Connexin Gap Junction Channels and Hemichannels: Insights from High-Resolution Structures.

Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αß fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a "ball-and-chain" fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein-protein and protein-lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation.

Free energy and kinetics of cAMP permeation through connexin26 via applied voltage and milestoning.

The connexin family is a diverse group of highly regulated wide-pore channels permeable to biological signaling molecules. Despite the critical roles of connexins in mediating selective molecular signaling in health and disease, the basis of molecular permeation through these pores remains unclear. Here, we report the thermodynamics and kinetics of binding and transport of a second messenger, adenosine-3',5'-cyclophosphate (cAMP), through a connexin26 hemichannel (Cx26). First, inward and outward fluxes of cAMP molecules solvated in KCl solution were obtained from 4 µs of ± 200 mV simulations. These fluxes data yielded a single-channel permeability of cAMP and cAMP/K+ permeability ratio consistent with experimentally measured values. The results from voltage simulations were then compared with the potential of mean force (PMF) and the mean first passage times (MFPTs) of a single cAMP without voltage, obtained from a total of 16.5 µs of Voronoi-tessellated Markovian milestoning simulations. Both the voltage simulations and the milestoning simulations revealed two cAMP-binding sites, for which the binding constants KD and dissociation rates koff were computed from PMF and MFPTs. The protein dipole inside the pore produces an asymmetric PMF, reflected in unequal cAMP MFPTs in each direction once within the pore. The free energy profiles under opposite voltages were derived from the milestoning PMF and revealed the interplay between voltage and channel polarity on the total free energy. In addition, we show how these factors influence the cAMP dipole vector during permeation, and how cAMP affects the local and nonlocal pore diameter in a position-dependent manner.

A novel voltage-clamp/dye uptake assay reveals saturable transport of molecules through CALHM1 and connexin channels.

Large-pore channels permeable to small molecules such as ATP, in addition to atomic ions, are emerging as important regulators in health and disease. Nonetheless, their mechanisms of molecular permeation and selectivity remain mostly unexplored. Combining fluorescence microscopy and electrophysiology, we developed a novel technique that allows kinetic analysis of molecular permeation through connexin and CALHM1 channels in Xenopus oocytes rendered translucent. Using this methodology, we found that (1) molecular flux through these channels saturates at low micromolar concentrations, (2) kinetic parameters of molecular transport are sensitive to modulators of channel gating, (3) molecular transport and ionic currents can be differentially affected by mutation and gating, and (4) N-terminal regions of these channels control transport kinetics and permselectivity. Our methodology allows analysis of how human disease-causing mutations affect kinetic properties and permselectivity of molecular signaling and enables the study of molecular mechanisms, including selectivity and saturability, of molecular transport in other large-pore channels.

The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening.

A group of human mutations within the N-terminal (NT) domain of connexin 26 (Cx26) hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. In this study, we explore the molecular basis by which N14K, an NT KID mutant, promotes gain of function. In macroscopic and single-channel recordings, we find that the N14K mutant favors the open conformation of hemichannels, shifts calcium and voltage sensitivity, and slows deactivation kinetics. Multiple copies of MD simulations of WT and N14K hemichannels, followed by the Kolmogorov-Smirnov significance test (KS test) of the distributions of interaction energies, reveal that the N14K mutation significantly disrupts pairwise interactions that occur in WT hemichannels between residue K15 of one subunit and residue E101 of the adjacent subunit (E101 being located at the transition between transmembrane segment 2 [TM2] and the cytoplasmic loop [CL]). Double mutant cycle analysis supports coupling between the NT and the TM2/CL transition in WT hemichannels, which is disrupted in N14K mutant hemichannels. KS tests of the α carbon correlation coefficients calculated over MD trajectories suggest that the effects of the N14K mutation are not confined to the K15-E101 pairs but extend to essentially all pairwise residue correlations between the NT and TM2/CL interface. Together, our data indicate that the N14K mutation increases hemichannel open probability by disrupting interactions between the NT and the TM2/CL region of the adjacent connexin subunit. This suggests that NT-TM2/CL interactions facilitate Cx26 hemichannel closure.

Electrical coupling and its channels.

As the physiology of synapses began to be explored in the 1950s, it became clear that electrical communication between neurons could not always be explained by chemical transmission. Instead, careful studies pointed to a direct intercellular pathway of current flow and to the anatomical structure that was (eventually) called the gap junction. The mechanism of intercellular current flow was simple compared with chemical transmission, but the consequences of electrical signaling in excitable tissues were not. With the recognition that channels were a means of passive ion movement across membranes, the character and behavior of gap junction channels came under scrutiny. It became evident that these gated channels mediated intercellular transfer of small molecules as well as atomic ions, thereby mediating chemical, as well as electrical, signaling. Members of the responsible protein family in vertebrates-connexins-were cloned and their channels studied by many of the increasingly biophysical techniques that were being applied to other channels. As described here, much of the evolution of the field, from electrical coupling to channel structure-function, has appeared in the pages of the Journal of General Physiology.

Intramolecular signaling in a cardiac connexin: Role of cytoplasmic domain dimerization.

Gap junctions, composed of connexins, mediate electrical coupling and impulse propagation in the working myocardium. In the human heart, the spatio-temporal regulation and distinct functional properties of the three dominant connexins (Cx43, Cx45, and Cx40) suggests non-redundant physiological roles for each isoform. There are substantial differences in gating properties, expression, and trafficking among these isoforms, however, little is known about the determinants of these different phenotypes. To gain insight regarding these determinants, we focused on the carboxyl-terminal (CT) domain because of its importance in channel regulation and large degree of sequence divergence among connexin family members. Using in vitro biophysical experiments, we identified a structural feature unique to Cx45: high affinity (KD~100nM) dimerization between CT domains. In this study, we sought to determine if this dimerization occurs in cells and to identify the biological significance of the dimerization. Using a bimolecular fluorescence complementation assay, we demonstrate that the CT domains dimerize at the plasma membrane. By inhibiting CT dimerization with a mutant construct, we show that CT dimerization is necessary for proper Cx45 membrane localization, turnover, phosphorylation status, and binding to protein partners. Furthermore, CT dimerization is needed for normal intercellular communication and hemichannel activity. Altogether, our results demonstrate that CT dimerization is a structural feature important for correct Cx45 function. This study is significant because discovery of how interactions mediated by the CT domains can be modulated would open the door to strategies to ameliorate the pathological effects of altered connexin regulation in the failing heart.

Genomic instability induced in distant progeny of bystander cells depends on the connexins expressed in the irradiated cells.

PURPOSE: To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation; and to investigate the impact of gap junction communication on genomic instability in distant progeny of bystander cells. MATERIALS AND METHODS: A layered cell culture system was developed to investigate the propagation of harmful effects from irradiated normal or tumor cells that express specific connexins to contiguous bystander normal human fibroblasts. Irradiated cells were exposed to moderate mean absorbed doses from 3.7 MeV α particle, 1000 MeV/u iron ions, 600 MeV/u silicon ions, or 137Cs γ rays. Following 5 h of co-culture, pure populations of bystander cells, unexposed to secondary radiation, were isolated and DNA damage and oxidative stress was assessed in them and in their distant progeny (20-25 population doublings). RESULTS: Increased frequency of micronucleus formation and enhanced oxidative changes were observed in bystander cells co-cultured with confluent cells exposed to either sparsely ionizing (137Cs γ rays) or densely ionizing (α particles, energetic iron or silicon ions) radiations. The irradiated cells propagated signals leading to biological changes in bystander cells within 1 h of irradiation, and the effect required cellular coupling by gap junctions. Notably, the distant progeny of isolated bystander cells also exhibited increased levels of spontaneous micronuclei. This effect was dependent on the type of junctional channels that coupled the irradiated donor cells with the bystander cells. Previous work showed that gap junctions composed of connexin26 (Cx26) or connexin43 (Cx43) mediate toxic bystander effects within 5 h of co-culture, whereas gap junctions composed of connexin32 (Cx32) mediate protective effects. In contrast, the long-term progeny of bystander cells expressing Cx26 or Cx43 did not display elevated DNA damage, whereas those coupled by Cx32 had enhanced DNA damage. CONCLUSIONS: In response to moderate doses from either sparsely or densely ionizing radiations, toxic and protective effects are rapidly communicated to bystander cells through gap junctions. We infer that bystander cells damaged by the initial co-culture (expressing Cx26 or Cx43) die or undergo proliferative arrest, but that the bystander cells that were initially protected (expressing Cx32) express DNA damage upon sequential passaging. Together, the results inform the roles that intercellular communication play under stress conditions, and aid assessment of the health risks of exposure to ionizing radiation. Identification of the communicated molecules may enhance the efficacy of radiotherapy and help attenuate its debilitating side-effects.

Endothelial cAMP deactivates ischemia-reperfusion-induced microvascular hyperpermeability via Rap1-mediated mechanisms.

Approaches to reduce excessive edema due to the microvascular hyperpermeability that occurs during ischemia-reperfusion (I/R) are needed to prevent muscle compartment syndrome. We tested the hypothesis that cAMP-activated mechanisms actively restore barrier integrity in postischemic striated muscle. We found, using I/R in intact muscles and hypoxia-reoxygenation (H/R, an I/R mimic) in human microvascular endothelial cells (HMVECs), that hyperpermeability can be deactivated by increasing cAMP levels through application of forskolin. This effect was seen whether or not the hyperpermeability was accompanied by increased mRNA expression of VEGF, which occurred only after 4 h of ischemia. We found that cAMP increases in HMVECs after H/R, suggesting that cAMP-mediated restoration of barrier function is a physiological mechanism. We explored the mechanisms underlying this effect of cAMP. We found that exchange protein activated by cAMP 1 (Epac1), a downstream effector of cAMP that stimulates Rap1 to enhance cell adhesion, was activated only at or after reoxygenation. Thus, when Rap1 was depleted by small interfering RNA, H/R-induced hyperpermeability persisted even when forskolin was applied. We demonstrate that 1) VEGF mRNA expression is not involved in hyperpermeability after brief ischemia, 2) elevation of cAMP concentration at reperfusion deactivates hyperpermeability, and 3) cAMP activates the Epac1-Rap1 pathway to restore normal microvascular permeability. Our data support the novel concepts that 1) different hyperpermeability mechanisms operate after brief and prolonged ischemia and 2) cAMP concentration elevation during reperfusion contributes to deactivation of I/R-induced hyperpermeability through the Epac-Rap1 pathway. Endothelial cAMP management at reperfusion may be therapeutic in I/R injury.NEW & NOTEWORTHY Here, we demonstrate that 1) stimulation of cAMP production deactivates ischemia-reperfusion-induced hyperpermeability in muscle and endothelial cells; 2) VEGF mRNA expression is not enhanced by brief ischemia, suggesting that VEGF mechanisms do not activate immediate postischemic hyperpermeability; and 3) deactivation mechanisms operate via cAMP-exchange protein activated by cAMP 1-Rap1 to restore integrity of the endothelial barrier.

Non-junctional Cx32 mediates anti-apoptotic and pro-tumor effects via epidermal growth factor receptor in human cervical cancer cells.

The role of connexin proteins (Cx), which form gap junctions (GJ), in progression and chemotherapeutic sensitivity of cervical cancer (CaCx), is unclear. Using cervix specimens (313 CaCx, 78 controls) and CaCx cell lines, we explored relationships among Cx expression, prognostic variables and mechanisms that may link them. In CaCx specimens, Cx32 was upregulated and cytoplasmically localized, and three other Cx downregulated, relative to controls. Cx32 expression correlated with advanced FIGO staging, differentiation and increased tumor size. In CaCx cell lines, Cx32 expression suppressed streptonigrin/cisplatin-induced apoptosis in the absence of functional GJ. In CaCx specimens and cell lines, expression of Cx32 upregulated epidermal growth factor receptor (EGFR) expression. Inhibition of EGFR signaling abrogated the anti-apoptotic effect of Cx32 expression. In conclusion, upregulated Cx32 in CaCx cells produces anti-apoptotic, pro-tumorigenic effects in vivo and vitro. Abnormal Cx32 expression/localization in CaCx appears to be both a mechanism and biomarker of chemotherapeutic resistance.

Mechanism of gating by calcium in connexin hemichannels.

Aberrant opening of nonjunctional connexin hemichannels at the plasma membrane is associated with many diseases, including ischemia and muscular dystrophy. Proper control of hemichannel opening is essential to maintain cell viability and is achieved by physiological levels of extracellular Ca2+, which drastically reduce hemichannel activity. Here we examined the role of conserved charged residues that form electrostatic networks near the extracellular entrance of the connexin pore, a region thought to be involved in gating rearrangements of hemichannels. Molecular dynamics simulations indicate discrete sites for Ca2+ interaction and consequent disruption of salt bridges in the open hemichannels. Experimentally, we found that disruption of these salt bridges by mutations facilitates hemichannel closing. Two negatively charged residues in these networks are putative Ca2+ binding sites, forming a Ca2+-gating ring near the extracellular entrance of the pore. Accessibility studies showed that this Ca2+-bound gating ring does not prevent access of ions or small molecules to positions deeper into the pore, indicating that the physical gate is below the Ca2+-gating ring. We conclude that intra- and intersubunit electrostatic networks at the extracellular entrance of the hemichannel pore play critical roles in hemichannel gating reactions and are tightly controlled by extracellular Ca2+ Our findings provide a general mechanism for Ca2+ gating among different connexin hemichannel isoforms.

Computational Studies of Molecular Permeation through Connexin26 Channels.

A signal property of connexin channels is the ability to mediate selective diffusive movement of molecules through plasma membrane(s), but the energetics and determinants of molecular movement through these channels have yet to be understood. Different connexin channels have distinct molecular selectivities that cannot be explained simply on the basis of size or charge of the permeants. To gain insight into the forces and interactions that underlie selective molecular permeation, we investigated the energetics of two uncharged derivatized sugars, one permeable and one impermeable, through a validated connexin26 (Cx26) channel structural model, using molecular dynamics and associated analytic tools. The system is a Cx26 channel equilibrated in explicit membrane/solvent, shown by Brownian dynamics to reproduce key conductance characteristics of the native channel. The results are consistent with the known difference in permeability to each molecule. The energetic barriers extend through most of the pore length, rather than being highly localized as in ion-specific channels. There is little evidence for binding within the pore. Force decomposition reveals how, for each tested molecule, interactions with water and the Cx26 protein vary over the length of the pore and reveals a significant contribution from hydrogen bonding and interaction with K(+). The flexibility of the pore width varies along its length, and the tested molecules have differential effects on pore width as they pass through. Potential sites of interaction within the pore are defined for each molecule. The results suggest that for the tested molecules, differences in hydrogen bonding and entropic factors arising from permeant flexibility substantially contribute to the energetics of permeation. This work highlights factors involved in selective molecular permeation that differ from those that define selectivity among atomic ions.

An electrostatic mechanism for Ca(2+)-mediated regulation of gap junction channels.

Gap junction channels mediate intercellular signalling that is crucial in tissue development, homeostasis and pathologic states such as cardiac arrhythmias, cancer and trauma. To explore the mechanism by which Ca(2+) blocks intercellular communication during tissue injury, we determined the X-ray crystal structures of the human Cx26 gap junction channel with and without bound Ca(2+). The two structures were nearly identical, ruling out both a large-scale structural change and a local steric constriction of the pore. Ca(2+) coordination sites reside at the interfaces between adjacent subunits, near the entrance to the extracellular gap, where local, side chain conformational rearrangements enable Ca(2+)chelation. Computational analysis revealed that Ca(2+)-binding generates a positive electrostatic barrier that substantially inhibits permeation of cations such as K(+) into the pore. Our results provide structural evidence for a unique mechanism of channel regulation: ionic conduction block via an electrostatic barrier rather than steric occlusion of the channel pore.

Glutathione release through connexin hemichannels: Implications for chemical modification of pores permeable to large molecules.

Cysteine-scanning mutagenesis combined with thiol reagent modification is a powerful method with which to define the pore-lining elements of channels and the changes in structure that accompany channel gating. Using the Xenopus laevis oocyte expression system and two-electrode voltage clamp, we performed cysteine-scanning mutagenesis of several pore-lining residues of connexin 26 (Cx26) hemichannels, followed by chemical modification using a methanethiosulfonate (MTS) reagent, to help identify the position of the gate. Unexpectedly, we observed that the effect of MTS modification on the currents was reversed within minutes of washout. Such a reversal should not occur unless reducing agents, which can break the disulfide thiol-MTS linkage, have access to the site of modification. Given the permeability to large metabolites of connexin channels, we tested whether cytosolic glutathione (GSH), the primary cell reducing agent, was reaching the modified sites through the connexin pore. Inhibition of gamma-glutamylcysteine synthetase by buthionine sulfoximine decreased the cytosolic GSH concentration in Xenopus oocytes and reduced reversibility of MTS modification, as did acute treatment with tert-butyl hydroperoxide, which oxidizes GSH. Cysteine modification based on thioether linkages (e.g., maleimides) cannot be reversed by reducing agents and did not reverse with washout. Using reconstituted hemichannels in a liposome-based transport-specific fractionation assay, we confirmed that homomeric Cx26 and Cx32 and heteromeric Cx26/Cx32 are permeable to GSH and other endogenous reductants. These results show that, for wide pores, accessibility of cytosolic reductants can lead to reversal of MTS-based thiol modifications. This potential for reversibility of thiol modification applies to on-cell accessibility studies of connexin channels and other channels that are permeable to large molecules, such as pannexin, CALHM, and VRAC.

Gap junctions modulate glioma invasion by direct transfer of microRNA.

The invasiveness of high-grade glioma is the primary reason for poor survival following treatment. Interaction between glioma cells and surrounding astrocytes are crucial to invasion. We investigated the role of gap junction mediated miRNA transfer in this context. By manipulating gap junctions with a gap junction inhibitor, siRNAs, and a dominant negative connexin mutant, we showed that functional glioma-glioma gap junctions suppress glioma invasion while glioma-astrocyte and astrocyte-astrocyte gap junctions promote it in an in vitro transwell invasion assay. After demonstrating that glioma-astrocyte gap junctions are permeable to microRNA, we compared the microRNA profiles of astrocytes before and after co-culture with glioma cells, identifying specific microRNAs as candidates for transfer through gap junctions from glioma cells to astrocytes. Further analysis showed that transfer of miR-5096 from glioma cells to astrocytes is through gap junctions; this transfer is responsible, in part, for the pro-invasive effect. Our results establish a role for glioma-astrocyte gap junction mediated microRNA signaling in modulation of glioma invasive behavior, and that gap junction coupling among astrocytes magnifies the pro-invasive signaling. Our findings reveal the potential for therapeutic interventions based on abolishing alteration of stromal cells by tumor cells via manipulation of microRNA and gap junction channel activity.

Motifs in the permeation pathway of connexin channels mediate voltage and Ca (2+) sensing.

Connexin channels mediate electrical coupling, intercellular molecular signaling, and extracellular release of signaling molecules. Connexin proteins assemble intracellularly as hexamers to form plasma membrane hemichannels. The docking of two hemichannels in apposed cells forms a gap junction channel that allows direct electrical and selective cytoplasmic communication between adjacent cells. Hemichannels and junctional channels are gated by voltage, but extracellular Ca (2+) also gates unpaired plasma membrane hemichannels. Unlike other ion channels, connexin channels do not contain discrete voltage- or Ca (2+)-sensing modules linked to a separate pore-forming module. All studies to date indicate that voltage and Ca (2+) sensing are predominantly mediated by motifs that lie within or are exposed to the pore lumen. The sensors appear to be integral components of the gates, imposing an obligatory structural linkage between sensing and gating not commonly present in other ion channels, in which the sensors are semi-independent domains distinct from the pore. Because of this, the structural and electrostatic features that define connexin channel gating also define pore permeability properties, and vice versa; analysis/mutagenesis of gating and of permeability properties are linked. This offers unique challenges and opportunities for elucidating mechanisms of ligand and voltage-driven gating.

Divalent regulation and intersubunit interactions of human connexin26 (Cx26) hemichannels.

Control of plasma membrane connexin hemichannel opening is indispensable, and is achieved by physiological extracellular divalent ion concentrations. Here, we explore the differences between regulation by Ca(2+) and Mg(2+) of human connexin26 (hCx26) hemichannels and the role of a specific interaction in regulation by Ca (2+). To effect hemichannel closure, the apparent affinity of Ca(2+) (0.33 mM) is higher than for Mg(2+) (1.8 mM). Hemichannel closure is accelerated by physiological Ca(2+) concentrations, but non-physiological concentrations of extracellular Mg(2+) are required for this effect. Our recent report provided evidence that extracellular Ca(2+) facilitates hCx26 hemichannel closing by disrupting a salt bridge interaction between positions D50 and K61 that stabilizes the open state. New evidence from mutant cycle analysis indicates that D50 also interacts with Q48. We find that the D50-Q48 interaction contributes to stabilization of the open state, but that it is relatively insensitive to disruption by extracellular Ca(2+) compared with the D50-K61 interaction.

Insights on the mechanisms of Ca(2+) regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y).

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca(2+) concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca(2+) regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca(2+) concentration, indicating that Ca(2+) facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca(2+)-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca(2+) insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca(2+), and (b) facilitating closing and stabilization of the closed state in the presence of Ca(2+). To explore the role of a negatively charged residue at position 50 in regulation by Ca(2+), this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)(-). D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES(-) restores the wild-type Ca(2+) regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca(2+) regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca(2+). Mutant cycle analysis supports a Ca(2+)-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca(2+) destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca(2+).

Connexin 43 is involved in the generation of human-induced pluripotent stem cells.

Although somatic cells can be successfully programmed to create pluripotent stem cells by ectopically expressing defined transcriptional factors, reprogramming efficiency is low and the reprogramming mechanism remains unclear. Previous reports have shown that almost all human connexin (CX) isoforms are expressed by human embryonic stem (hES) cells and that gap junctional intercellular communication (GJIC) is important for ES cell survival and differentiation. However, the CX expression profiles in human induced pluripotent stem (iPS) cells and the role of CXs in the process of reprogramming back to iPS cells remains unknown. Here, we determined the expression levels of most forms of CX in human embryonic fibroblasts (hEFs) and in the hEF-derived iPS cells. A scrape loading/dye transfer assay showed that human iPS cells contained functional gap junctions (GJs) that could be affected by pharmacological inhibitors of GJ function. We found that CX43 was the most dramatically upregulated CX following reprogramming. Most importantly, the ectopic expression of CX43 significantly enhanced the reprogramming efficiency, whereas shRNA-mediated knockdown of endogenous CX43 expression greatly reduced the efficiency. In addition, we found that CX43 overexpression or knockdown affected the expression of E-CADHERIN, a marker of the mesenchymal-to-epithelial transition (MET), during reprogramming. In conclusion, our data indicate that CX43 expression is important for reprogramming and may mediate the MET that is associated with the acquisition of pluripotency.

Human cell responses to ionizing radiation are differentially affected by the expressed connexins.

In multicellular organisms, intercellular communication is essential for homeostatic functions and has a major role in tissue responses to stress. Here, we describe the effects of expression of different connexins, which form gap junction channels with different permeabilities, on the responses of human cells to ionizing radiation. Exposure of confluent HeLa cell cultures to (137)Cs γ rays, 3.7 MeV α particles, 1000 MeV protons or 1000 MeV/u iron ions resulted in distinct effects when the cells expressed gap junction channels composed of either connexin26 (Cx26) or connexin32 (Cx32). Irradiated HeLa cells expressing Cx26 generally showed decreased clonogenic survival and reduced metabolic activity relative to parental cells lacking gap junction communication. In contrast, irradiated HeLa cells expressing Cx32 generally showed enhanced survival and greater metabolic activity relative to the control cells. The effects on clonogenic survival correlated more strongly with effects on metabolic activity than with DNA damage as assessed by micronucleus formation. The data also showed that the ability of a connexin to affect clonogenic survival following ionizing radiation can depend on the specific type of radiation. Together, these findings show that specific types of connexin channels are targets that may be exploited to enhance radiotherapeutic efficacy and to formulate countermeasures to the harmful effects of specific types of ionizing radiation.

Monocyte-endothelial adhesion is modulated by Cx43-stimulated ATP release from monocytes.

Adhesion of circulating monocytes to vascular endothelial cells is a crucial event in development of vascular inflammatory conditions, including atherosclerosis. We investigated the roles of connexin43 (Cx43) and ATP release on monocyte-endothelial adhesion. Cx43 function and expression were manipulated by connexin channel inhibitors, overexpression and siRNA. Connexin channel inhibitors rapidly decreased ATP release from U937 monocytes and increased adhesion to human umbilical vein endothelial cells (HUVEC). Monocyte ATP release correlated with Cx43 expression, not with Cx37 expression. Exogenous adenosine (ADO) or ATP decreased adhesion, and inhibition of ATP conversion to ADO increased adhesion. We infer that monocyte Cx43 channel activity causes ATP release, likely via Cx43-containing hemichannels, and that ATP decreases adhesion via conversion to ADO. Inhibition of HUVEC connexin channel activity did not affect ATP release or adhesion. In contrast, expression of Cx43 protein in U937 cells enhanced adhesion. Thus, Cx43 channel function and expression have opposite effects: Cx43 channel function in monocytes, but not in HUVEC, rapidly decreases adhesion via ATP release and conversion to ADO, whereas Cx43 expression itself enhances adhesion. These studies suggest that local regulation of monocyte Cx43 activity within the vasculature can dynamically modulate the monocyte-endothelial adhesion that is an initiating event in vascular inflammatory pathologies, with the baseline adhesion set by Cx43 expression levels. This balance of rapid and tonic influences may be crucial in development of vascular pathologies.