Gap junctions allow the direct and bidirectional transfer of small molecules between cells. Polyamine sensitivity, which has been observed for a certain gap junction in vitro, confers rectification property to gap junction. Here we report that the polyamine sensitivity of gap junctions in vivo is crucial for skin pattern formation in zebrafish. Transgenic experiments have revealed that several connexin genes were able to rescue the spot phenotype of mutant zebrafish. Mutational analyses of the N-terminal region of connexins revealed that the ExxxE motif, a hypothetical polyamine-binding site, was important for connexin’s role in pattern formation. Ectopic expression of spermidine/spermine N(1)-acetyltransferase (SSAT), a polyamine metabolic enzyme, also caused stripe pattern changes, which further indicates that the polyamine sensitivity of gap junctions is crucial. This is the first report to show that polyamine sensitivity has a physiologically relevant function and is related to skin pattern formation in animals.
Gap junctions facilitate exchange of small molecules between adjacent cells, serving a crucial function for the maintenance of cellular homeostasis. Mutations in connexins, the basic unit of gap junctions, are associated with several human hereditary disorders. For example, mutations in connexin26 (Cx26) cause both non-syndromic deafness and syndromic deafness associated with skin abnormalities such as keratitis-ichthyosis-deafness (KID) syndrome. These mutations can alter the formation and function of gap junction channels through different mechanisms, and in turn interfere with various cellular processes leading to distinct disorders. The KID associated Cx26 mutations were mostly shown to result in elevated hemichannel activities. However, the effects of these aberrant hemichannels on cellular processes are recently being deciphered. Here, we assessed the effect of two Cx26 mutations associated with KID syndrome, Cx26I30N and D50Y, on protein biosynthesis and channel function in N2A and HeLa cells.
The coordination of complex tumor processes requires cells to rapidly modify their phenotype and is achieved by direct cell-cell communication through gap junction channels composed of connexins. Previous reports have suggested that gap junctions are tumor suppressive based on connexin 43 (Cx43), but this does not take into account differences in connexin-mediated ion selectivity and intercellular communication rate that drive gap junction diversity. We find that glioblastoma cancer stem cells (CSCs) possess functional gap junctions that can be targeted using clinically relevant compounds to reduce self-renewal and tumor growth. Our analysis reveals that CSCs express Cx46, while Cx43 is predominantly expressed in non-CSCs. During differentiation, Cx46 is reduced, while Cx43 is increased, and targeting Cx46 compromises CSC maintenance. The difference between Cx46 and Cx43 is reflected in elevated cell-cell communication and reduced resting membrane potential in CSCs. Our data demonstrate a pro-tumorigenic role for gap junctions that is dependent on connexin expression.
Nasal mucosal epithelial cells express connexins, the prototypical gap junction proteins, and pannexins, a new family of channel proteins homologous to the invertebrate gap junction proteins. The physiological and pathophysiological roles of these transmembrane proteins in nasal mucosa are largely still unknown.
Extracellular pH is an important physiological determinant of vascular tone that is normally maintained within 7.35-7.45. Any change outside this range leads to severe pathological repercussions. We investigated the unknown effects of extracellular acidosis on relaxation in the superior mesenteric artery (SMA) of goat. SMA rings were employed to maintain isometric contractions at extracellular pH (pHo) 7.4 and 6.8. We analyzed the effect of acidosis (pHo6.8) compared to physiological pH (pHo7.4) on three signaling mediators of endothelium-dependent hyperpolarization: nitric oxide (NO), prostaglandin I2(PGI2), and myoendothelial gap junctions (MEGJ). NO and cyclic guanosine monophosphate (cGMP) levels were compared between normal and acidic pH. Quantitative real-time PCR (qPCR) studies determined the change in expression of vascular connexin (Cx), Cx37, Cx40, and Cx43. Under acidosis, acetyl choline-induced relaxation was augmented in an endothelium-dependent manner via eNOS-NO-cGMP signaling. Conversely, at normal pH, acetyl choline-induced vasorelaxation was mediated primarily via COX-PGI2pathway. The functional activity of MEGJ was increased under acidosis as evident from increased sensitivity of connexin blockers and upregulated gene and protein expression of connexins. In conclusion, acetyl choline-induced augmented vasorelaxation under acidosis is mediated by NOS-NO-cGMP, with a partial role of MEGJ as EDH mediators in the SMA. Present data suggest a novel role of connexin as therapeutic targets to attenuate the detrimental effect of acidosis on vascular tone.
Gap junctions confer interconnectivity of the cytoplasm in neighboring cells via docking of two connexons expressed in each of the adjacent membranes. Undocked connexons, referred to as hemichannels, may open and connect the cytoplasm with the extracellular fluid. The hemichannel configuration of connexins (Cxs) displays isoform-specific permeability profiles that are not directly determined by the size and charge of the permeant. To further explore Ca(2+)-mediated gating and permeability features of connexin hemichannels, we heterologously expressed Cx30 hemichannels in Xenopus laevis oocytes. The sensitivity toward divalent cation-mediated gating differed between small atomic ions (current) and fluorescent dye permeants, indicating that these permeants are distinctly gated. Three aspartate residues in Cx30 (D50, D172 and D179) have previously been implicated in the Ca(2+) sensitivity of other hemichannel isoforms. While the aspartate at position D50 was indispensable for divalent cation-dependent gating of Cx30 hemichannels, substitutions of the two other residues had no significant effect on gating, illustrating differences in the gating mechanisms between connexin isoforms. Using the substituted-cysteine accessibility method (SCAM), we evaluated the role of possible pore-lining residues in the permeation of ions and ethidium through Cx30 hemichannels. Of the cysteine-substituted residues, interaction of a proposed pore-lining cysteine at position 37 with the positively charged compound [2-(trimethylammonium)ethyl] methane thiosulfonate bromide (MTS-ET) increased Cx30-mediated currents, with unperturbed ethidium permeability. In summary, our results demonstrate that the permeability of hemichannels is regulated in a permeant-specific manner, and underscores that hemichannels are selective rather than non-discriminating and freely-diffusable pores.
Gap junctions are cellular contact sites composed of clustered connexin transmembrane proteins that act in dual capacities as channels for direct intercellular exchange of small molecules and as structural adhesion complexes known as gap junction nexuses. Depending on the connexin isoform, the cluster of channels (the gap junction plaque) can be stably or fluidly arranged. Here we used confocal microscopy and mutational analysis to identify the residues within the connexin proteins that determine gap junction plaque stability. We found that stability is altered by changing redox balance using a reducing agent indicating gap junction nexus stability is modifiable. Stability of the arrangement of connexins is thought to regulate intercellular communication by establishing an ordered supramolecular platform. By identifying the residues that establish plaque stability, these studies lay the groundwork for exploration of mechanisms by which gap junction nexus stability modulates intercellular communication.
While gap junctions support the exchange of a number of molecules between neighboring cells, connexin hemichannels provide communication between the cytosol and the extracellular environment of an individual cell. The latter equally holds true for channels composed of pannexin proteins, which display an architecture reminiscent of connexin hemichannels. In physiological conditions, gap junctions are usually open, while connexin hemichannels and, to a lesser extent, pannexin channels are typically closed, yet they can be activated by a number of pathological triggers. Several agents are available to inhibit channels built up by connexin and pannexin proteins, including alcoholic substances, glycyrrhetinic acid, anesthetics and fatty acids. These compounds not always strictly distinguish between gap junctions, connexin hemichannels and pannexin channels, and may have effects on other targets as well. An exception lies with mimetic peptides, which reproduce specific amino acid sequences in connexin or pannexin primary protein structure. In this paper, a state-of-the-art overview is provided on inhibitors of cellular channels consisting of connexins and pannexins with specific focus on their mode-of-action and therapeutic potential.
Most of the early studies on gap junction (GJ) channel function and docking compatibility were on rodent connexins, while recent research on GJ channels gradually shifted from rodent to human connexins largely due to the fact that mutations in many human connexin genes are found to associate with inherited human diseases. The studies on human connexins have revealed some key differences from those found in rodents, calling for a comprehensive characterization of human GJ channels. Functional studies revealed that docking and formation of functional GJ channels between two hemichannels are possible only between docking-compatible connexins. Two groups of docking-compatible rodent connexins have been identified. Compatibility is believed to be due to their amino acid residue differences at the extracellular loop domains (E1 and E2). Sequence alignment of the E1 and E2 domains of all connexins known to make GJs revealed that they are highly conserved and show high sequence identity with human Cx26, which is the only connexin with near atomic resolution GJ structure. We hypothesize that different connexins have a similar structure as that of Cx26 at the E1 and E2 domains and use the corresponding residues in their E1 and E2 domains for docking. Based on the Cx26 GJ structure and sequence analysis of well-studied connexins, we propose that the E1-E1 docking interactions are staggered with each E1 interacting with two E1s on the docked connexon. The putative E1 docking residues are conserved in both docking-compatible and -incompatible connexins, indicating that E1 does not likely serve a role in docking compatibility. However, in the case of E2-E2 docking interactions, the putative docking residues are only conserved within the docking-compatible connexins, suggesting the E2 is likely to serve the function of docking compatibility. Docking compatibility studies on human connexins have attracted a lot of attention due to the fact that putative docking residues are mutational hotspots for several connexin-linked human diseases. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.
Enhanced expression and function of gap junctions and pannexin (Panx) channels has been associated with both peripheral and central mechanisms of pain sensitization. At the level of the sensory ganglia, evidence includes augmented gap junction and pannexin1 expression in glial cells and neurons in inflammatory and neuropathic pain models and increased synchrony and enhanced cross-excitation among sensory neurons by gap junction-mediated coupling. In spinal cord and in suprapinal areas, evidence is largely limited to increased expression of relevant proteins, although in several rodent pain models, hypersensitivity is reduced by treatment with gap junction/Panx1 channel blocking compounds. Moreover, targeted modulation of Cx43 expression was shown to modulate pain thresholds, albeit in somewhat contradictory ways, and mice lacking Panx1 expression globally or in specific cell types show depressed hyperalgesia. We here review the evidence for involvement of gap junctions and Panx channels in a variety of animal pain studies and then discuss ways in which gap junctions and Panx channels may mediate their action in pain processing. This discussion focusses on spread of signals among satellite glial cells, in particular intercellular Ca(2+) waves, which are propagated through both gap junction and Panx1-dependent routes and have been associated with the phenomenon of spreading depression and the malady of migraine headache with aura.