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Gap junction channels made of different connexins have distinct permeability to second messengers, which could affect many cell processes, including lens epithelial cell division. Here, we have compared the permeability of IP3 and Ca2+ through channels made from two connexins, Cx43 and Cx50, that are highly expressed in vertebrate lens epithelial cells. Solute transfer was measured while simultaneously monitoring junctional conductance via dual whole-cell/perforated patch clamp. HeLa cells expressing Cx43 or Cx50 were loaded with Fluo-8, and IP3 or Ca2+ were delivered via patch pipette to one cell of a pair, or to a monolayer while fluorescence intensity changes were recorded. Cx43 channels were permeable to IP3 and Ca2+. Conversely, Cx50 channels were impermeable to IP3, while exhibiting high permeation of Ca2+. Reduced Cx50 permeability to IP3 could play a role in regulating cell division and homeostasis in the lens.

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of gf (pacemaker conductance), gK1 (background K+ conductance), and gj (intercellular coupling conductance) in biological pacing. We studied gf and gK1 in single HEK293 cells expressing cardiac sodium current channel Nav1.5 (SCN5A). At fixed gf, increasing gK1 hyperpolarized the cell and initiated pacing. As gK1 increased, rate increased, then decreased, finally ceasing at membrane potentials near EK. At fixed gK1, increasing gf depolarized the cell and initiated pacing. With increasing gf, rate increased reaching a plateau, then decreased, ceasing at a depolarized membrane potential. We studied gj via virtual coupling with two non-adjacent cells, a driver (HEK293 cell) in which gK1 and gf were injected without SCN5A and a follower (HEK293 cell), expressing SCN5A. At the chosen values of gK1 and gf oscillations initiated in the driver, when gj was increased synchronized pacing began, which then decreased by about 35% as gj approached 20 nS. Virtual uncoupling yielded similar insights into gj. We also studied subthreshold oscillations in physically and virtually coupled cells. When coupling was insufficient to induce pacing, passive spread of the oscillations occurred in the follower. These results show a non-monotonic relationship between gK1, gf, gj, and pacing. Further, oscillations can be generated by gK1 and gf in the absence of SCN5A.

One promising approach to cancer therapeutics is to induce changes in gene expression that either reduce cancer cell proliferation or induce cancer cell death. Therefore, delivering oligonucleotides (siRNA/miRNA) that target specific genes or gene programs might have a potential therapeutic benefit. The aim of this study was to examine the potential of cell-based delivery of oligonucleotides to cancer cells via two naturally occurring intercellular pathways: gap junctions and vesicular/exosomal traffic. We utilized human mesenchymal stem cells (hMSCs) as delivery cells and chose to deliver in vitro two synthetic oligonucleotides, AllStars HS Cell Death siRNA and miR-16 mimic, as toxic (therapeutic) oligonucleotides targeting three cancer cell lines: prostate (PC3), pancreatic (PANC1) and cervical (HeLa). Both oligonucleotides dramatically reduced cell proliferation and/or induced cell death when transfected directly into target cells and delivery hMSCs. The delivery and target cells we chose express gap junction connexin 43 (Cx43) endogenously (PC3, PANC1, hMSC) or via stable transfection (HeLaCx43). Co-culture of hMSCs (transfected with either toxic oligonucleotide) with any of Cx43 expressing cancer cells induced target cell death (~20% surviving) or senescence (~85% proliferation reduction) over 96 hours. We eliminated gap junction-mediated delivery by using connexin deficient HeLaWT cells or knocking out endogenous Cx43 in PANC1 and PC3 cells via CRISPR/Cas9. Subsequently, all Cx43 deficient target cells co-cultured with the same toxic oligonucleotide loaded hMSCs proliferated, albeit at significantly slower rates, with cell number increasing on average ~2.2-fold (30% of control cells) over 96 hours. Our results show that both gap junction and vesicular/exosomal intercellular delivery pathways from hMSCs to target cancer cells deliver oligonucleotides and function to either induce cell death or significantly reduce their proliferation. Thus, hMSC-based cellular delivery is an effective method of delivering synthetic oligonucleotides that can significantly reduce tumor cell growth and should be further investigated as a possible approach to cancer therapy.

Gap junction channels mediate the direct intercellular passage of small ions as well as larger solutes such as second messengers. A family of proteins called connexins make up the subunits of gap junction channels in chordate animals. Each individual connexin forms channels that exhibit distinct permeability to molecules that influence cellular signaling, such as calcium ions, cyclic nucleotides, or inositol phosphates. In this review, we examine the permeability of connexin channels containing Cx43, Cx46, and Cx50 to signaling molecules and attempt to relate the observed differences in permeability to possible in vivo consequences that were revealed by studies of transgenic animals where these connexin genes have been manipulated. Taken together, these data suggest that differences in the permeability of individual connexin channels to larger solutes like 3′,5′-cyclic adenosine monophosphate (cAMP) and inositol 1,4,5-trisphosphate (IP3) could play a role in regulating epithelial cell division, differentiation, and homeostasis in organs like the ocular lens.

We previously demonstrated that a two-cell syncytium, composed of a ventricular myocyte and an mHCN2 expressing cell, recapitulated most properties of in vivo biological pacing induced by mHCN2-transfected hMSCs in the canine ventricle. Here, we use the two-cell syncytium, employing dynamic clamp, to study the roles of g

Cell-to-cell communication between bone, cartilage and the synovial membrane is not fully understood and it is only attributed to the diffusion of substances through the extracellular space or synovial fluid. In this study, we found for the first time that primary bone cells (BCs) including osteocytes, synovial cells (SCs) and chondrocytes (CHs) are able to establish cellular contacts and to couple through gap junction (GJ) channels with connexin43 (Cx43) being dominant. Transwell co-culture and identification by mass spectrometry revealed the exchange of essential amino acids, peptides and proteins including calnexin, calreticulin or CD44 antigen between contacting SCs, BCs and CHs. These results reveal that CHs, SCs and BCs are able to establish intercellular connections and to communicate through GJ channels, which provide a selective signalling route by the direct exchange of potent signalling molecules and metabolites.

This review focuses on the biophysical properties and structure of the pore and vestibule of homotypic gap junction channels as they relate to channel permeability and selectivity. Gap junction channels are unique in their sole role to connect the cytoplasm of two adjacent cells. In general, these channels are considered to be poorly selective, possess open probabilities approximating unity, and exhibit mean open times ranging from milliseconds to seconds. These properties suggest that such channels can function as delivery pathways from cell to cell for solutes that are significantly larger than monovalent ions. We have taken quantitative data from published works concerning unitary conductance, ion flux, and permeability for homotypic connexin 43 (Cx43), Cx40, Cx26, Cx50, and Cx37, and performed a comparative analysis of conductance and/or ion/solute flux versus diffusion coefficient. The analysis of monovalent cation flux portrays the pore as equivalent to an aqueous space where hydrogen bonding and weak interactions with binding sites dominate. For larger solutes, size, shape and charge are also significant components in determining the permeation rate. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Purpose Gap junction channels exhibit connexin specific biophysical properties, including the selective intercellular passage of larger solutes, such as second messengers. Here, we have examined the cyclic nucleotide permeability of the lens connexins, which could influence events like epithelial cell division and differentiation. Methods We compared the cAMP permeability through channels composed of Cx43, Cx46, or Cx50 using simultaneous measurements of junctional conductance and intercellular transfer. For cAMP detection, the recipient cells were transfected with a cAMP sensor gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents were recorded from the other cell of a pair that expressed SpIH. cAMP permeability was also directly visualized in transfected cells using a chemically modified fluorescent form of the molecule. Results cAMP transfer was observed for homotypic Cx43 channels over a wide range of junctional conductance. Homotypic Cx46 channels also transferred cAMP, but permeability was reduced compared with Cx43. In contrast, homotypic Cx50 channels exhibited extremely low permeability to cAMP, when compared with either Cx43, or Cx46. Conclusions These data show that channels made from Cx43 and Cx46 result in the intercellular delivery of cAMP in sufficient quantity to activate cyclic nucleotide-modulated channels. The data also suggest that the greatly reduced cAMP permeability of Cx50 channels could play a role in the regulation of cell division in the lens.

Alterations in the permeability and conductance of mutant atrial connexins may contribute to reentry arrhythmias., Gap junctions ensure the rapid propagation of the action potential throughout the myocardium. Three mutant forms of connexin40 (Cx40; A96S, M163V, and G38D), the primary component of the atrial gap junction channel, are associated with atrial fibrillation and retain the ability to form functional channels. We determined the biophysical properties of these mutant gap junctions in transiently transfected HeLa and N2A cells. All three mutants showed macroscopic junctional conductances over the range of 0.5 to 40 nS, and voltage dependences comparable to those of wild-type (WT) Cx40. However, the unitary conductance of G38D channels was ∼1.6-fold higher than that of WT Cx40 channels (∼220 vs. ∼135 pS), whereas the unitary conductances of the A96S and M163V mutants were similar to that of WT Cx40. Furthermore, the M163V and G38D channels exhibited approximately two- and approximately fivefold higher permeability to the anionic dye Lucifer yellow (LY) relative to K+ (LY/K+) compared with that of WT Cx40, whereas A96S LY transfer was similar to that of WT (G38D > M163V > A96S ≈ Cx40WT). In contrast, G38D channels were almost impermeable to cationic ethidium bromide (EtBr), suggesting that G38D alters channel selectivity. Conversely, A96S and M163V channels showed enhanced EtBr permeability relative to WT Cx40, with the following permeability order: M163V > A96S > Cx40WT > G38D. Altered conductive and permeability properties of mutant channels suggest an essential role for Cx40-mediated biochemical and electrical coupling in cardiac tissues. The altered properties of the three single-base substitution mutants may play a role in mechanisms of reentry arrhythmias.

ObjectiveThis study investigated whether chondrocytes within the cartilage matrix have the capacity to communicate through intercellular connections mediated by voltage-gated gap junction (GJ) channels.MethodsFrozen cartilage samples were used for immunofluorescence and immunohistochemistry assays. Samples were embedded in cacodylate buffer before dehydration for scanning electron microscopy. Co-immunoprecipitation experiments and mass spectrometry (MS) were performed to identify proteins that interact with the C-terminal end of Cx43. GJ communication was studied through in situ electroporation, electrophysiology and dye injection experiments. A transwell layered culture system and MS were used to identify and quantify transferred amino acids.ResultsMicroscopic images revealed the presence of multiple cellular projections connecting chondrocytes within the matrix. These projections were between 5 and 150 µm in length. MS data analysis indicated that the C-terminus of Cx43 interacts with several cytoskeletal proteins implicated in Cx trafficking and GJ assembly, including α-tubulin and β-tubulin, actin, and vinculin. Electrophysiology experiments demonstrated that 12-mer oligonucleotides could be transferred between chondrocytes within 12 min after injection. Glucose was homogeneously distributed within 22 and 35 min. No transfer was detected when glucose was electroporated into A549 cells, which have no GJs. Transwell layered culture systems coupled with MS analysis revealed connexins can mediate the transfer of L-lysine and L-arginine between chondrocytes.ConclusionsThis study reveals that intercellular connections between chondrocytes contain GJs that play a key role in cell–cell communication and a metabolic function by exchange of nutrients including glucose and essential amino acids. A three-dimensional cellular network mediated through GJs might mediate metabolic and physiological homeostasis to maintain cartilage tissue.

Osteoarthritis (OA) is the most common joint disease and involves progressive degeneration of articular cartilage. The aim of this study was to investigate if chondrocytes from human articular cartilage express gap junction proteins called connexins (Cxs). We show that human chondrocytes in tissue express Cx43, Cx45, Cx32, and Cx46. We also find that primary chondrocytes from adults retain the capacity to form functional voltage-dependent gap junctions. Immunohistochemistry experiments in cartilage from OA patients revealed significantly elevated levels of Cx43 and Cx45 in the superficial zone and down through the next approximately 1000 μm of tissue. These zones corresponded with regions damaged in OA that also had high levels of proliferative cell nuclear antigen. An increased number of Cxs may help explain the increased proliferation of cells in clusters that finally lead to tissue homeostasis loss. Conversely, high levels of Cxs in OA cartilage reflect the increased number of adjacent cells in clusters that are able to interact directly by gap junctions as compared with hemichannels on single cells in normal cartilage. Our data provide strong evidence that OA patients have a loss of the usual ordered distribution of Cxs in the damaged zones and that the reductions in Cx43 levels are accompanied by the loss of correct Cx localization in the nondamaged areas.

Background— After the recent cloning of light-sensitive ion channels and their expression in mammalian cells, a new field, optogenetics, emerged in neuroscience, allowing for precise perturbations of neural circuits by light. However, functionality of optogenetic tools has not been fully explored outside neuroscience, and a nonviral, nonembryogenesis-based strategy for optogenetics has not been shown before. Methods and Results— We demonstrate the utility of optogenetics to cardiac muscle by a tandem cell unit (TCU) strategy, in which nonexcitable cells carry exogenous light-sensitive ion channels, and, when electrically coupled to cardiomyocytes, produce optically excitable heart tissue. A stable channelrhodopsin2 (ChR2)-expressing cell line was developed, characterized, and used as a cell delivery system. The TCU strategy was validated in vitro in cell pairs with adult canine myocytes (for a wide range of coupling strengths) and in cardiac syncytium with neonatal rat cardiomyocytes. For the first time, we combined optical excitation and optical imaging to capture light-triggered muscle contractions and high-resolution propagation maps of light-triggered electric waves, found to be quantitatively indistinguishable from electrically triggered waves. Conclusions— Our results demonstrate feasibility to control excitation and contraction in cardiac muscle by light, using the TCU approach. Optical pacing in this case uses less energy, offers superior spatiotemporal control and remote access and can serve not only as an elegant tool in arrhythmia research but may form the basis for a new generation of light-driven cardiac pacemakers and muscle actuators. The TCU strategy is extendable to (nonviral) stem cell therapy and is directly relevant to in vivo applications.

Gap junction channels formed by different connexins exhibit specific permeability to a variety of larger solutes including second messengers, polypeptides, and small interfering RNAs. Here, we report the permeability of homotypic connexin26 (Cx26), Cx40, Cx43, and Cx45 gap junction channels stably expressed in HeLa cells to solutes with different size and net charge. Channel permeability was determined using simultaneous measurements of junctional conductance and the cell-cell flux of a fluorescent probe. All four connexins allowed passage of both cationic and anionic probes, but the transfer rates were connexin dependent. The negatively charged probes [Lucifer yellow (LY; median axial diameter 9.9 Å, charge −2), carboxyfluorescein (CF; 8.2 Å; −2), and Alexa Fluor350 (AF350, 5.4 Å; −1)] exhibited the following permeability order: Cx43 > Cx45 > Cx26 > Cx40. In contrast, for the positively charged species permeability, the orders were as follows: Cx26 ≈ Cx43 ≈ Cx40 ≈ Cx45 for N, N, N-trimethyl-2-[methyl-(7-nitro-2,1,3-benzoxadiol-4-yl) amino] ethanaminium (NBD-m-TMA; 5.5 Å, +1) and Cx26 ≥ Cx43 ≈ Cx40 > Cx45 for ethidium bromide (10.3 Å, +1). Comparison of probe permeability relative to K+revealed that Cx43 and Cx45 exhibited similar permeability for NBD-m-TMA and AF350, indicating weak charge selectivity. However, lesser transfer of CF and LY through Cx45 relative to Cx43 channels suggests stronger size-dependent discrimination of solute. The permeability of NBD-m-TMA for Cx40 and Cx26 channels was approximately three times higher than to anionic AF350 despite the fact that both have similar minor diameters, suggesting charge selectivity. In conclusion, these results confirm that channels formed from individual connexins can discriminate for solutes based on size and charge, suggesting that channel selectivity may be a key factor in cell signaling.

We examined whether coupling of a ventricular myocyte to a non-myocyte cell expressing HCN2 could create a two-cell syncytium capable of generating sustained pacing. Three non-myocyte cell types were transfected with the mHCN2 gene and used as sources of mHCN2-induced currents. They were human mesenchymal stem cells and HEK293 cells, both of which express connexin43 (Cx43), and HeLa cells transfected with Cx43. Cell–cell coupling between heterologous pairs increased with time in co-culture, and hyperpolarization of the myocyte induced HCN2 currents, indicating current transfer from the mHCN2-expressing cell to the myocyte via gap junctions. The magnitude of the HCN2 currents recorded in myocytes increased with increasing junctional conductance. Once a critical level of electrical cell–cell coupling between myocytes and mHCN2 transfected cells was exceeded spontaneous action potentials were generated at frequencies of ∼0.6 to 1.7 Hz (1.09 ± 0.05 Hz). Addition of carbenoxolone (200 μm), a gap junction channel blocker, to the media stopped spontaneous activity in heterologous cell pairs. Carbenoxolone washout restored activity. Blockade of HCN2 currents by 100 μm 9-amino-1,2,3,4-tetrahydroacridine (THA) stopped spontaneous activity and subsequent washout restored it. Neither THA nor carbenoxolone affected electrically stimulated action potentials in isolated single myocytes. In summary, the inward current evoked in the genetically engineered (HCN2-expressing) cell was delivered to the cardiac myocyte via gap junctions and generated action potentials such that the cell pair could function as a pacemaker unit. This finding lays the groundwork for understanding cell-based biological pacemakersin vivo once an understanding of delivery and target cell geometry is defined.

Cellular delivery of small interfering RNAs to target cells of a tissue has the potential to travel by two intercellular pathways. For intimately apposed cells gap junctions allow transport exclusive of the extracellular space. For cells not in intimate contact, exocytotic release of vesicular contents and subsequent retrieval via endocytosis of exosomes and other vesicular contents represent an alternative intercellular delivery system that utilizes the extracellular space. Previous studies have shown siRNA/miRNA transfer from a delivery cell to a target cell via gap junction channels. We hypothesized that siRNA can be delivered via gap junctions and downregulate the expression of a reporter gene, the cyclic nucleotide-gated cation channel gene (mHCN2), in the recipient cells of cell pairs. Whole-cell patch clamp was used to measure the mHCN2-induced current and junctional conductance. The target cells were HEK293 cells that endogenously express Cx43 or HeLaCx43 cells, both transfected with mHCN2. The source cells were HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA targeting mHCN2. We found that siRNA targeting mHCN2 resulted in significant downregulation of mHCN2 currents both in single cells and the recipient cell of a cell pair. In addition we also documented downregulation in target cells that were not in contact with source cells suggesting an extracellular-mediated delivery. To test further for extracellular delivery HEK293/HCN2 or HeLaCx43/HCN2 cells were cultured in medium collected from HEK293 or HeLaCx43 cells transfected with fluorescent-labeled siRNA or fluorescent-labeled morpholino designed to target HCN2. After 24 h single HEK293/HCN2 or HeLaCx43cells showed accumulation of siRNA. The mHCN2 currents were also down regulated in cells with siRNA uptake. Application of 200 nmol/L Bafilomycin A1, which has been shown to affect endosome acidification and endocytotic activity, resulted in a smaller accumulation of fluorescent-labeled siRNA in single target cells. In distinction to siRNA, morpholinos targeting HCN2 exhibited greatly reduced extracellularly mediated transfer while in cell pairs, target cells exhibited reduced HCN2 currents consistent with effective gap junction-mediated delivery.

Human HeLa cells expressing mouse connexin30 were used to study the electrical properties of gap junction channel substates. Experiments were performed on cell pairs using a dual voltage-clamp method. Single-channel currents revealed discrete levels attributable to a main state, a residual state, and five substates interposed, suggesting the operation of six subgates provided by the six connexins of a gap junction hemichannel. Substate conductances, gamma(j,substate), were unevenly distributed between the main-state and the residual-state conductance (gamma(j,main state) = 141 pS, gamma(j,residual state) = 21 pS). Activation of the first subgate reduced the channel conductance by approximately 30%, and activation of subsequent subgates resulted in conductance decrements of 10-15% each. Current transitions between the states were fast (

Purpose: Intercellular communication through gap junctions was assessed in 8 to 10-week STZ diabetic rats to evaluate diabetes related effects on gap junctional conductance and permeability in short-term cultures of corporeal myocytes. Materials and Methods: Rats were made diabetic by a single intraperitoneal injection of STZ. Eight to 10 weeks later erectile function was evaluated in vivo and corporeal tissue was harvested to isolate corporeal myocytes. Dual whole cell patch clamp studies of intercellular communication through connexin43 (Cx43) derived gap junction channels were done in short-term, ie passages 0 to 2, cultured corporeal myocytes excised from STZ diabetic rats with documented erectile impairment as well as in myocytes from age matched control rats. Results: No differences in macroscopic junctional conductance, single channel conductance or open probability were detected between myocytes from age matched control and STZ diabetic rats, confirming the lack of diabetes related alterations in Cx43 gating or conductance. However, fluorescence dye transfer experiments revealed a marked 3-fold increase in Cx43 mediated junctional permeability in the absence of any detectable change in Cx43 protein expression. Conclusions: These data suggest that an alteration in the selectivity filter of Cx43 in diabetic animals affects the permeability of specifically sized and charged solutes. To our knowledge these studies provide the first evidence of a diabetes related increase in intercellular permselectivity in corporeal myocytes and, thus, they may have important implications for diabetes related erectile dysfunction.

We demonstrate that the Src kinase can augment gap junctional communication between cells derived from homozygous null Cx43 knockout mice. The total conductance between Src transformed cells was nearly twice that of nontransformed cells. In addition, the unitary conductance of the majority of single channel events between transformed cells was about 35% greater than that of nontransformed cells. Analysis showed that both nontransformed and transformed cells expressed at least two populations of channels, suggesting that Src increased junctional conductance by up-regulating one population and/or by increasing the unitary conductance of another population of channels. Interestingly, the conductance displayed by heterologous pairs of transformed and nontransformed cells resembled that of nontransformed cells. The majority of single channel events between heterologous pairs shifted back to lower conductances that were exhibited by nontransformed cells. Thus, nontransformed cells can effectively "normalize" the conductance of gap junction channels expressed by adjacent tumor cells.

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels ( Ihc) that were activated and increased by stepwise depolarization of membrane potential ( Vm) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance ( γhc: 250–320 pS). The γhc of Cx35 hemichannels exhibited a pronounced Vm dependence; i.e., γhc increased/decreased with relative hyperpolarization/depolarization ( γhc was 72 pS at Vm = −100 mV and 35 pS at Vm = 100 mV). Extrapolation to Vm = 0 mV predicted a γhc of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of ∼24 pS. Channel gating was also Vm dependent: open time declined with negative Vm and increased with positive Vm. The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo.

Human mesenchymal stem cells (hMSCs) are a multipotent cell population with the potential to be a cellular repair or delivery system provided that they communicate with target cells such as cardiac myocytes via gap junctions. Immunostaining revealed typical punctate staining for Cx43 and Cx40 along regions of intimate cell-to-cell contact between hMSCs. The staining patterns for Cx45 rather were typified by granular cytoplasmic staining. hMSCs exhibited cell-to-cell coupling to each other, to HeLa cells transfected with Cx40, Cx43 and Cx45 and to acutely isolated canine ventricular myocytes. The junctional currents (Ij) recorded between hMSC pairs exhibited quasi-symmetrical and asymmetrical voltage (Vj) dependence. Ij records from hMSC–HeLaCx43 and hMSC–HeLaCx40 cell pairs also showed symmetrical and asymmetrical Vj dependence, while hMSC–HeLaCx45 pairs always produced asymmetrical Ij with pronounced Vj gating when the Cx45 side was negative. Symmetrical Ij suggests that the dominant functional channel is homotypic, while the asymmetrical Ij suggests the activity of another channel type (heterotypic, heteromeric or both). The hMSCs exhibited a spectrum of single channels with transition conductances (γj) of 30–80pS. The macroscopic Ij obtained from hMSC–cardiac myocyte cell pairs exhibited asymmetrical Vj dependence, while single channel events revealed γj of the size range 40–100pS. hMSC coupling via gap junctions to other cell types provides the basis for considering them as a therapeutic repair or cellular delivery system to syncytia such as the myocardium.

Gap junctions mediate the transfer of small cytoplasmic molecules between adjacent cells. A family of gap junction proteins exist that form channels with unique properties, and differ in their ability to mediate the transfer of specific molecules. Mutations in a number of individual gap junction proteins, called connexins, cause specific human diseases. Therefore, it is important to understand how gap junctions selectively move molecules between cells. Rules that dictate the ability of a molecule to travel through gap junction channels are complex. In addition to molecular weight and size, the ability of a solute to transverse these channels depends on its net charge, shape, and interactions with specific connexins that constitute gap junctions in particular cells. This review presents some data and interpretations pertaining to mechanisms that govern the differential transfer of signals through gap junction channels.

Background: Many stimuli can successfully protect the heart against ischemia. We investigated whether gap junction uncoupling before ischemia was myoprotective. We also studied the function of the adenosine triphosphate-dependent potassium channel, which has been implicated in the mechanism of pharmacologic preconditioning, with respect to gap junction physiology. Methods: Twenty-eight rabbit hearts were placed on a Langendorff perfusion apparatus. Five were given a 5-minute infusion of 1 mmol/L heptanol (a gap junction uncoupler), 5 were given 10 μmol/L 2,3-butanedione monoxime (an electromechanical uncoupler), and 6 were given no drug. The left anterior descending coronary artery was then occluded for 1 hour and reperfused for 2 hours. Six hearts received 10 μmol/L glybenclamide before heptanol to evaluate the role of the adenosine triphosphate-dependent potassium channel. Six hearts underwent ischemic preconditioning with 2 cycles of 5 minutes of global ischemia and reperfusion. Action-potential duration of the ischemic zone, left ventricular developed pressure, and coronary flow were measured continuously. Infarct size was determined at the end of reperfusion. Results: Heptanol significantly reduced infarct size (from 46% ± 2% to 22% ± 5%, P

Several proteins including connexin40 (Cx40) and connexin43 (Cx43) form gap junctions between cells of the heart; they may be found separately or may be coexpressed. These connexins form channels with differing conductance and permeability properties. Cx40 and Cx43 are each required for normal electrical conduction between cells in different regions of the heart. We hypothesized that the major difference between these connexins might be in their selective intercellular passage of small molecules such as second messengers, which can be assessed using biologically inert fluorescent probes. Therefore, we designed experimental paradigms to quantitate the permeability properties of these cardiac connexins using simultaneous measurement of junctional conductance ( g j ) by the double whole-cell patch-clamp technique and intercellular transfer of Lucifer Yellow (LY) by fluorescence microscopy. These studies were performed in HeLa cells stably transfected with Cx40 or Cx43 or cotransfected with both connexins. We found that homotypic Cx43 channels were about 5 times more permeable to LY than homotypic Cx40 channels (flux of ≈1560 versus ≈300 molecules/channel per second). Channels between heterotypic (Cx40-Cx43) cell pairs and between pairs of coexpressing cells exhibited intermediate LY permeability. The permeability ratio for LY relative to monovalent cation (K + ) ranged from 0.0025 for Cx40 to 0.028 for Cx43. These permeability ratios suggest that the connexins are highly selective for solutes in the size and charge range of many second messengers. Moreover, the data indicate that coexpression of connexins does not generate unique permeability characteristics, but rather results in an intermediate permeability for solutes involved in metabolic/biochemical coupling.

Hyperhomocysteinemia is an established cause of defective vasorelaxation. Gene expression screening of human umbilical vein endothelial cells (HUVEC) treated with homocysteine (Hcy) revealed that connexin43 (Cx43) was upregulated. Expression of Cx43 was increased more than twofold in Hcy-treated HUVEC. Gap junctional communication (Lucifer yellow and whole cell patch clamp) was not enhanced in Hcy-treated HUVEC. HUVEC expressing chimeric Cx43-green fluorescent protein exhibited it at cell-cell contacts in control but showed redistribution to the intracellular compartment(s) in Hcy-treated cells. Confocal microscopy of HUVEC stained with anti-Cx43, mitochondrial, and endoplasmic reticulum fluorescent markers showed the localization of Cx43 to the plasma membrane of control cells and its colocalization with the mitochondrial marker in Hcy-treated HUVEC. Studies of isolated mitochondria confirmed overexpression of Cx43 in the mitochondria of Hcy-treated HUVEC. Microdissected renal interlobar arteries, which normally exhibit endothelium-derived hyperpolarizing factor-induced vasorelaxation, showed reduced nitric oxide synthase- and cyclooxygenase-independent vasorelaxation to acetylcholine after pretreatment with Hcy. In summary, Hcy-induced upregulation of Cx43 transcript and protein expression are associated with unaltered intercellular communication, redistribution of Cx43 in HUVEC, and reduced nitric oxide- and prostanoid-independent vascular responses to acetylcholine in Hcy-treated arteries.

In glands such as the liver and pancreas, gap junctions containing connexin 26 and 32 (Cx26 and Cx32, respectively) couple the secretory cells. Uncoupling these junctions compromises the secretory function of these glands. Lacrimal glands also contain extensive arrays of gap junctions consisting of Cx26 and Cx32. We wanted to determine the role of these junctions in fluid secretion. In Cx32-deficient mice, immunocytochemistry showed that, in the male lacrimal gland, the remaining Cx26 was found evenly distributed in the membrane whereas there was little in the membranes of female glands. Western blot analysis of Cx26 showed that female Cx32-deficient mice expressed Cx26. Patch-clamp analyses of acinar cell coupling showed that the cell pairs from male glands were coupled whereas those from female glands were not. Stimulated fluid production by the glands from Cx32-deficient mice was abnormally low in female glands compared with controls at low topical doses of carbachol. The protein secretory response to different doses of carbachol was the same in all animals. These data suggest that gap junctions are essential for optimal fluid secretion in lacrimal glands.

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o)0.11, V(m)-50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m)50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.

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 ( gj,ss) in coexpressing cell pairs showed a reduced sensitivity to the voltage between cells ( Vj) compared with homotypic gap junctions and/or an asymmetrical Vjdependence 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.

HeLa cells stably transfected with mouse cDNA coding for connexin30 (Cx30) were used to study the electrical properties of gap junction channels. The experiments involved the measurement of intercellular currents (Ij) from cell pairs using dual whole-cell recording with the patch-clamp method. The aim was to compare Ij from cell pairs whose gap junctions consisted of a single channel and cell pairs whose gap junctions consisted of many channels. We found that both the ensemble average currents gained from single-channel records and the currents obtained from multichannel records inactivated exponentially with time. However, the former inactivated significantly slower than the latter. At ajunctional voltage (Vj) of 50 mV, the time constants of inactivation (tau(i)) were 8.1 s and 1.6 s, respectively. Moreover, the ratio tau(i)(single-channel)/tau(i)(multichannel) turned out to be voltage sensitive, i.e. it decreased with increasing V(j) These observations suggest that the operation of Cx30 gap junction channels in the multichannel configuration involves co-operative interactions.

Cx43 is widely expressed in many different cell types, and many of these cells also express other connexins. If these connexins are capable of mixing, the functional properties of channels containing heteromeric connexons may substantially influence intercellular communication between such cells. We used biochemical strategies (sedimentation through sucrose gradients, co-immunoprecipitation, or co-purification by Ni-NTA chromatography) to examine heteromeric mixing of Cx43 with other connexins (including Cx26, Cx37, Cx40, Cx45, and Cx56) in transfected cells. These analyses showed that all of the tested connexins except Cx26 formed heteromeric connexons with Cx43. We used the double whole-cell patch-camp technique to analyze the electrophysiological properties of gap junction channels in pairs of co-expressing cells. Cx37 and Cx45 made a large variety of functional heteromeric combinations with Cx43 based on detection of many different single channel conductances. Most of the channel event sizes observed in cells co-expressing Cx40 and Cx43 were similar to those of homomeric Cx43 or Cx40 hemichannels in homo- or hetero-typic configurations. Our data suggest several different possible consequences of connexin co-expression: (1) some combinations of connexins may form heteromeric connexons with novel proeprties; (2) some connexins may form heteromeric channels that do not have unique properties, and (3) some connexins may be incompatible for heteromeric mixing.

Myocytes were isolated from neonatal rat hearts using an enzymatic procedure. Cell pairs were used to control the junctional voltage, V j, and to measure the transjunctional current, I j, using the dual voltage-clamp method. V j gradients provoked I j signals with voltage-dependent inactivation. During voltage pulses, I j remained virtually constant at |V j| 40 mV, it inactivated with time to a residual level. The inactivation followed a single exponential. The time constant of I j inactivation, τi, and the size of I j at steady state, I j,ss, were both sensitive to the ions in the pipette solution. I j,ss was smaller in the presence of tetraethylammonium aspartate (TEA+ aspartate–) than KCl, while τi was smaller in the presence of KCl than TEA+ aspartate–. The modification of I j,ss is readily explained by a change in the residual conductance of the gap junction channels, γj,residual. The alterations in τi are correlated with a change in β, the rate constant that describes the transition of the channel from the main state to the residual state. Pipette solutions may affect the kinetics of gap junction currents by altering the conductive and/or kinetic parameters. Computer simulations revealed a substantial influence of the latter, but only a marginal effect of the former. Conceivably, ions of the pipette solution may affect the kinetics of gap junction channels by screening surface charges of the channel wall.

Human HeLa cells transfected with mouse connexin Cx30, Cx46 or Cx50 were used to study the electrical properties of gap junction hemichannels. With no extracellular Ca2+, whole-cell recording revealed currents arising from hemichannels. Multichannel currents showed a time-dependent inactivation sensitive to voltage, Vm. Plots of the instantaneous conductance, ghc,inst, versus Vm were constant; plots of the steady-state conductance, ghc,ss, versus Vm were bell-shaped. Single-channel currents showed two conductances, gammahc,main and gammahc,residual, the latter approximately or approximately equals=1/6 of the former. Single-channel currents exhibited fast transitions (1-2 ms) between the main state and residual state. Late during wash-in and early during wash-out of 2 mM heptanol, single-hemichannel currents showed slow transitions between an open state and closed state. The open channel probability, Po, was Vm-dependent. It declined from approximately =1 at Vm= 0 mV to 0 at large Vm of either polarity. Hemichannel currents showed a voltage-dependent gammahc,main, i.e., it increased/decreased with hyperpolarization/depolarization. Extrapolation to Vm=0 mV led to a gammahc,main of 283, 250 and 352 pS for Cx30, Cx46 and Cx50, respectively. The hemichannels possess two gating mechanisms. Gating with positive voltage reflects Vj-gating of gap junction channels, gating with negative voltage reflects a property inherent to hemichannels, i.e., Vm or "loop" gating. We conclude that Cx30, Cx46 and Cx50 form voltage-sensitive hemichannels in single cells which are closed under physiological conditions.

Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254Delta293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254Delta293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics.

Myocytes isolated from neonatal rat hearts were grown in culture dishes. Cell pairs were selected to examine the mode of action of arachidonic acid (AA) on gap junctions. The dual voltage-clamp method was used to measure intercellular currents and determine the gap junction conductance, gj. Exposure of cell pairs to 10 microM AA produced reversible uncoupling. Pretreatment with 10 microM POCA (sodium-2-[5-(4-chlorophenyl)-pentyl]-oxirane-2-carboxylate; which inhibits mitochondrial beta-oxidation) did not prevent AA-dependent uncoupling. Thus, it seems that metabolites of beta-oxidation are not involved in AA-induced impairment of gj. Pre-exposure to 10 microM indomethacin (which blocks the cyclooxygenase pathway of the AA-cascade) had no effect on AA-dependent uncoupling. This suggests that cyclooxygenase products such as prostaglandins or thromboxanes play no role in gj modulation. Exposure to 5 microM NDGA (nordihydroguaiaretic acid; which inhibits the 5-lipoxygenase pathway) or 10 microM ETYA (5,8,11,14-eicosatetrynoic acid: which inhibits the 12- and 15-lipoxygenase pathway) led to a reversible decrease in gj. Pre-treatment with 4-BPB (4-bromophenacyl bromide: which inhibits phospholipase A2) did not prevent the effects on gj by NDGA or ETYA. This renders it unlikely that gj is regulated by eicosanoids. Also, accumulation of endogenous AA cannot be responsible for NDGA- and ETYA-dependent uncoupling. Exposure to 75 microM SKF-525A (inhibits the epoxygenase pathway) reversibly impaired gj. This is consistent with a direct action of SKF-525A on gj, but leaves open the possibility of an involvement of epoxides. The data gathered will be discussed in terms of molecular mechanisms. Due to their amphipathic character. AA, NDGA, ETYA and SKF-525A may interfere with gj by disturbing the lipid-protein interface of the cell membranes and thereby impair gap junction channels.

Longitudinal resistance is a key factor in determining cardiac action potential propagation. Action potential conduction velocity has been shown to be proportional to the square root of longitudinal resistance. A major determinant of longitudinal resistance in myocardium is the gap junction channel, comprised of connexin proteins. Within the ventricular myocardium connexin 43 (Cx43) is the dominantly expressed connexin. Reduced numbers of gap junction channels will result in an increase in longitudinal resistance creating the possibility of slowed conduction velocity while increased numbers of channels would potentially result in an increase in conduction velocity. We sought to determine if inhibition of histone deacetylase (HDAC) by 4-phenylbutyrate (4-PB), a known inhibitor of HDAC resulted in an increase in junctional conductance and permeability, which is not the result of changes in single channel unitary conductance. These experiments were performed using HEK-293 cells and HeLa cells stably transfected with Cx43. Following treatment with increasing concentrations of 4-PB up-regulation of Cx43 was observed via Western blot analysis. Junctional (gj) conductance and unitary single channel conductance were measured via whole-cell patch clamp. In addition intercellular transfer of Lucifer Yellow (LY) was determined by fluorescence microscopy. The data in this study indicates that 4-PB is able to enhance functional Cx43 gap junction coupling as indicated by LY dye transfer and multichannel and single channel data along with Western blot analysis. As a corollary, pharmacological agents such as 4-PB have the potential, by increasing intercellular coupling, to reduce the effect of ischemia. It remains to be seen whether drugs like 4-PB will be effective in preventing cardiac maladies.

Cyclic adenosine monophosphate (cAMP) is a well-known intracellular and intercellular second messenger. The membrane permeability of such molecules has potential importance for autocrine-like or paracrine-like delivery. Here experiments have been designed to demonstrate whether gap junction hemichannels, composed of connexins, are a possible entrance pathway for cyclic nucleotides into the interior of cells. HeLa cells stably expressing connexin43 (Cx43) and connexin26 (Cx26) were used to study the cyclic nucleotide permeability of gap junction hemichannels. For the detection of cAMP uptake, the cells were transfected using the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH) as the cAMP sensor. SpIH derived currents (I m) were recorded in whole-cell/perforated patch clamp configuration. Perfusion of the cells in an external K(+) aspartate(-) (KAsp) solution containing 500 μM cAMP and no extracellular Ca(2) (+), yielded a five to sevenfold increase in the I m current level. The SpIH current increase was associated with detectable hemichannel current activity. Depolarization of cells in Ca(2) (+)-free NaCl perfusate with 500 μM cAMP also induced a SpIH current increase. Elevating extracellular Ca(2) (+) to mM levels inhibited hemichannel activity. Perfusion with a depolarizing KAsp solution containing 500 μM cAMP and 2 mM Ca(2) (+) did not increase SpIH currents. The addition of the gap junction blocker carbenoxolone to the external solution inhibited cAMP uptake. Both cell depolarization and lowered extracellular Ca(2) (+) increase the open probability of non-junctional hemichannels. Accordingly, the SpIH current augmentation was induced by the uptake of extracellular cAMP via open membrane hemichannels in Cx43 and Cx26 expressing cells. The data presented here show that hemichannels of Cx43 and Cx26 are permeable to cAMP, and further the data suggest that hemichannels are, in fact, a potential pathway for cAMP mediated cell-to-cell signaling.

s / Osteoarthritis and Cartilage 20 (2012) S10–S53 S11 3 LONG-DISTANCE PHYSICAL CONNECTIONS BETWEEN CHONDROCYTES; CELL-TO-CELL COMMUNICATION WITHIN HYALINE CARTILAGE M. Mayan , R. Gago , P. Carpintero , P. Filgueira-Fernandez , N. Goyanes , V. Valiunas , P. Brink , F.J. Blanco . Osteoarticular and Aging Res. Lab. INIBIC-CHUAC, A Coruna, Spain; Dept. of Physiology and Biophysics, State University of New York, Stony Brook, NY, USA Purpose: Even chondrocytes are metabolically active cells, responsible of the maintenance of the hyaline cartilage during the whole adult life, it is highly accepted in the field that chondrocytes in cartilage do not connect each other, as they are isolated inside their lacunae separated from each other by a distance between 5 to 60mm. In the same lacuna can co-exit several chondocytes interacting between them. However, how the chondrocytes from different lacuna interact between each other and timely respond to physical or chemicals stimuli, are largely unknown. Thus far, the unique communication between chondrocytes in the superficial layer with chondrocytes in themiddle and deeper layers it is accepted that only occurs through diffusion. Nevertheless, the intercellular communication confers to a tissue the ability to responduniformly to localized stimuli. The purpose of this study was to investigate how chondrocytes communicate each other within the matrix and find for some alteration that could explain the degeneration of the matrix that occurs in patients with OA. Methods: In situ cartilage from human and Sus scrofa were fixed and frozen immediately using Tissue-Tek O.C.T. and isopentanol in liquid nitrogen. Samples were fixed with acetone for confocal optical microscopy assays. Samples were embedded in cacodylate buffer before dehydratation for Scanning Electron Microscope (SEM). Gold was use for coating. Frozen samples were also used for immunofluorescence and immunohistochemistry assays. For total RNA isolation, the proteins and DNA were removed from cell extracts using TRIZOL Reagent (Invitrogene) before using the RNeasy Kit (Qiagen) which includes DNase treatment (RNAse-Free DNAse Set). Quantifications were done using SYBr green real-time PCR. Virgin Valiunas performed dual voltageclamp method and whole-cell/perforated patch using chondrocytes from healthy donors and patients with OA. Results: By using confocal optical microscopy and Scanning Electron Microscopy (SEM), we have found that chondrocytes are physical connected between each other across the extracellular matrix through large cytoplasmic projections. The projections are between 5 to 150 mm in length in human healthy cartilage and young cartilage from Sus scrofa. However condrocytes projections found in OA cartilage are between 10 to 200 mm in length. High levels of expression for the gap junction protein connexin 43 have been detected by RT-PCR and immunohistochemical experiments. We have also found expression of Cx32 and Cx45, however electrophysiology experiments demonstrated that healthy and OA chondrocytes show voltage-dependent behaviour for channels mainly formed by Cx43. We have performed patch clamp experiments to study the interchange of Lucifer Yellow and small molecules of RNA. Our results demonstrated that healthy and OA chondrocytes are able to interchange Lucifer Yellow and small molecules of RNA through Cx43 channels. In fact, our preliminary results showed that inhibition of channels affect the synthesis of collagen type II, MMP3 andMMP9 suggesting that the activity of channels are actively involved with the maintenance of the cartilage matrix. Conclusions:Wehavefoundcell/cellphysicalconnectionsthroughall layers of cartilage between chondrocytes located at different distance within cartilage. Intercellular communication occurs through channels voltagedependent formedby Cx43. Cytoplasmic projections fromOAchondrocytes are longer than from healthy chondrocytes, therefore the channel activity can be affected by the electrical depolarization of the membrane in OA chondrocytes. We have found that chondrocytes interchange small RNAs and inhibition of channels affect the expression of matrix-related genes. 4 SYNERGISTIC INDUCTION OF ELF3. IN VITRO EFFECT OF LEPTIN AND IL-1 IN HUMAN CHONDROCYTES J. Conde , M. Scotece , M. Otero , R. Gomez , J.J. Gomez-Reino , M.B. Goldring , O. Gualillo . 1 SERGAS, Santiago Univ. Clinical Hosp. NEIRID LAB, Santiago de Compostela, Spain; Hosp. for Special Surgery

Dominant Cx26 mutations that cause keratitis-ichthyosis-deafness syndrome (KIDS) show increased hemichannel activity. Transgenic expression of these mutations recapitulates human skin disease in mice. Excess hemichannel activity persists in diseased epidermis from the transgenic mice. Thus hemichannel activity may be a novel therapeutic target in the treatment of KIDS., Mutations in the GJB2 gene (Cx26) cause deafness in humans. Most are loss-of-function mutations and cause nonsyndromic deafness. Some mutations produce a gain of function and cause syndromic deafness associated with skin disorders, such as keratitis-ichthyosis-deafness syndrome (KIDS). Cx26-G45E is a lethal mutation linked to KIDS that forms constitutively active connexin hemichannels. The pathomechanism(s) by which mutant Cx26 hemichannels perturb normal epidermal cornification are poorly understood. We created an animal model for KIDS by generating an inducible transgenic mouse expressing Cx26-G45E in keratinocytes. Cx26-G45E mice displayed reduced viability, hyperkeratosis, scaling, skin folds, and hair loss. Histopathology included hyperplasia, acanthosis, papillomatosis, increased cell size, and osteal plugging. These abnormalities correlated with human KIDS pathology and were associated with increased hemichannel currents in transgenic keratinocytes. These results confirm the pathogenic nature of the G45E mutation and provide a new model for studying the role of aberrant connexin hemichannels in epidermal differentiation and inherited connexin disorders.

In vivo delivery of small interfering RNAs (siRNAs) to target cells via the extracellular space has been hampered by dilution effects and immune responses. Gap junction-mediated transfer between cells avoids the extracellular space and its associated limitations. Because of these advantages cell based delivery via gap junctions has emerged as a viable alternative for siRNA or miRNA delivery. Here we discuss the advantages and disadvantages of extracellular delivery and cell to cell delivery via gap junction channels composed of connexins. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Intercellular communication is important for cochlear homeostasis because connexin26 (Cx26) mutations are the leading cause of hereditary deafness. Gap junctions formed by different connexins have unique selectivity to large molecules, so compensating for the loss of one isoform can be challenging in the case of disease causing mutations. We compared the properties of Cx26 mutants T8M and N206S with wild-type channels in transfected cells using dual whole cell voltage clamp and dye flux experiments. Wild-type and mutant channels demonstrated comparable ionic coupling, and their average unitary conductance was ∼106 and ∼60 pS in 120 mM K+-aspartate−and TEA+-aspartate−solution, respectively, documenting their equivalent permeability to K+and TEA+. Comparison of cAMP, Lucifer Yellow (LY), and ethidium bromide (EtBr) transfer revealed differences in selectivity for larger anionic and cationic tracers. cAMP and LY permeability to wild-type and mutant channels was similar, whereas the transfer of EtBr through mutant channels was greatly reduced compared with wild-type junctions. Altered permeability of Cx26 to large cationic molecules suggests an essential role for biochemical coupling in cochlear homeostasis.

Gap junction channels exhibit connexin dependent biophysical properties, including selective intercellular passage of larger solutes, such as second messengers and siRNA. Here, we report the determination of cyclic nucleotide (cAMP) permeability through gap junction channels composed of Cx43, Cx40, or Cx26 using simultaneous measurements of junctional conductance and intercellular transfer of cAMP. For cAMP detection the recipient cells were transfected with a reporter gene, the cyclic nucleotide-modulated channel from sea urchin sperm (SpIH). cAMP was introduced via patch pipette into the cell of the pair that did not express SpIH. SpIH-derived currents (I(h)) were recorded from the other cell of a pair that expressed SpIH. cAMP diffusion through gap junction channels to the neighboring SpIH-transfected cell resulted in a five to sixfold increase in I(h) current over time. Cyclic AMP transfer was observed for homotypic Cx43 channels over a wide range of conductances. However, homotypic Cx40 and homotypic Cx26 exhibited reduced cAMP permeability in comparison to Cx43. The cAMP/K(+) permeability ratios were 0.18, 0.027, and 0.018 for Cx43, Cx26, and Cx40, respectively. Cx43 channels were approximately 10 to 7 times more permeable to cAMP than Cx40 or Cx26 (Cx43 > Cx26 > or = Cx40), suggesting that these channels have distinctly different selectivity for negatively charged larger solutes involved in metabolic/biochemical coupling. These data suggest that Cx43 permeability to cAMP results in a rapid delivery of cAMP from cell to cell in sufficient quantity before degradation by phosphodiesterase to trigger relevant intracellular responses. The data also suggest that the reduced permeability of Cx26 and Cx40 might compromise their ability to deliver cAMP rapidly enough to cause functional changes in a recipient cell.

Cataracts are a major cause of impaired vision and blindness throughout the world.1 Congenital cataracts are responsible for 10% of childhood blindness and are a common treatable cause of childhood visual impairment.2 A substantial fraction of isolated (nonsyndromic) childhood cataracts are inherited. Inheritance is most commonly autosomal dominant, although autosomal recessive and X-linked forms have also been reported. Hereditary congenital cataracts have been linked to mutations of several different genes, including the gap junction proteins connexin46 (CX46) and connexin50 (CX50).3-17 Maintenance of a transparent lens depends on gap junction-mediated communication between its cells. Gap junctions are plasma membrane specializations containing clusters of intercellular channels. These channels allow intercellular transfer of ions and molecules of ≤1000 Da. Within the lens, gap junction channels form a network of cell-to-cell channels that facilitate exchange of water, ions, nutrients, and metabolites, especially between fiber cells.18,19 A gap junction channel contains 12 subunit proteins (connexins) distributed among two coaxially aligned hexameric hemichannels located in the plasma membranes of the apposing cells. Lens fiber cells primarily express CX5020 and CX46,21 whereas the lens epithelial cells mainly express connexin43 (CX43).22,23 Large numbers of gap junction channels may cluster together within the appositional membranes to form plaques. Genetic data from rodents have also emphasized the importance of alterations of the lens fiber connexins in cataractogenesis. Several strains of mice or rats that develop cataracts harbor mutations of the Gja3 (CX46) or Gja8 (CX50) genes.24-28 Targeted ablation of the murine Gja3 (CX46)29 or Gja8 (CX50)30,31 genes also causes cataracts. In fact, not only loss of CX50, but also its overexpression, can result in cataracts.32 Investigators in several previous studies have examined the behavior of cataract-associated mutant connexins in expression systems, to elucidate cellular and physiological abnormalities that may lead to cataract formation. Some lens connexin mutants exhibit loss of function-for example, small or no gap junction conductances were detected when they were expressed in paired Xenopus oocytes.17,28,33,34 When coexpressed with wild-type connexins, some mutants exert a dominant-negative effect abolishing conductance,15,35,36 whereas some other mutants do not eliminate coupling, but may affect channel gating properties.37 Several mutants do not form gap junction plaques.15,36 In one of these cases a unique sequence in the mutant protein (generated by a frame shift) impairs trafficking to the plasma membrane.38 The human mutation, CX50R23T (which leads to the substitution of a threonine for arginine-23), was first described in members of an Iranian family with autosomal dominant, bilateral, congenital nuclear cataract that progressed to cause visual impairment by the second decade of life.9 The mechanisms responsible for the development of this cataract are not understood. However, this mutation affects a critical position within the connexin molecule at the predicted interface between the N-terminal cytoplasmic domain and the first transmembrane domain (Fig. 1). Structural models suggest the importance of this residue for maintaining associations of transmembrane domains.39 Substitutions of this amino acid have been identified in the mutant forms of at least three other connexins associated with other diseases.40-44 The present experiments were designed to elucidate the mechanism of cataract formation in patients carrying the mutation CX50R23T by examining the cellular distribution and physiological function of the CX50R23T mutant and of site-directed mutants containing other amino acid substitutions at this position. FIGURE 1 Diagram of the membrane topology of human CX50 showing the position of the R23T substitution.

Mutations in GJB2 and GJB6, the genes that encode the human gap junction proteins connexin26 (Cx26) and connexin30 (Cx30), respectively, cause hearing loss. Cx26 and Cx30 are both expressed in the cochlea, leading to the potential formation of heteromeric hemichannels and heterotypic gap junction channels. To investigate their interactions, we expressed human Cx26 and Cx30 individually or together in HeLa cells. When they were expressed together, Cx26 and Cx30 appeared to interact directly (by their colocalization in gap junction plaques, by coimmunoprecipitation, and by fluorescence resonance energy transfer). Scrape-loading cells that express either Cx26 or Cx30 demonstrated that Cx26 homotypic channels robustly transferred both cationic and anionic tracers, whereas Cx30 homotypic channels transferred cationic but not anionic tracers. Cells expressing both Cx26 and Cx30 also transferred both cationic and anionic tracers by scrape loading, and the rate of calcein (an anionic tracer) transfer was intermediate between their homotypic counterparts by fluorescence recovery after photobleaching. Fluorescence recovery after photobleaching also showed that Cx26 and Cx30 form functional heterotypic channels, allowing the transfer of calcein, which did not pass the homotypic Cx30 channels. Electrophysiological recordings of cell pairs expressing different combinations of Cx26 and/or Cx30 demonstrated unique gating properties of cell pairs expressing both Cx26 and Cx30. These results indicate that Cx26 and Cx30 form functional heteromeric and heterotypic channels, whose biophysical properties and permeabilities are different from their homotypic counterparts.

Connexins (Cx) form gap junction channels mediating direct intercellular communication. To study the role of amino acids within the cytoplasmic loop, we produced a recombinant adenovirus containing Cx43 with a deletion of amino acids 130-136 (Cx43del(130-136)). Cx43del(130-136) expressed alone in HeLa cells localized within the cytoplasm and did not allow transfer of ions, neurobiotin or Lucifer yellow. When co-expressed with wild type Cx43, Cx43del(130-136) blocked electrical coupling and transfer of neurobiotin or Lucifer yellow. Cx43del(130-136) and Cx43 co-localized by immunofluorescence and were co-purified from Triton X-100-solubilized cell extracts. Intercellular transfer mediated by Cx37 and Cx45 (but not Cx26 or Cx40) was inhibited when co-expressed with Cx43del(130-136). Cx43del(130-136) co-localized with Cx37, Cx40, or Cx45, but not Cx26. These data suggest that Cx43del(130-136) produces connexin-specific inhibition of intercellular communication through formation of heteromeric connexons that are non-functional and/or retained in the cytoplasm.

Non-stationary fluctuation analysis was applied to macroscopic records of junctional currents arising from homotypic Cx37 and Cx43 gap junction channels expressed in RIN cells. The data were analyzed by a modification of existing analytical methods that takes endemic uncoupling into account. The results are consistent with both channels having open probabilities ranging from 0.7 to near unity for low transjunctional voltages. The analysis also yielded estimates of single-channel conductances for the two channel types similar to those seen in single-channel recordings. The results presented here show that fluctuation analysis can be used to extract single-channel gap junctional conductances from macroscopic double whole-cell recordings. These results also constitute empirically determined estimates of the open probability that are not model-dependent.

Many cells contain two (or more) gap junction proteins that are able to oligomerize with each other to form heteromeric gap junction channels and influence the properties of intercellular communication. Cx26 and Cx43 are found together in a number of cell types, but previous data have suggested that they might not form heteromeric connexons. We studied the possible interactions of these connexins by co-expression in three different cell lines. Analysis of N2aCx26/Cx43 cell pairs by double whole-cell patch-clamp methods showed that these cells were coupled, but contained only a small number of sizes of single channels consistent with those formed by homomeric Cx26 or Cx43 channels. Immunofluorescence studies showed that both connexins localized to appositional membranes, but in largely distinct domains. Analysis of Triton X-100-solubilized connexons from co-expressing cells by centrifugation through sucrose gradients or by affinity purification using a Ni-NTA column showed no evidence of mixing of Cx26 and Cx43. These results contrast with our observations in cells co-expressing other connexins with Cx43 and suggest that Cx26 and Cx43 do not form heteromeric hemichannels. Moreover, the incorporation of Cx26 and Cx43 into oligomers and into the membrane were similarly affected by treatment of co-expressing cells with brefeldin A or nocodazole, suggesting that the lack of mixing is due to incompatibility of these connexins, not to differences in biosynthetic trafficking.