated the Cx43-elevating effects of Cd²⁺.

Conclusions

Collectively, our results thus indicate that Cd²⁺-induced upregulation of Cx43 is through activation of nonjunctional Cx43 hemichannels. Our findings thus support the existence of a hemichannel-mediated self-regulation of Cx43 and provide novel insights into the molecular mechanisms of Cx43 expression and function.     

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 InsP₃ 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.     

Inducible Coexpression of Connexin37 or Connexin40 with Connexin43 Selectively Affects Intercellular Molecular Transfer

Many tissues express multiple gap junction proteins, or connexins (Cx); for example, Cx43, Cx40, and Cx37 are coexpressed in vascular cells. This study was undertaken to elucidate the consequences of coexpression of Cx40 or Cx37 with Cx43 at different ratios. EcR-293 cells (which endogenously produce Cx43) were transfected with ecdysone-inducible plasmids encoding Cx37 or Cx40. Immmunoblotting showed a ponasterone dose-dependent induction of Cx37 or Cx40 while constant levels of Cx43 were maintained. The coexpressed connexins colocalized at appositional membranes. Double whole-cell patch clamp recordings showed no significant change in total junctional conductances in cells treated with 0, 0.5, or 4?μM ponasterone; however, they did show a diversity of unitary channel sizes consistent with the induced connexin expression. In cells with induced expression of either Cx40 or Cx37, intercellular transfer of microinjected Lucifer yellow was reduced, but transfer of NBD-TMA (2-(4-nitro-2,1,3-benzoxadiol-7-yl)[aminoethyl]trimethylammonium) was not affected. In cocultures containing uninduced EcR cells together with cells induced to coexpress Cx37 or Cx40, Lucifer yellow transfer was observed only between the cells expressing Cx43 alone. These data show that induced expression of either Cx37 or Cx40 in Cx43-expressing cells can selectively alter the intercellular exchange of some molecules without affecting the transfer of others.     

Role of Connexin37 and Connexin40 in Vascular Development

Mice lacking both connexin37 (Cx37) and connexin40 (Cx40), gap junction proteins expressed in vascular endothelium, die perinatally with pronounced vascular abnormalities. Early vasculogenesis proceeds normally, but by E18.5 Cx37^?/?Cx40^?/?animals display vessel dilatation and congestion as well as localized hemorrhages in skin, testis, intestines, and lungs. Abnormal vascular channels are present in the testis, often forming cavernous hemangioma-like defects. Unusually large, distended vessels are also present in the submucosa and lamina propria of the intestine. Ablation of Cx40 has a greater effect on endothelial dye-transfer than ablation of Cx37, and the effect of Cx40 ablation is age-dependent. Only in embryonic aortas lacking both Cx37 and Cx40 is there a complete loss of endothelial coupling. Surprisingly, elimination of Cx40 results in a large drop in aortic endothelial Cx37 on western blots, and deletion of Cx37 also reduces endothelial Cx40 levels. In contrast, in the medial layer, both Cx37 and Cx43 increase when Cx40 is ablated. These studies indicate that Cx37 and Cx40 are collectively critical for endothelial communication and provide evidence of an important role for gap junctions in vascular development. In addition, Cx37 and Cx40 appear to be mutually dependent on each other for normal expression in vascular endothelium.     

Mouse Horizontal Cells do not Express Connexin26 or Connexin36

Gap junctions between neurons function as electrical synapses, and are present in all layers of mammalian and teleost retina. These synapses are largest and most prominent between horizontal cells where they function to increase the receptive field of a single neuron beyond the width of its dendrites. Receptive field size and the extent of gap junctional coupling between horizontal cells is regulated by ambient light levels and may mediate light/dark adaptation. Furthermore, teleost horizontal cell gap junction hemichannels may facilitate a mechanism of feedback inhibition between horizontal cells and cone photoreceptors. As a prelude to using mouse genetic models to study horizontal cell gap junctions and hemichannels, we sought to determine the connexin complement of mouse horizontal cells. Cx36, Cx37, Cx43, Cx45 and Cx57 mRNA could be detected in mouse retina by RT-PCR. Microscopy was used to further examine the distribution of Cx26 and Cx36. Cx26 immunofluorescence and a β-gal reporter under regulatory control of the Cx36 promoter did not colocalize with a horizontal cell marker, indicating that these genes are not expressed by horizontal cells. The identity of the connexin(s) forming electrical synapses between mouse horizontal cells and the connexin that may form hemichannels in the horizontal cell telodendria remains unknown.     

Differential Oligomerization of Endoplasmic Reticulum-Retained Connexin43/Connexin32 Chimeras

To examine early events in connexin oligomerization, we made connexin constructs containing a C-terminal di-lysine based endoplasmic reticulum (ER) retention/retrieval signal (HKKSL). Previously, we found that both Cx32-HKKSL and Cx43-HKKSL were retained in the ER. However, Cx32-HKKSL oligomerized into hexameric hemichannels, but Cx43-HKKSL was retained as an apparent monomer. To define elements that prevent Cx43-HKKSL oligomerization in the ER, we made a series of HKKSL-tagged Cx43/Cx32 chimeras. When expressed by HeLa cells, some chimeras were retained in the ER as apparent monomers, whereas others oligomerized in the ER. To date, the second and third transmembrane domains and the cytoplasmic loop domain provide the minimal sufficient Cx43 element to inhibit ER oligomerization.     

缝隙连接蛋白43 研究进展

缝隙连接蛋白(Connexin,Cx)组成缝隙连接通道发挥其通讯功能,其本身也对细胞的生长、分化、凋亡、肿瘤具有调节作用。缝隙连接蛋白43(Cx43)在肺泡上皮和肺血管内皮高表达,在多种肺损伤的病理过程中起重要作用,因此成为研究热点。Cx43的表达和功能受多种因素的调节,丝裂原激活的蛋白激酶(MAPKs)是主要的调节途径。本文就Cx43在肺的病理生理过程中的作用及MAPK对其调节作用进行综述。     

Permeation Pathway of Homomeric Connexin 26 and Connexin 30 Channels Investigated by Molecular Dynamics

Abstract

Mutations in the genes GJB2 and GJB6 encoding human connnexin26 (hCx26) and connexin30 (hCx30), respectively, are the leading cause of non-syndromic prelingual deafness in several human populations. In this work, we exploited the high degree (77%) of sequence similarity shared by hCx26 and hCx30 to create atomistic models of homomeric hCx26 and hCx30 connexons starting from the X-ray crystallographic structure of an intercellular channel formed by hCx26 protomers at 3.5-Å resolution. The equilibrium dynamics of the two protein complexes was followed for 40 ns each by Molecular Dynamics (MD) simulations. Our results indicate that, in hCx26, positively charged Lys41 residues establish a potential barrier within the fully open channel, hindering ion diffusion in the absence of an electrochemical gradient. A similar role is played, in hCx30, by negatively charged Glu49 residues. The different position and charge of these two ion sieves account for the differences in unitary conductance observed experimentally. Our results are discussed in terms of present models of voltage gating in connexin channels.     

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 γ_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 γ_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.     

连接蛋白基因突变与疾病

连接蛋白是构成缝隙连接的结构分子,由多基因家族编码。目前在啮齿类中已发现至少有１３各连接蛋白。由于缝隙连接在细胞的生长和分化过程中具有重要作用,连接蛋白基因突变与人类疾病之间的关系已成 一人新的研究领域。迄今为止,已在三种人类疾病中发现有连接蛋白基因突变。     

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_jand γ_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_jin Cx40/Cx40 pairs, but decreased g_jin 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_jsuggest that a single subunit of Cx43 is sufficient to confer PDGF sensitivity. The effect of PDGF on g_jinvolved a decrease in both γ_jand 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.     

Connexin 33 Impairs Gap Junction Functionality by Accelerating Connexin 43 Gap Junction Plaque Endocytosis

Connexin 33 (Cx33) is a testis-specific gap junction protein. We previously reported that Cx33 exerts dominant-negative effect on gap junction intercellular communication by sequestering Cx43 within early endosomes in Sertoli cells. However, the molecular mechanisms that drive this process are unknown. The present study analyzed: (i) the trafficking of Cx33 and Cx43 in wild-type Sertoli cells transfected with Cx33-DsRed2 and Cx43-green fluorescent protein vectors; (ii) the formation of heteromeric Cx33/Cx43 hemi-channels and their incorporation into gap junction plaques. Fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer and videomicroscopy studies demonstrated that Cx33 and Cx43 associated to form heteromeric oligomers that trafficked along microtubules to the plasma membrane. However, the plaques containing Cx33 were not functional. Immunoprecipitation experiments revealed that zonula occludens-1 (ZO-1), a scaffold protein proposed to secure Cx in gap junction plaques at the cell–cell boundary, associated with Cx33 in testis extracts. In cells expressing Cx33, Cx33 and ZO-1 specifically interacted with P₁ phosphorylated and P₀ unphosphorylated isoforms of Cx43, and the ZO-1 membranous signal level was reduced. It is suggested that alteration of Cx43/ZO-1 association by Cx33 could be one mechanism by which Cx33 exerts its dominant-negative effect on gap junction plaque.     

Connexin and gap junction degradation

Many of the subunit proteins (connexins) that form gap junctions are rather dynamic, with half-lives of only a few hours. Thus, alterations in connexin turnover and degradation may represent significant mechanisms for the regulation of intercellular communication. We describe a pharmacological approach to determining pathways of connexin degradation. Cell cultures are left untreated or treated with inhibitors of lysosomal or proteasomal proteolysis. Changes in connexin levels, localization, or decay curves (derived from pulse-chase experiments) are assessed by immunoblotting, immunofluorescence, and immunoprecipitation, respectively. Such experiments have provided evidence that connexin43 degradation involves both the lysosome and the proteasome.     

Influence of V5/6-His Tag on the Properties of Gap Junction Channels Composed of Connexin43, Connexin40 or Connexin45

HeLa cells expressing wild-type connexin43, connexin40 or connexin45 and connexins fused with a V5/6-His tag to the carboxyl terminus (CT) domain (Cx43-tag, Cx40-tag, Cx45-tag) were used to study connexin expression and the electrical properties of gap junction channels. Immunoblots and immunolabeling indicated that tagged connexins are synthesized and targeted to gap junctions in a similar manner to their wild-type counterparts. Voltage-clamp experiments on cell pairs revealed that tagged connexins form functional channels. Comparison of multichannel and single-channel conductances indicates that tagging reduces the number of operational channels, implying interference with hemichannel trafficking, docking and/or channel opening. Tagging provoked connexin-specific effects on multichannel and single-channel properties. The Cx43-tag was most affected and the Cx45-tag, least. The modifications included (1) V _j-sensitive gating of I _j (V _j, gap junction voltage; I _j, gap junction current), (2) contribution and (3) kinetics of I _j deactivation and (4) single-channel conductance. The first three reflect alterations of fast V _j gating. Hence, they may be caused by structural and/or electrical changes on the CT that interact with domains of the amino terminus and cytoplasmic loop. The fourth reflects alterations of the ion-conducting pathway. Conceivably, mutations at sites remote from the channel pore, e.g., 6-His-tagged CT, affect protein conformation and thus modify channel properties indirectly. Hence, V5/6-His tagging of connexins is a useful tool for expression studies in vivo. However, it should not be ignored that it introduces connexin-dependent changes in both expression level and electrophysiological properties.     

Connexin Mediated Cataract Prevention in Mice

Cataracts, named for any opacity in the ocular lens, remain the leading cause of vision loss in the world. Non-surgical methods for cataract prevention are still elusive. We have genetically tested whether enhanced lens gap junction communication, provided by increased α3 connexin (Cx46) proteins expressed from α8(Kiα3) knock-in alleles in Gja8^tm1(Gja3)Tww mice, could prevent nuclear cataracts caused by the γB-crystallin S11R mutation in Crygb^S11R/S11R mice. Remarkably, homozygous knock-in α8(Kiα3/Kiα3) mice fully prevented nuclear cataracts, while single knock-in α8(Kiα3/−) allele mice showed variable suppression of nuclear opacities in Crygb^S11R/S11R mutant mice. Cataract prevention was correlated with the suppression of many pathological processes, including crystallin degradation and fiber cell degeneration, as well as preservation of normal calcium levels and stable actin filaments in the lens. This work demonstrates that enhanced intercellular gap junction communication can effectively prevent or delay nuclear cataract formation and suggests that small metabolites transported through gap junction channels protect the stability of crystallin proteins and the cytoskeletal structures in the lens core. Thus, the use of an array of small molecules to promote lens homeostasis may become a feasible non-surgical approach for nuclear cataract prevention in the future.     

Connexin36 and pancreatic beta-cell functions

Most cell types are functionally coupled by connexin (Cx) channels, i.e. exchange cytoplasmic ions and small metabolites through gap junction domains of their membrane. This form of direct cell-to-cell communication occurs in all existing animals, whatever their position in the phylogenetic scale, and up to humans. Pancreatic beta-cells are no exception, and normally cross-talk with their neighbors via channels made of Cx36. These exchanges importantly contribute to coordinate and synchronize the function of individual cells within pancreatic islets, particularly in the context of glucose-induced insulin secretion. Compelling evidence now indicates that Cx36-mediated coupling, and/or the Cx36 protein per se, play significant regulatory roles in various beta-cell functions, ranging from the biosynthesis, storage and release of insulin. Recent preliminary data further suggest that the protein may also be implicated in the balance of beta-cell growth versus necrosis and apoptosis, and in the regulated expression of specific genes. Here, we review this evidence, discuss the possible involvement of Cx36 in the pathophysiology of diabetes, and evaluate the relevance of this connexin in the therapeutic approaches to the disease.     

Connexin and pannexin channels in cancer

Communication among cells via direct cell-cell contact by connexin gap junctions, or between cell and extracellular environment via pannexin channels or connexin hemichannels, is a key factor in cell function and tissue homeostasis. Upon malignant transformation in different cancer types, the dysregulation of these connexin and pannexin channels and their effect in cellular communication, can either enhance or suppress tumorigenesis and metastasis. In this review, we will highlight the latest reports on the role of the well characterized connexin family and its ability to form gap junctions and hemichannels in cancer. We will also introduce the more recently discovered family of pannexin channels and our current knowledge about their involvement in cancer progression.