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Molecular models of myoinositol and inositol phosphates. Molecular models of myoinositol and inositol phosphates used were made with Spartan 04. Tracer.pdb files were retrieved from a data base of small heteromolecules or built using Spartan 04
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Previous work has shown that channels formed by both connexin (Cx)26 and Cx32 (heteromeric Cx26/Cx32 hemichannels) are selectively permeable to cAMP and cGMP. To further investigate differential connexin channel permeability among second messengers, and the influence of connexin channel composition on the selectivity, the permeability of inositol p...
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Citations
... [37][38][39] The connexin types determine the permeability of channels to different molecules: For instance, homotypic Cx32 gap junctions had a higher permeability to IP 3 than Cx43 or Cx26 ones and they also showed a higher permeability to adenosine while Cx43 channels had higher permeability to ATP. 40 Moreover, heteromeric combinations can also alter the selectivity of channels compared to homomeric arrangements; while homomeric Cx26 or Cx32 hemichannels were permeable to myoinositol and all inositol phosphates, Cx26/Cx32 heteromeric hemichannels had selective permeability to inositol phosphates. 41 Therefore, different subunit and connexon combinations can lead to diverse hemichannel and gap junction channel functions and contribute to the complexity of communication mediated by connexins. During development and adult life, connexin oligomerization can be altered based on the cells/ tissues' need. ...
In spite of clinical advancements and improved diagnostic techniques, breast cancers are the leading cause of cancer-associated deaths in women worldwide. Although 70% of early breast cancers can be cured, there are no efficient therapies against metastatic breast cancers. Several factors including connexins and gap junctions play roles in breast tumorigenesis. Connexins are critical for cellular processes as a linkage between connexin mutations and hereditary disorders demonstrated their importance for tissue homeostasis. Further, alterations in their expression, localization and channel activities were observed in many cancers including breast cancer. Both channel-dependent and independent functions of connexins were reported in initiation and progression of cancers. Unlike initial reports suggesting tumor suppressor functions, connexins and gap junctions have stage, context and isoform dependent effects in breast cancers similar to other cancers. In this review, we tried to describe the current understanding of connexins in tumorigenesis specifically in breast cancers.
... The relevant literature describes different connexon combinations and GJ types: homomeric homotypic, heteromeric homotypic, homomeric heterotypic, and heteromeric heterotypic (Fig. 1b). The functional properties (permeability and selectivity for different molecules and ions) in heterotypic channels formed by two different connexins can be different from the respective properties of homotypic channels [48]. For example, heterotypic GJs and their functional difference from homotypic GJs are discussed by Lin et al. [49], who have shown that the GJs of heart ventricles contain only Cx43, while atrial GJs consist of Cx40 and Cx43. ...
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Numerous data obtained in the last 20 years indicate that all parts of the mature central nervous system, from the retina and olfactory bulb to the spinal cord and brain, contain cells connected by gap junctions (GJs). The morphological basis of the GJs is a group of joined membrane hemichannels called connexons, the subunit of each connexon is the protein connexin. In the central nervous system, connexins show specificity and certain types of them are expressed either in neurons or in glial cells. Connexins and GJs of neurons, combining certain types of inhibitory hippocampal and neocortical neuronal ensembles, provide synchronization of local impulse and rhythmic activity, thalamocortical conduction, control of excitatory connections, which reflects their important role in the processes of perception, concentration of attention and consolidation of memory, both on the cellular and at the system level. Connexins of glial cells are ubiquitously expressed in the brain, and the GJs formed by them provide molecular signaling and metabolic cooperation and play a certain role in the processes of neuronal migration during brain development, myelination, tissue homeostasis, and apoptosis. At the same time, mutations in the genes of glial connexins, as well as a deficiency of these proteins, are associated with such diseases as congenital neuropathies, hearing loss, skin diseases, and brain tumors. This review summarizes the existing data of numerous molecular, electrophysiological, pharmacological, and morphological studies aimed at progress in the study of the physiological and pathophysiological significance of glial and neuronal connexins and GJs for the central nervous system.
... One consequence of gap junction formation by two or more connexins is that that cells form gap junction channels with unique permeability characteristics. For instance, Cx26 has a fairly restrictive permeability and forms heteromeric gap junction channels with Cx32 (Ayad et al., 2006). Cx32 is fully permeable to cAMP and cGMP. ...
Connexins are transmembrane proteins that form gap junction channels. Gap junctions interconnect cells to allow the diffusion of signaling molecules and metabolites between cells. Cells in the respiratory system exhibit unique patterns of connexin expression which determine whether they are interconnected with neighboring cells and the types of signals transmitted between them. Connexin expression in the lung is altered in response to injury as a compensatory mechanism to alter the extent of cell-cell coupling. Gap junctional communication between type II and type I cells plays a critical role in integrating the alveolus to enable type I cells to act as mechanical sensors to regulate pulmonary surfactant secretion by type II cells. By regulating intercellular signaling, controlling the flow of metabolites and restricting the flow of toxic agents, connexins enable the cells of the lung to act as integrated systems.
... The permeability of different connexins to IP 3 was tested using a variety of experimental approaches, most often for Cx26 channels [14,[53][54][55][56][57]. One frequent approach used IP 3 -mediated ER calcium release [58,59] and Ca 2+ -sensitive fluorescent dyes to detect IP 3 permeation through connexin channels. ...
... Gap junction channels were first reported to have poor selectivity for molecules smaller than 1000 Da [85,86]; however, experiments quantitatively measuring the movement of ions and small tracer molecules between coupled cells have shown profound differences in permeation between connexin family members [33,[87][88][89]. Improved technical approaches have extended this quantitative analysis of permeation to signaling molecules like cAMP and IP 3 [3,[36][37][38]53,54,57]. These studies firmly established that each connexin channel type has distinct permeability and conductance properties [3][4][5][6]38]. ...
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.
... On the other hand, permeability studies using HeLa cells co-transfected with Cx26 and Cx30 showed that the heteromeric channels only transport cations, unlike Cx26 homomeric channels which can transport both cations and anions (8), providing further evidence that heteromeric channels exhibit different transport selectivity and biophysical properties. The high molecular selectivity that heteromeric channels can present was shown by Ayad et al. 2006, who compared the permeability of homomeric (Cx26 or Cx32) and heteromeric (Cx26/Cx32) channels to different inositol phosphates. They demonstrated that heteromeric channels are highly selective, able to discriminate among different isomers of inositol phosphate, suggesting that this selective permeability is due to different heteromeric conformations (9). ...
... The high molecular selectivity that heteromeric channels can present was shown by Ayad et al. 2006, who compared the permeability of homomeric (Cx26 or Cx32) and heteromeric (Cx26/Cx32) channels to different inositol phosphates. They demonstrated that heteromeric channels are highly selective, able to discriminate among different isomers of inositol phosphate, suggesting that this selective permeability is due to different heteromeric conformations (9). ...
... Heteromeric (30). This is consistent with a variety of permeabilities observed in reconstituted heteromeric Cx26/Cx32 hemichannels (9). Interestingly, our 3Cx26/3Cx30 subunit arrangements can be theoretically calculated via LTP approach, which demonstrates that the hetero-oligomerization process could occur randomly via a monomeric assembly, where subunit interfacial features would be conserved. ...
Connexin (Cx) protein forms hemichannels and gap junctional channels, which play diverse and profound roles in human physiology and diseases. Gap junctions are arrays of intercellular channels formed by the docking of two hemichannels from adjacent cells. Each hexameric hemichannel contains the same or different Cx isoform. While homomeric Cxs forms have been largely described functionally and structurally, the stoichiometry and arrangement of heteromeric Cx channels remain unknown. The latter, however, are widely expressed in human tissues and variation might have important implications on channel function. Investigating properties of heteromeric Cx channels is challenging considering the high number of potential subunit arrangements and stoichiometries, even when only combining two Cx isoforms. To tackle this problem, we engineered an HA tag onto Cx26 or Cx30 subunits and imaged hemichannels that were liganded by Fab-epitope antibody fragments via atomic force microscopy (AFM). For Cx26-HA/Cx30 or Cx30-HA/Cx26 heteromeric channels, the Fab-HA binding distribution was binomial with a maximum of 3 Fab-HA bound. Furthermore, imaged Cx26/Cx30-HA triple liganded by Fab-HA showed multiple arrangements which can be derived from the law of total probabilities. AFM imaging of ring-like structures of Cx26/Cx30-HA hemichannels confirmed these findings and also detected a polydisperse distribution of stoichiometries. Our results indicate a dominant subunit stoichiometry of 3Cx26:3Cx30 with the most abundant subunit arrangement of Cx26-Cx26-Cx30-Cx26-Cx30-Cx30. To our knowledge, this is the first time that the molecular architecture of heteromeric Cx channels has been revealed, thus providing the basis to explore the functional effect of these channels in biology.
... Cx43 channels are permeable to IP 3 . Permeability of connexin channels to IP 3 has been documented in a number of experimental systems, most frequently for channels composed of Cx26 [21][22][23][42][43][44] . Many of these studies utilized IP 3 mediated ER calcium release 45,46 and Ca 2+ sensitive fluorescent dyes to detect IP 3 permeation through gap junction channels. ...
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.
... [11][12][13][14] Innovative technical approaches have allowed this type of analysis to be extended to signaling molecules like IP 3 and cAMP. [15][16][17][18][19] Thus, different connexin channels are functionally distinct in terms of their conductance and permeability to small molecules. 15,16,[20][21][22] Genetic studies in mice have documented that such functional differences between gap junction channel types are important, because the loss of one connexin cannot be compensated for by replacement with other connexins. ...
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.
... So far, HC permeability studies have concentrated on homomeric HCs (Figure 1), however, HC composition strongly determines permeability [32]. Heteromeric Cx26/Cx32 HCs exhibit lower permeability to, for example, IP 3 , when compared with their homomeric counterparts [33]. ...
Connexins, in particular connexin 43 (Cx43), function as gap junction channels (GJCs) and hemichannels (HCs). Only recently, specific tools have been developed to study their pleiotropic functions. Based on various protein interaction sites, distinct connexin-mimetic peptides have been established that enable discrimination between the function of HCs and GJCs. Although the precise mechanism of action of most of these peptides is still a matter of debate, an increasing number of studies report on important effects of those compounds in disease models. In this review, we summarize the structure, life cycle, and the most important physiological and pathological functions of both connexin GJCs and HCs. We provide a critical overview on the use of connexin-targeting peptides, in particular targeting Cx43, with a special focus on the remaining questions and hurdles to be taken in the research field of connexin channels.
... Depending on their composition, connexin channels can discriminate between highly similar second messengers (e.g. cAMP and cGMP, and among inositol triphosphates (Bevans et al. 1998;Locke et al. 2004;Ayad et al. 2006). Therefore, it is not surprising that functional deletion of a particular connexin isoform produces a specific and distinct pathology (Brisset et al. 2009). ...
Purpose: i) To examine the time window during which intercellular signaling though gap junctions mediates non-targeted (bystander) effects induced by moderate doses of ionizing radiation. ii) 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 one hour 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.
... Fluorescent dyes were a vital tool in such studies with Lucifer yellow (LY) being the most commonly used for dye diffusion studies (Verselis et al., 1986;Cao et al., 1998;Martinez et al., 2002;Valiunas et al., 2002;Beltramello et al., 2003;Dong et al., 2006;Eckert, 2006;Rackauskas et al., 2007;Yum et al., 2007). Gap junctions serve as conduits for intercellular diffusion and transport studies of physiologically relevant molecules and ions were also conducted (Verselis et al., 1986;Veenstra et al., 1994;Goldberg et al., 1999Goldberg et al., , 2002Niessen et al., 2000;Beyer et al., 2001;Veenstra, 2001;Martinez et al., 2002;Bedner et al., 2003Bedner et al., , 2006Locke et al., 2004;Valiunas et al., 2005;Ayad et al., 2006;Bukauskas et al., 2006;Harris, 2007). Our research focused on homotypic and heterotypic gap junction channels involving Cx43 and Cx45, which are abundantly present in the heart (van Kempen et al., 1995;Vozzi et al., 1999;Severs et al., 2001). ...
... Molecular permeability of biological molecules in gap junction has been extensively studied for key functional cytoplasmic molecules, including secondary messengers like IP 3 , cAMP, cGMP, and Ca 2+ (Bevans et al., 1998;Niessen et al., 2000;Bedner et al., 2003Bedner et al., , 2006Locke et al., 2004;Ayad et al., 2006), nucleotides like RNA and siRNA (Valiunas et al., 2005), and glucose and its metabolites like ATP, ADP and glutamate (Goldberg et al., 1999(Goldberg et al., , 2002. These studies reported 3 to 33-fold differences in permeabilities between different molecules in different connexin types. ...
Gap junction channels play a vital role in intercellular communication by connecting cytoplasm of adjoined cells through arrays of channel-pores formed at the common membrane junction. Their structure and properties vary depending on the connexin isoform(s) involved in forming the full gap junction channel. Lack of information on the molecular structure of gap junction channels has limited the development of computational tools for single channel studies. Currently, we rely on cumbersome experimental techniques that have limited capabilities. We have earlier reported a simplified Brownian dynamics gap junction pore model and demonstrated that variations in pore shape at the single channel level can explain some of the differences in permeability of heterotypic channels observed in in vitro experiments. Based on this computational model, we designed simulations to study the influence of pore shape, particle size and charge in homotypic and heterotypic pores. We simulated dye diffusion under whole cell voltage clamping. Our simulation studies with pore shape variations revealed a pore shape with maximal flux asymmetry in a heterotypic pore. We identified pore shape profiles that match the in silico flux asymmetry results to the in vitro results of homotypic and heterotypic gap junction formed out of Cx43 and Cx45. Our simulation results indicate that the channel's pore-shape established flux asymmetry and that flux asymmetry is primarily regulated by the sizes of the conical and/or cylindrical mouths at each end of the pore. Within the set range of particle size and charge, flux asymmetry was found to be independent of particle size and directly proportional to charge magnitude. While particle charge was vital to creating flux asymmetry, charge magnitude only scaled the observed flux asymmetry. Our studies identified the key factors that help predict asymmetry. Finally, we suggest the role of such flux asymmetry in creating concentration imbalances of messenger molecules in cardiomyocytes. We also assess the potency of fibroblasts in aggravating such imbalances through Cx43-Cx45 heterotypic channels in fibrotic heart tissue.
