Search

Connexin hemichannels were identified as the first members of the eukaryotic large-pore channel family that mediate permeation of both atomic ions and small molecules between the intracellular and extracellular environments. The conventional view is that their pore is a large passive conduit through which both ions and molecules diffuse in a similar manner. In stark contrast to this notion, we demonstrate that the permeation of ions and of molecules in connexin hemichannels can be uncoupled and differentially regulated. We find that human connexin mutations that produce pathologies and were previously thought to be loss-of-function mutations due to the lack of ionic currents are still capable of mediating the passive transport of molecules with kinetics close to those of wild-type channels. This molecular transport displays saturability in the micromolar range, selectivity, and competitive inhibition, properties that are tuned by specific interactions between the permeating molecules and the N-terminal domain that lies within the pore-a general feature of large-pore channels. We propose that connexin hemichannels and, likely, other large-pore channels, are hybrid channel/transporter-like proteins that might switch between these two modes to promote selective ion conduction or autocrine/paracrine molecular signaling in health and disease processes.

Connexins are membrane channels that form intercellular gap junctions for direct intercellular communications or assemble as unpaired hemichannels, which mediate diffusional secretion of molecules with a molecular weight of ~1 kDa. Besides their primary gap junction forming role, connexins are also essential for brain homeostasis and synaptic function, which suggests their involvement in the pathogenesis of neuropathic pain. Opening of astrocytic Cx43 hemichannels contributes to the pathogenesis of neuropathic pain through the release of signaling molecules, which promote neuronal hyperactivity and instruct a neuroinflammatory environment. Upregulation of neuronal Cx36 augments gap junction communication in both the peripheral and central nervous system (PNS and CNS). In the PNS, enhanced neuronal gap junctional connectivity causes the spreading of intracellular injury-related signals to uninjured neurons, contributing to abnormal neuronal firing. Cx43 hemichannel blockers and Cx36 gap junction blockers reduce allodynia in preclinical studies, making connexins promising therapeutic agents for the treatment of neuropathic pain. Neuropathic pain is a chronic debilitating condition caused by nerve injury or a variety of diseases. At the core of neuropathic pain lies the aberrant neuronal excitability in the peripheral and/or central nervous system (PNS and CNS). Enhanced connexin expression and abnormal activation of connexin-assembled gap junctional channels are prominent in neuropathic pain along with reactive gliosis, contributing to neuronal hypersensitivity and hyperexcitability. In this review, we delve into the current understanding of how connexin expression and function contribute to the pathogenesis and pathophysiology of neuropathic pain and argue for connexins as potential therapeutic targets for neuropathic pain management. [ABSTRACT FROM AUTHOR]

Simple Summary: Gap junction channels are composed of an assembly of connexin proteins and allow direct communication between cells. They are highly conserved across vertebrates and form wide pores in cell membranes for the passage of ions and metabolites. Junctional channels are formed from the end-to-end docking of hemichannels, and both junctional channels and hemichannels are vital for many physiological activities. Several medical conditions are associated with problems in gap junction communication, ranging from deafness to fatal cardiac arrhythmias. Many connexin channel diseases can be linked to genetic mutations, and nearly 1000 have been identified in connexin genes. Prior to 2009, atomic-level structural details of gap junction channels were essentially non-existent. This information is critical for understanding channel function and to assess the pathological nature of disease-causing mutations. Fortunately, since 2009, the powerful tools of X-ray crystallography and electron cryomicroscopy have yielded over 50 high-resolution structures of connexin channels. This review aims to provide a comprehensive summary of this astounding 15-year period of structural discovery in the gap junction field. Divided into eight distinct sections, we describe key details found in this compendium of structures, such as conserved features in the design of connexin channels, insights into channel gating and surprises regarding where membrane lipids are bound to the channels. In addition, we highlight areas in which we need more information, such as the structure of highly flexible regions within connexin channels that have so far resisted visualization. Furthermore, targeting connexins for drug discovery is still in its infancy, and much more structural data are needed to pursue this end. Connexins (Cxs) are a family of integral membrane proteins, which function as both hexameric hemichannels (HCs) and dodecameric gap junction channels (GJCs), behaving as conduits for the electrical and molecular communication between cells and between cells and the extracellular environment, respectively. Their proper functioning is crucial for many processes, including development, physiology, and response to disease and trauma. Abnormal GJC and HC communication can lead to numerous pathological states including inflammation, skin diseases, deafness, nervous system disorders, and cardiac arrhythmias. Over the last 15 years, high-resolution X-ray and electron cryomicroscopy (cryoEM) structures for seven Cx isoforms have revealed conservation in the four-helix transmembrane (TM) bundle of each subunit; an αβ fold in the disulfide-bonded extracellular loops and inter-subunit hydrogen bonding across the extracellular gap that mediates end-to-end docking to form a tight seal between hexamers in the GJC. Tissue injury is associated with cellular Ca2+ overload. Surprisingly, the binding of 12 Ca2+ ions in the Cx26 GJC results in a novel electrostatic gating mechanism that blocks cation permeation. In contrast, acidic pH during tissue injury elicits association of the N-terminal (NT) domains that sterically blocks the pore in a "ball-and-chain" fashion. The NT domains under physiologic conditions display multiple conformational states, stabilized by protein–protein and protein–lipid interactions, which may relate to gating mechanisms. The cryoEM maps also revealed putative lipid densities within the pore, intercalated among transmembrane α-helices and between protomers, the functions of which are unknown. For the future, time-resolved cryoEM of isolated Cx channels as well as cryotomography of GJCs and HCs in cells and tissues will yield a deeper insight into the mechanisms for channel regulation. The cytoplasmic loop (CL) and C-terminal (CT) domains are divergent in sequence and length, are likely involved in channel regulation, but are not visualized in the high-resolution X-ray and cryoEM maps presumably due to conformational flexibility. We expect that the integrated use of synergistic physicochemical, spectroscopic, biophysical, and computational methods will reveal conformational dynamics relevant to functional states. We anticipate that such a wealth of results under different pathologic conditions will accelerate drug discovery related to Cx channel modulation. [ABSTRACT FROM AUTHOR]

Connexins allow intercellular communication by forming gap junction channels (GJCs) between juxtaposed cells. Connexin26 (Cx26) can be regulated directly by CO2. This is proposed to be mediated through carbamylation of K125. We show that mutating K125 to glutamate, mimicking the negative charge of carbamylation, causes Cx26 GJCs to be constitutively closed. Through cryo-EM we observe that the K125E mutation pushes a conformational equilibrium towards the channel having a constricted pore entrance, similar to effects seen on raising the partial pressure of CO2. In previous structures of connexins, the cytoplasmic loop, important in regulation and where K125 is located, is disordered. Through further cryo-EM studies we trap distinct states of Cx26 and observe density for the cytoplasmic loop. The interplay between the position of this loop, the conformations of the transmembrane helices and the position of the N-terminal helix, which controls the aperture to the pore, provides a mechanism for regulation.

Gap junction channels (GJCs) mediate intercellular communication by connecting two neighbouring cells and enabling direct exchange of ions and small molecules. Cell coupling via connexin-43 (Cx43) GJCs is important in a wide range of cellular processes in health and disease (Churko and Laird, 2013; Liang et al., 2020; Poelzing and Rosenbaum, 2004), yet the structural basis of Cx43 function and regulation has not been determined until now. Here, we describe the structure of a human Cx43 GJC solved by cryo-EM and single particle analysis at 2.26 Å resolution. The pore region of Cx43 GJC features several lipid-like densities per Cx43 monomer, located close to a putative lateral access site at the monomer boundary. We found a previously undescribed conformation on the cytosolic side of the pore, formed by the N-terminal domain and the transmembrane helix 2 of Cx43 and stabilized by a small molecule. Structures of the Cx43 GJC and hemichannels (HCs) in nanodiscs reveal a similar gate arrangement. The features of the Cx43 GJC and HC cryo-EM maps and the channel properties revealed by molecular dynamics simulations suggest that the captured states of Cx43 are consistent with a closed state.

Mature oligodendrocytes are myelin forming glial cells which are responsible for myelination of neuronal axons in the white matter of the central nervous system. Myelin pathology is a major feature of severe neurological disorders. Oligodendrocyte-specific gene mutations and/or white matter alterations have also been addressed in a variety of mental disorders. Breakdown of myelin integrity and demyelination is associated with severe symptoms, including impairments in motor coordination, breathing, dysarthria, perception (vision and hearing), and cognition. Furthermore, there is evidence indicating that myelin sheath defects and white matter pathology contributes to the affective and cognitive symptoms of patients with mental disorders. Oligodendrocytes express the connexins GJC2; mCx47 [human (GJC2) and mouse (mCx47) connexin gene nomenclature according to Söhl and Willecke (2003) ], GJB1; mCx32, and GJD1; mCx29 in both white and gray matter. Preclinical findings indicate that alterations in connexin expression in oligodendrocytes and astrocytes can induce myelin defects. GJC2; mCx47 is expressed at early embryonic stages in oligodendrocyte precursors cells which precedes central nervous system myelination. In adult humans and animals GJC2, respectively mCx47 expression is essential for oligodendrocyte function and ensures adequate myelination as well as myelin maintenance in the central nervous system. In the past decade, evidence has accumulated suggesting that mental disorders can be accompanied by changes in connexin expression, myelin sheath defects and corresponding white matter alterations. This dual pathology could compromise inter-neuronal information transfer, processing and communication and eventually contribute to behavioral, sensory-motor, affective and cognitive symptoms in patients with mental disorders. The induction of myelin repair and remyelination in the central nervous system of patients with mental disorders could help to restore normal neuronal information propagation and ameliorate behavioral and cognitive symptoms in individuals with mental disorders. [ABSTRACT FROM AUTHOR]

Simple Summary: Congenital heart disease is associated with substantial mortality and morbidity as well as socioeconomic burden, and increasing research underscores the genetic basis for it. In this study, a genome-wide genotyping with microsatellite markers followed by linkage analysis was performed in a Chinese family suffering congenital heart disease and arrhythmias, and a novel genetic locus linked to the disease was located on chromosome 17q21.31-q21.33. Sequencing assays of the candidate genes at the locus revealed a novel heterozygous mutation in the GJC1 gene coding for Cx45, NM_005497.4:c.550A>G;p.R184G, which was in co-segregation with the disease in the whole family and was not detected in 516 control individuals. Electrophysiological analyses showed that the mutation significantly diminished the coupling conductance in homomeric cell pairs (R184G/R184G) and in cell pairs expressing R184G/Cx45 or R184G/Cx43, in a dominant-negative mode. Fluorescent dye uptake experiments demonstrated no apparent change of the R184G mutation on the Cx45 hemichannels. These findings define a new genetic locus for congenital heart disease and arrhythmias on chromosome 17q21.31-q21.33 and indicate GJC1 as a new gene predisposing to the disease. As the most prevalent type of birth malformation, congenital heart disease (CHD) gives rise to substantial mortality and morbidity as well as a socioeconomic burden. Although aggregating investigations highlight the genetic basis for CHD, the genetic determinants underpinning CHD remain largely obscure. In this research, a Chinese family suffering from autosomal dominant CHD (atrial septal defect) and arrhythmias was enrolled. A genome-wide genotyping with microsatellite markers followed by linkage assay as well as sequencing analysis was conducted. The functional effects of the discovered genetic mutation were characterized by dual patch-clamp electrophysiological recordings in N2A cells and propidium iodide uptake assays in HeLa cells. As a result, a novel genetic locus for CHD and arrhythmias was located on chromosome 17q21.31-q21.33, a 4.82-cM (5.12 Mb) region between two markers of D17S1861 and D17S1795. Sequencing assays of the genes at the mapped locus unveiled a novel heterozygous mutation in the GJC1 gene coding for connexin 45 (Cx45), NM_005497.4:c.550A>G;p.R184G, which was in co-segregation with the disease in the whole family and was not observed in 516 unrelated healthy individuals or gnomAD. Electrophysiological analyses revealed that the mutation significantly diminished the coupling conductance in homomeric cell pairs (R184G/R184G) and in cell pairs expressing either R184G/Cx45 or R184G/Cx43. Propidium iodide uptake experiments demonstrated that the Cx45 R184G mutation did not increase the Cx45 hemichannel function. This investigation locates a new genetic locus linked to CHD and arrhythmias on chromosome 17q21.31-q21.33 and indicates GJC1 as a novel gene predisposing to CHD and arrhythmias, implying clinical implications for prognostic risk assessment and personalized management of patients affected with CHD and arrhythmias. [ABSTRACT FROM AUTHOR]

Ginsenoside Rg1, a traditional Chinese medicine monomer, has been shown to have antidepressant effects. We previously found that Rg1 exerts antidepressant effects by improving the gap junction channels (GJCs) dysfunction; however, the downstream mechanisms through which Rg1 ameliorates GJC dysfunction remain unclear. Since hemichannels directly release glutamate, GJC dysfunction decreases the expression levels of glutamate transporters in astrocytes, and glutamatergic system dysfunction plays an essential role in the pathogenesis of depression. The glutamatergic system may be a potential downstream target of Rg1 that exerts antidepressant effects. Therefore, in this study, we aimed to determine the downstream mechanisms by which Rg1 ameliorated GJC dysfunction and exerted its antidepressant effects. Corticosterone (CORT) is used to mimic high glucocorticoid levels in patients with depression in vitro. Primary cortical astrocytes were isolated and phosphorylation of connexin43 (Cx43) as well as the functions of hemichannels, GJCs, and the glutamatergic system were evaluated after drug treatment. Rg1 pretreatment reversed the anomalous activation of Cx43 phosphorylation as well as the dysfunction of hemichannels, GJCs, and the glutamatergic system induced by CORT. These results suggest that Rg1 can ameliorate CORT-induced dysfunction of the glutamatergic system in astrocytes by potentially reducing Cx43 phosphorylation and inhibiting opening of hemichannels, thereby improving GJC dysfunction. [ABSTRACT FROM AUTHOR]

CO 2 directly modifies the gating of connexin26 (Cx26) gap junction channels and hemichannels. This gating depends upon Lys125, and the proposed mechanism involves carbamylation of Lys125 to allow formation of a salt bridge with Arg104 on the neighbouring subunit. We demonstrate via carbamate trapping and tandem mass spectrometry that five Lys residues within the cytoplasmic loop, including Lys125, are indeed carbamylated by CO 2 . The cytoplasmic loop appears to provide a chemical microenvironment that facilitates carbamylation. Systematic mutation of these Lys residues to Arg shows that only carbamylation of Lys125 is essential for hemichannel opening. By contrast, carbamylation of Lys108 and Lys125 is essential for gap junction closure to CO 2 . Chicken (Gallus gallus) Cx26 gap junction channels lack Lys108 and do not close to CO 2 , as shown by both a dye transfer assay and a high-resolution cryogenic electron microscopy structure. The mutation Lys108Arg prevents CO 2 -dependent gap junction channel closure in human Cx26. Our findings directly demonstrate carbamylation in connexins, provide further insight into the differential action of CO 2 on Cx26 hemichannels and gap junction channels, and increase support for the role of the N-terminus in gating the Cx26 channel. KEY POINTS: Direct evidence of carbamylation of multiple lysine residues in the cytoplasmic loop of Cx26. Concentration-dependent carbamylation at lysines 108, 122 and 125. Only carbamylation of lysine 125 is essential for hemichannel opening to CO 2 . Carbamylation of lysine 108 along with lysine 125 is essential for CO 2 -dependent gap junction channel closure., (© 2025 The Author(s). The Journal of Physiology published by John Wiley & Sons Ltd on behalf of The Physiological Society.)

Gap junction (GJ) intercellular communication is crucial in many physiological and pathological processes. A GJ channel is formed by head-to-head docking of two hexameric hemichannels from two neighboring cells. Heterotypic GJ channels formed by two different homomeric connexin hemichannels often display rectification properties in the current-voltage relationship while the underlying mechanisms are not fully clear. Here we studied heterotypic Cx46/Cx50 GJs at a single GJ channel level. Our data showed unitary Cx46/Cx50 GJ channel conductance (γ j ) rectification when 5 mmol/L Mg 2+ was included in the patch pipette solution, while no γ j rectification was observed when no Mg 2+ was added. Including 5 mmol/L Mg 2+ in pipette solution significantly decreased the γ j of homotypic Cx46 GJ with little change in homotypic Cx50 γ j . A missense point variant in Cx46 (E43F) reduced the Mg 2+ -dependent reduction in γ j of Cx46 E43F GJ, indicating that E43 might be partially responsible for Mg 2+ -dependent decrease in γ j of Cx46. A comprehensive understanding of Mg 2+ modulation of GJ at the individual channel level is useful in understanding factors in modulating GJ-mediated intercellular communication in health and diseases., Competing Interests: Honghong Chen and Donglin Bai declare that they have no conflict of interest., (© The Author(s) 2024.)

Connexins are a family of proteins that can form two distinct types of channels: hemichannels and gap junction channels. Hemichannels are composed of six connexin subunits and when open allow for exchanges between the cytoplasm and the extracellular milieu. Gap junction channels are formed by head-to-head docking of two hemichannels in series, each one from one of two adjacent cells. These channels allow for exchanges between the cytoplasms of contacting cells. The lens is a transparent structure located in the eye that focuses light on the retina. The transparency of the lens depends on its lack of blood irrigation and the absence of organelles in its cells. To survive such complex metabolic scenario, lens cells express Cx43, Cx46 and Cx50, three connexins isoforms that form hemichannels and gap junction channels that allow for metabolic cooperation between lens cells. This review focuses on the roles of Cx46 hemichannels and gap junction channels in the lens under physiological conditions and in the formation of cataracts, with emphasis on the modulation by posttranslational modifications.

Connexins are a family of proteins that can form two distinct types of channels: hemichannels and gap junction channels. Hemichannels are composed of six connexin subunits and when open allow for exchanges between the cytoplasm and the extracellular milieu. Gap junction channels are formed by head-to-head docking of two hemichannels in series, each one from one of two adjacent cells. These channels allow for exchanges between the cytoplasms of contacting cells. The lens is a transparent structure located in the eye that focuses light on the retina. The transparency of the lens depends on its lack of blood irrigation and the absence of organelles in its cells. To survive such complex metabolic scenario, lens cells express Cx43, Cx46 and Cx50, three connexins isoforms that form hemichannels and gap junction channels that allow for metabolic cooperation between lens cells. This review focuses on the roles of Cx46 hemichannels and gap junction channels in the lens under physiological conditions and in the formation of cataracts, with emphasis on the modulation by posttranslational modifications. [ABSTRACT FROM AUTHOR]

Key points: Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.Recent structural studies by cryo‐electron microscopy have produced high‐resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure–function comparison.Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform‐specific differences in Cx46 and Cx50 intercellular channel function.We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N‐terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.The results of this study establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease‐causing variants. Connexins form intercellular communication channels, known as gap junctions (GJs), that facilitate diverse physiological roles, from long‐range electrical and chemical coupling to coordinating development and nutrient exchange. GJs formed by different connexin isoforms harbour unique channel properties that have not been fully defined mechanistically. Recent structural studies on Cx46 and Cx50 defined a novel and stable open state and implicated the amino‐terminal (NT) domain as a major contributor for isoform‐specific functional differences between these closely related lens connexins. To better understand these differences, we constructed models corresponding to wildtype Cx50 and Cx46 GJs, NT domain swapped chimeras, and point variants at the 9th residue for comparative molecular dynamics (MD) simulation and electrophysiology studies. All constructs formed functional GJ channels, except the chimeric Cx46‐50NT variant, which correlated with an introduced steric clash and increased dynamical behaviour (instability) of the NT domain observed by MD simulation. Single channel conductance correlated well with free‐energy landscapes predicted by MD, but resulted in a surprisingly greater degree of effect. Additionally, we observed significant effects on transjunctional voltage‐dependent gating (Vj gating) and/or open state dwell times induced by the designed NT domain variants. Together, these studies indicate intra‐ and inter‐subunit interactions involving both hydrophobic and charged residues within the NT domains of Cx46 and Cx50 play important roles in defining GJ open state stability and single channel conductance, and establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating GJ channel mechanisms and the molecular basis of cataract‐linked connexin variants. Key points: Gap junctions formed by different connexins are expressed throughout the body and harbour unique channel properties that have not been fully defined mechanistically.Recent structural studies by cryo‐electron microscopy have produced high‐resolution models of the related but functionally distinct lens connexins (Cx50 and Cx46) captured in a stable open state, opening the door for structure–function comparison.Here, we conducted comparative molecular dynamics simulation and electrophysiology studies to dissect the isoform‐specific differences in Cx46 and Cx50 intercellular channel function.We show that key determinants Cx46 and Cx50 gap junction channel open stability and unitary conductance are shaped by structural and dynamic features of their N‐terminal domains, in particular the residue at the 9th position and differences in hydrophobic anchoring sites.The results of this study establish the open state Cx46/50 structural models as archetypes for structure–function studies targeted at elucidating the mechanism of gap junction channels and the molecular basis of disease‐causing variants. [ABSTRACT FROM AUTHOR]

Background: Atrial fibrillation (AF) represents the most common clinical cardiac arrhythmia and substantially increases the risk of cerebral stroke, heart failure, and death. Although causative genes for AF have been identified, the genetic determinants for AF remain largely unclear.Objective: This study aimed to investigate the molecular basis of AF in a Chinese kindred.Methods: A 4-generation family with autosomal-dominant AF and other arrhythmias (atrioventricular block, sinus bradycardia, and premature ventricular contractions) was recruited. Genome-wide scan with microsatellite markers and linkage analysis as well as whole-exome sequencing analysis were performed. Electrophysiological characteristics and subcellular localization of the AF-linked mutant were analyzed using dual whole-cell patch clamps and confocal microscopy, respectively.Results: A novel genetic locus for AF was mapped to chromosome 17q21.3, a 3.23-cM interval between markers D17S951 and D17S931, with a maximum 2-point logarithm of odds score of 4.2144 at marker D17S1868. Sequencing analysis revealed a heterozygous mutation in the mapping region, NM_005497.4:c.703A>T;p.(M235L), in the GJC1 gene encoding connexin45 (Cx45). The mutation cosegregated with AF in the family and was absent in 632 control individuals. The mutation decreased the coupling conductance in cell pairs (M235L/M235L, M235L/Cx45, M235L/Cx43, and M235L/Cx40), likely because of impaired subcellular localization.Conclusion: This study defines a novel genetic locus for AF on chromosome 17q21.3 and reveals a loss-of-function mutation in GJC1 (Cx45) contributing to AF and other cardiac arrhythmias. [ABSTRACT FROM AUTHOR]

Extracellular ATP (eATP) is a potent singling molecule in activation of fish innate immunity while the molecular determinants for eATP release in fish were not completely understood. Connexin32 (Cx32) is a member of gap junction protein family that plays important immunological functions in mammals. However, the immune relevance of Cx32 and its role in ATP release in fish has not been investigated. Here, we identified, characterized three Cx32 isoform genes (Cx32.2 , Cx32.2x and Cx32.7) from the Japanese flounder Paralichthys olivaceus , and investigated their role in inflammation-induced ATP release in fish. Expression analysis revealed that even though all the three Cx32 genes are constitutively expressed in all examined Japanese flounder tissues, Cx32.2 and Cx32.2x are dominantly expressed in liver, and Cx32.7 is highly expressed in intestine and head kidney macrophages. In addition, we showed that gene expression of all the three Cx32 isoforms was modulated by cAMP stimulation and inflammatory challenges. Furthermore, we revealed that Cx32 expression was upregulated in TNF-alpha overexpressed Japanese flounder FG-9307 cells. Moreover, overexpression of the three Cx32 isoforms significantly reduced the gene expression level of LPS-induced pro-inflammatory cytokine IL-8 and TNF-alpha , indicating that Cx32 is involved in modulating inflammatory response in fish. Finally, we showed that inflammation-induced ATP release was significantly increased in Cx32-overexpressed Japanese flounder FG-9307 cells, and this increased ATP release could be attenuated by pre-incubation with gap junction protein blocker carbenoxolone. Taken together, we for the first time reported the involvement of Cx32 in fish immunity. Our findings suggested that in addition to Cx43 and pannexin1 channels, Cx32 also plays a role in inflammation-induced ATP release in fish. • Three Cx32 isoform genes were expressed in Japanese flounder Paralichthys olivaceus. • Cx32 expression was modulated by PAMPs challenge and cAMP stimulation. • TNF-alpha overexpression up-regulated Cx32 expression. • Cx32 down-regulated LPS-induced pro-inflammatory cytokine expression. • Cx32 is an immune response gene involved in inflammation-induced ATP release in fish. [ABSTRACT FROM AUTHOR]

Gap junction (GJ) channels formed by Cx45 exist in nodal cells in the heart where the action potential propagation is the slowest. The cellular mechanisms of slow propagation speed (or longer junctional delay) in nodal cells could be a combination of several factors, including lack of voltage-gated sodium channels, smaller cell size, and a lower GJ coupling conductance of Cx45. Compared to other cardiac GJs, Cx45 GJs possess not only the lowest unitary channel conductance, but also the highest extent and the fastest kinetics of the transjunctional voltage-dependent gating (V j -gating) together with a slow recovery. These unique gating properties could make Cx45 GJs more vulnerable for dynamic uncoupling to a much lower coupling level, especially when junctional delay is lengthened and/or the heart rate is elevated. The molecular mechanisms determining the V j -gating properties of Cx45 (a connexin belongs to γ group) GJs have not been studied. Previous functional studies on the amino terminal (NT) domain chimeras or point variants of other connexins belong to α or β group showed that their NT domains played an important role in determining their V j -gating properties. The crystal and cryo-electron microscope structures of homologous connexin GJs showed that the NT domain lines the GJ pore, a position that could serve a role in V j -sensing and gating. We hypothesize that the residues in the NT domain of Cx45 are important for its V j -gating properties. Protein sequence alignment of human Cx45 NT domain with the connexins in the α and β groups revealed that the second and the eighth residues in Cx45 are different from most of these connexins. We generated a total of 14 variants on these two residues and studied their ability to form functional GJs and their V j -gating properties in model cells. Our results revealed an important role of these two residues on fast V j -gating kinetics and formation of morphological and functional GJ channels. In contrast, no V j -gating change was observed on a GFP tagged Cx45 at its carboxyl terminus. • Variants in amino terminal (NT) of human Cx45 were studied in model cells. • Gap junction V j -gating kinetics of the Cx45 NT variants were slowed. • Gap junction of tagged Cx45 (Cx45-GFP) showed no change in V j -gating properties. • Cx45 NT domain is important for V j -gating kinetics and gap junction formation. [ABSTRACT FROM AUTHOR]

Mutations in more than half of human connexin genes encoding gap junction (GJ) subunits have been linked to inherited human diseases. Functional studies of human GJ channels are essential for revealing mechanistic insights into the etiology of disease-linked connexin mutants. However, the commonly used Xenopus oocytes, N2A, HeLa, and other model cells for recombinant expression of human connexins have different and significant limitations. Here we developed a human cell line (HEK293) with each of the endogenous connexins (Cx43 and Cx45) knocked out using the CRISPR-Cas9 system. Double knockout HEK293 cells showed no background GJ coupling, were easily transfected with several human connexin genes (such as those encoding Cx46, Cx50, Cx37, Cx45, Cx26, and Cx36) which successfully formed functional GJs and were readily accessible for dual patch clamp analysis. Single knockout Cx43 or Cx45 HEK cell lines could also be used to characterize human GJ channels formed by Cx45 or Cx43, respectively, with an expression level suitable for studying macroscopic and single channel GJ channel properties. A cardiac arrhythmia linked Cx45 mutant R184G failed to form functional GJs in DKO HEK293 cells with impaired localizations. These genetically engineered HEK293 cells are well suited for patch clamp study of human GJ channels., (© 2024 The Author(s).)

Connexins are the proteins that form the gap junction channels that are essential for cell-to-cell communication. These channels are formed by head-to-head docking of hemichannels (each from one of two adjacent cells). Free "undocked" hemichannels at the plasma membrane are mostly closed, although they are still important under physiological conditions. However, abnormal and sustained increase in hemichannel activity due to connexin mutations or acquired conditions can produce or contribute to cell damage. For example, mutations of Cx26, a connexin isoform, can increase hemichannel activity and cause deafness. Studies using purified isolated systems under well-controlled conditions are essential for a full understanding of molecular mechanisms of hemichannel function under normal conditions and in disease, and here, we present methodology for the expression, purification, and functional analysis of hemichannels formed by Cx26., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Connexins are polytopic domain membrane proteins that form hexameric hemichannels (HCs) which can assemble into gap junction channels (GJCs) at the interface of two neighboring cells. The HCs may be involved in ion and small-molecule transport across the cellular plasma membrane in response to various stimuli. Despite their importance, relatively few structures of connexin HCs are available to date, compared to the structures of the GJCs. Here, we describe a protocol for expression, purification, and nanodisc reconstitution of connexin-43 (Cx43) HCs, which we have recently structurally characterized using cryo-EM analysis. Application of similar protocols to other connexin family members will lead to breakthroughs in the understanding of the structure and function of connexin HCs., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gap junction channels facilitate the intercellular exchange of ions and small molecules, a process that is critical for the function of many different kinds of cells and tissues. Recent crystal structures of channels formed by one connexin isoform (connexin26) have been determined, and they have been subjected to molecular modeling. These studies have provided high-resolution models to gain insights into the mechanisms of channel conductance, molecular permeability, and gating. The models share similarities, but there are some differences in the conclusions reached by these studies. Many unanswered questions remain to allow an atomic-level understanding of intercellular communication mediated by connexin26. Because some domains of the connexin polypeptides are highly conserved (like the transmembrane regions), it is likely that some features of the connexin26 structure will apply to other members of the family of gap junction proteins. However, determination of high-resolution structures and modeling of other connexin channels will be required to account for the diverse biophysical properties and regulation conferred by the differences in their sequences.

Abstract Human vascular connexins (Cx37, Cx40, Cx43, and Cx45) can form various types of gap junction channels to synchronize vasodilation/constriction to control local circulation. Most of our knowledge on heterotypic gap junctions of these vascular connexins was from studies on rodent connexins. In human vasculature, the same four homolog connexins exist, but whether these human connexins can form heterotypic GJs as those of rodents have not been fully studied. Here we used in vitro expression system to study the coupling status and GJ channel properties of human heterotypic Cx37/Cx40, Cx37/Cx43, and Cx37/Cx45 GJs. Our results showed that Cx37/Cx43 and Cx37/Cx45 GJs, but not Cx37/Cx40 GJs, were functional and each with unique rectifying channel properties. The failure of docking between Cx37 and Cx40 could be rescued by designed Cx40 variants. Characterization of the heterotypic Cx37/Cx43 and Cx37/Cx45 GJs may help us in understanding the intercellular communication at the myoendothelial junction. [ABSTRACT FROM AUTHOR]

Abstract Gap junctions (GJs) are intercellular channels directly linking neighbouring cells and are dodecamers of connexins. In the human heart, connexin40 (Cx40), Cx43, and Cx45 are expressed in different regions of the heart forming GJs ensuring rapid propagation of action potentials in the myocardium. Two of these connexins, Cx40 and Cx45, formed functional GJs with prominent transjunctional voltage-dependent gating (V j -gating), which can be a mechanism to down regulate coupling conductance (G j). It is not clear the effects of temperature on V j -gating properties. We expressed Cx40 or Cx45 in N2A cells to study the V j -gating extent, the kinetics of deactivation, and the recovery time course from deactivation at 22 °C, 28 °C, and 32 °C. Dynamic uncoupling between cell pairs were evaluated at different temperatures, junctional delays, and/or repeating frequencies. Cx40 or Cx45 GJs showed little changes in the extent of V j -gating, but in both cases with a faster deactivation kinetics at high temperatures. The recovery from deactivation was faster at higher temperatures for Cx45 GJs, but not for Cx40 GJs. Cx45 GJs, but not Cx40 GJs, were dynamically uncoupled when sufficient junctional delays and/or repeating frequency in all tested temperatures. Gap junction specific dynamic uncoupling could play an important role in regulating action potential propagation speed in Cx45 enriched nodal cells in the heart. [ABSTRACT FROM AUTHOR]

Atrial fibrillation (AF) is the most common form of cardiac arrhythmia. Recently, four novel heterozygous Cx40 mutations--K107R, L223M, Q236H, and I257L--were identified in 4 of 310 unrelated AF patients and a followup genetic analysis of themutant carriers' families showed that themutantswere present in all the affected members. To study possible alterations associated with these Cx40 mutants, including their cellular localization and gap junction (GJ) function, we expressed GFP-tagged and untaggedmutants in connexin-deficientmodel cells. All four Cx40mutants showed clustered localization at cell-cell junctions similar to that observed of wildtype Cx40. However, cell pairs expressing Cx40 Q236H, but not the other individual mutants, displayed a significantly lower GJ coupling conductance (Gj) than wildtype Cx40. Similarly, co-expression of Cx40 Q236H with Cx43 resulted in a significantly lower Gj. Transjunctional voltage-dependent gating (Vj gating) properties were also altered in the GJs formed by Q236H. Reduced GJ function and altered Vj gating may play a role in promoting the Q236H carriers to AF. [ABSTRACT FROM AUTHOR]

In this review article, we summarize the current insight on the role of Connexin- and Pannexin-based channels as modulators of sensory neurons. The somas of sensory neurons are located in sensory ganglia (i.e., trigeminal and nodose ganglia). It is well known that within sensory ganglia, sensory neurons do not form neither electrical nor chemical synapses. One of the reasons for this is that each soma is surrounded by glial cells, known as satellite glial cells (SGCs). Recent evidence shows that connexin43 (Cx43) hemichannels and probably pannexons located at SGCs have an important role in paracrine communication between glial cells and sensory neurons. This communication may be exerted via the release of bioactive molecules from SGCs and their subsequent action on receptors located at the soma of sensory neurons. The glio-neuronal communication seems to be relevant for the establishment of chronic pain, hyperalgesia and pathologies associated with tissue inflammation. Based on the current literature, it is possible to propose that Cx43 hemichannels expressed in SGCs could be a novel pharmacological target for treating chronic pain, which need to be directly evaluated in future studies.

Connexins are membrane proteins that form hemichannels and gap junction channels at the plasma membrane. Through these channels connexins participate in autocrine and paracrine intercellular communication. Connexin-based channels are tightly regulated by membrane potential, phosphorylation, pH, redox potential, and divalent cations, among others, and the imbalance of this regulation have been linked to many acquired and genetic diseases. Concerning the redox potential regulation, the nitric oxide (NO) has been described as a modulator of the hemichannels and gap junction channels properties. However, how NO regulates these channels is not well understood. In this mini-review, we summarize the current knowledge about the effects of redox potential focused in NO on the trafficking, formation and functional properties of hemichannels and gap junction channels. [ABSTRACT FROM AUTHOR]

Gap junction channels are essential for mediating intercellular communication in most multicellular organisms. Two gene families encode gap junction channels, innexin and connexin. Although the sequence similarity between these two families based on bioinformatics is not conclusively determined, the gap junction channels encoded by these two gene families are structurally and functionally analogous. We recently reported an atomic structure of an invertebrate innexin gap junction channel using single-particle cryo-electron microscopy. Our findings revealed that connexin and innexin families share several structural properties with regard to their monomeric and oligomeric structures, while simultaneously suggesting a diversity of gap junction channels in nature. This review summarizes cutting-edge progress toward determining an innexin gap junction channel structure, as well as essential tips for preparing cryo-electron microscopy samples for high-resolution structural analysis of an innexin gap junction channel. [ABSTRACT FROM AUTHOR]

In this review article, we summarize the current insight on the role of Connexin- and Pannexin-based channels as modulators of sensory neurons. The somas of sensory neurons are located in sensory ganglia (i.e., trigeminal and nodose ganglia). It is well known that within sensory ganglia, sensory neurons do not form neither electrical nor chemical synapses. One of the reasons for this is that each soma is surrounded by glial cells, known as satellite glial cells (SGCs). Recent evidence shows that connexin43 (Cx43) hemichannels and probably pannexons located at SGCs have an important role in paracrine communication between glial cells and sensory neurons. This communication may be exerted via the release of bioactive molecules from SGCs and their subsequent action on receptors located at the soma of sensory neurons. The glio-neuronal communication seems to be relevant for the establishment of chronic pain, hyperalgesia and pathologies associated with tissue inflammation. Based on the current literature, it is possible to propose that Cx43 hemichannels expressed in SGCs could be a novel pharmacological target for treating chronic pain, which need to be directly evaluated in future studies. [ABSTRACT FROM AUTHOR]

Gap junction (GJ) channels form low resistance passages between cardiomyocytes and play a role in the rapid propagation of action potentials in the heart. A GJ channel is formed by two properly docked hemichannels and each hemichannel is a hexamer of connexins. Connexin40 (Cx40) and Cx43 are the dominant connexins in atrial myocytes, while Cx45 is mostly expressed in the sinoatrial (SA) and atrioventricular (AV) nodes which directly connect nodal cells with atrial myocytes, possibly via heterotypic Cx40/Cx45 and/or Cx43/Cx45 GJs. However, the functional status and channel properties of human heterotypic Cx40/Cx45 or Cx43/Cx45 GJs have not been studied. Here we investigated human Cx40/Cx45 and Cx43/Cx45 heterotypic GJs by recombinant expression in GJ deficient cells. Unlike the finding on rodent connexins, cell pairs expressing human Cx40 in one and Cx45 in the other failed to form morphological and functional GJs. Modifications in human Cx40 with designed variants (D55N or P193Q, but not P193K) are sufficient to establish morphological and functional heterotypic GJs with Cx45. In contrast, heterotypic human Cx43/Cx45 GJs are functional similar to that described for rodent Cx43/Cx45 GJs. Detailed kinetic characterizations of human heterotypic Cx43/Cx45 GJs revealed a rapid asymmetric V j -gating and a much slower recovery, which could reduce the GJ conductance in a junctional delay, action potential frequency, and direction dependent manner. Dynamic uncoupling in Cx45-containing GJs might contribute to a slower action potential propagation in the AV node. [ABSTRACT FROM AUTHOR]

Pathogenic variants in Gap Junction Protein Beta 1 (GJB1) cause X-linked Charcot-Marie-Tooth (CMT) disease type 1 (CMTX1), which is a common hereditary motor and sensory neuropathy. A 45-year-old man presented with progressive muscle weakness, atrophy, sensory disturbance of all limbs from childhood, and visual field defects in both eyes at 40 years old. A segregation analysis revealed a novel variant, c.173C>A (p.P58H), in the GJB1 gene. Patients with variants at codon 58 in GJB1 showed clinically varied phenotypes, ranging from demyelinating neuropathy to cerebellar ataxia. This patient may represent one of the various clinical phenotypes of GJB1 variants.

Connexins form intercellular communication channels, known as gap junctions (GJs), in many tissues/organs. Mutations in connexin genes are found to be linked to various inherited diseases, but the mechanisms are not fully clear. The Arg76 (R76) in Cx50 is fully conserved across the entire connexin family and is a hotspot for five connexin-linked inherited diseases, including Cx50 and Cx46-linked congenital cataract, Cx43-linked oculodentodigital dysplasia, and Cx45-linked cardiac arrhythmias. To better understand the molecular and cellular mechanism of dysfunction caused by R76/75 mutations, we examined the functional status and properties of GJs containing R76 mutations in Cx50 (R76H/C), Cx43 (R76H/S/C), and Cx45 (R75H) with an emphasis on heterotypic GJs in connexin-deficient model cells. All tested mutants showed an impairment of homotypic GJ function reflected by a decreased coupling% and conductance, except for Cx43 R76H/S. These connexin mutants also showed impaired GJ function when paired with a docking-compatible connexin, such as Cx50/Cx46 or Cx45/Cx43, except for all mutants on Cx43 which formed functional heterotypic GJs with Cx45. Localization studies on fluorescent protein tagged connexin mutants revealed that Cx45 R75H and Cx43 R76C showed impaired localization. Our homology structure models indicated that mutations of R76/75 in these GJs led to a loss of intra- and/or inter-connexin non-covalent interactions (salt bridges) at the sidechain of this residue, which could contribute to the observed GJ impairments underlying diseases. It is interesting that unlike those disease-linked variants in Cx50 and Cx45, Cx43 can tolerate some variations at R76., (© 2023 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

In this mini-review, we briefly summarize the current knowledge about the effects of fatty acids (FAs) on connexin-based channels, as well as discuss the limited information about the impact FAs may have on pannexins (Panxs). FAs regulate diverse cellular functions, some of which are explained by changes in the activity of channels constituted by connexins (Cxs) or Panxs, which are known to play critical roles in maintaining the functional integrity of diverse organs and tissues. Cxs are transmembrane proteins that oligomerize into hexamers to form hemichannels (HCs), which in turn can assemble into dodecamers to form gap junction channels (GJCs). While GJCs communicate the cytoplasm of contacting cells, HCs serve as pathways for the exchange of ions and small molecules between the intra and extracellular milieu. Panxs, as well as Cx HCs, form channels at the plasma membrane that enable the interchange of molecules between the intra and extracellular spaces. Both Cx- and Panx-based channels are controlled by several post-translational modifications. However, the mechanism of action of FAs on these channels has not been described in detail. It has been shown however that FAs frequently decrease GJC-mediated cell-cell communication. The opposite effect also has been described for HC or Panx-dependent intercellular communication, where, the acute FA effect can be reversed upon washout. Additionally, changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (e.g., length of carbon chain and/or degree of saturation), but this possible link remains poorly understood. [ABSTRACT FROM AUTHOR]

communication and are composed of two docked hemichannels (connexin oligomers). It is well documented that the docking and formation of GJs are possible only between compatible hemichannels (or connexins). The mechanisms of heterotypic docking compatibility are not fully clear. We aligned the protein sequences of docking-compatible and -incompatible connexins with that of connexin26 (Cx26). We found that two docking hydrogen bond (HB)-forming residues on the second extracellular domain (E2) of Cx26 and their equivalent residues are well conserved within docking-compatible connexins, but different between docking-incompatible connexins. Replacing one or both of these residues of Cx26 into the corresponding residues in the docking incompatible connexins (K168V, N176H or K168V-N176H) increased the formation of morphological and functional heterotypicGJs with connexin43 (Cx43) or connexin40 (Cx40), indicating that these two residues are important for docking incompatibility between Cx26 and these connexins. Our homology structure models predict that both HBs and hydrophobic interactions at the E2 docking interface are important docking mechanisms in heterotypic Cx26 K168V-N176H/Cx43 GJs and probably other docking compatible connexins. Revealing the key residues and mechanisms of heterotypic docking compatibility will assist us in understanding why these putative docking residues are hotspots of disease-linked mutants. [ABSTRACT FROM AUTHOR]

Innexins are invertebrate-specific gap junction proteins with four transmembrane helices. These proteins oligomerize to constitute intercellular channels that allow for the passage of small signaling molecules associated with neural and muscular electrical activity. In contrast to the large number of structural and functional studies of connexin gap junction channels, few structural studies of recombinant innexin channels are reported. Here we show the three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. The N-terminal deleted INX-6 proteins are crystallized in lipid bilayers. The three-dimensional reconstruction determined by cryo-electron crystallography reveals that a single INX-6 gap junction channel comprises 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which comprise 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26. Two bulb densities are observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. These findings imply a structural diversity of gap junction channels among multicellular organisms. [ABSTRACT FROM AUTHOR]

A gap junction (GJ) channel is formed by docking of two GJ hemichannels and each of these hemichannels is a hexamer of connexins. All connexin genes have been identified in human, mouse, and rat genomes and their homologous genes in many other vertebrates are available in public databases. The protein sequences of these connexins align well with high sequence identity in the same connexin across different species. Domains in closely related connexins and several residues in all known connexins are also well-conserved. These conserved residues form signatures (also known as sequence logos) in these domains and are likely to play important biological functions. In this review, the sequence logos of individual connexins, groups of connexins with common ancestors, and all connexins are analyzed to visualize natural evolutionary variations and the hot spots for human disease-linked mutations. Several gap junction domains are homologous, likely forming similar structures essential for their function. The availability of a high resolution Cx26 GJ structure and the subsequently-derived homology structure models for other connexin GJ channels elevated our understanding of sequence logos at the three-dimensional GJ structure level, thus facilitating the understanding of how disease-linked connexin mutants might impair GJ structure and function. This knowledge will enable the design of complementary variants to rescue disease-linked mutants. [ABSTRACT FROM AUTHOR]

Gap junction (GJ) channels provide low resistance passages for rapid action potential propagation in the heart. Both connexin40 (Cx40) and Cx43 are abundantly expressed in and frequently co-localized between atrial myocytes, possibly forming heterotypic GJ channels. However, conflicting results have been obtained on the functional status of heterotypic Cx40/Cx43 GJs. Here we provide experimental evidence that the docking and formation of heterotypic Cx40/Cx43 GJs can be substantially increased by designed Cx40 variants on the extracellular domains (E1 and E2). Specifically, Cx40 D55N and P193Q, substantially increased the probability to form GJ plaque-like structures at the cell–cell interfaces with Cx43 in model cells. More importantly the coupling conductance (G j ) of D55N/Cx43 and P193Q/Cx43 GJ channels are significantly increased from the G j of Cx40/Cx43 in N2A cells. Our homology models indicate the electrostatic interactions and surface structures at the docking interface are key factors preventing Cx40 from docking to Cx43. Improving heterotypic G j of these atrial connexins might be potentially useful in improving the coupling and synchronization of atrial myocardium. [ABSTRACT FROM AUTHOR]

Damage to peripheral nerves or the spinal cord is often accompanied by neuropathic pain, which is a complex, chronic pain state. Increasing evidence indicates that alterations in the expression and activity of gap junction channels in the spinal cord are involved in the development of neuropathic pain. Thus, this review briefly summarizes evidence that regulation of the expression, coupling, and activity of spinal gap junction channels modulates pain signals in neuropathic pain states induced by peripheral nerve or spinal cord injury. We particularly focus on connexin 43 and pannexin 1 because their regulation vastly attenuates symptoms of neuropathic pain. We hope that the study of gap junction channels eventually leads to the development of a suitable treatment tool for patients with neuropathic pain. [ABSTRACT FROM AUTHOR]

Connexins and pannexins form connexons, pannexons and membrane channels, which are critically involved in many aspects of cardiovascular physiology. For that reason, a vast number of studies have addressed the role of connexins and pannexins in the arterial and venous systems as well as in the heart. Moreover, a role for connexins in lymphatics has recently also been suggested. This review provides an overview of the current knowledge regarding the involvement of connexins and pannexins in cardiovascular physiology. [ABSTRACT FROM AUTHOR]

Bone adaptation to changes in mechanical stimuli occurs by adjusting bone formation and resorption by osteoblasts and osteoclasts, to maintain optimal bone mass. Osteocytes coordinate the actions of these cells on the bone surface by sensing mechanical forces and producing cytokines that increase or prevent osteoblast and osteoclast differentiation and function. Channels formed by connexins (Cxs) and, in particular, connexin 43 (Cx43) in osteoblasts and osteocytes are central part of this mechanism to control bone mass. Cx43 hemichannels are opened by fluid flow and mediate the antiapoptotic effect of mechanical stimulation in vitro, suggesting that Cx43 participates in mechanotransduction. However, mice lacking Cx43 in osteoblasts and/or osteocytes show an increased anabolic response to loading and decreased catabolic response to unloading. This evidence suggests that Cx43 channels expressed in osteoblastic cells are not required for the response to mechanical stimulation, but mediate the consequence of lack thereof. The molecular basis of these unexpected responses to mechanical stimulation is currently under investigation. [ABSTRACT FROM AUTHOR]

Communication through gap junction channels is essential for synchronized and coordinated cellular activities. The gap junction channel pore size, its switch control for opening/closing, and the modulations by chemicals can be different depending on the connexin subtypes that compose the channel. Recent structural and functional studies provide compelling evidence that the amino terminal (NT) domains of several connexins line the pore of gap junction channels and play an important role in single channel conductance (γj) and transjunctional voltage-dependent gating (Vj-gating). This article reviews recent studies conducted on a series of mutations/chimeras in the NT domain of connexin50 (Cx50). Functional examination of the gap junction channels formed by these mutants/chimeras shows the net charge number at the NT domain to be an important factor in γj and in Vj-gating. Furthermore, with an increase in the net negative charge at the NT domain, we observed an increase in the γj, as well as changes in the parameters of the Boltzmann fit of the normalized steady-state conductance and Vj relationship. Our data are consistent with a structural model where the NT domain of Cx50 lines the gap junction pore and plays an important role in sensing Vj and in the subsequent conformational changes leading to gating, as well as in limiting the rate of ion permeation.

Abstract: The gap junctions (GJs) formed by Cx40 and Cx43 provide a low resistance passage allowing for rapid propagation of action potentials. Sporadic somatic mutations in GJA5 (encoding Cx40) have been identified in lone atrial fibrillation (AF) patients. More recently germline autosomal dominantly inherited mutations in GJA5 have been found in early onset lone AF patients in several families over generations. Characterizations of these AF-linked Cx40 mutants in model cells and in patient tissues revealed that some of the mutants reduced GJ channel function due to an impaired trafficking or channel formation. While others showed a gain-of-function in hemichannels. These functional alterations in GJs or hemichannel may play an important role in the pathogenesis of AF in the mutant carriers. [Copyright &y& Elsevier]

GJ (gap junction) channels mediate direct intercellular communication and play an important role in many physiological processes. Six connexins oligomerize to form a hemichannel and two hemichannels dock together end-to-end to form a GJ channel. Connexin extracellular domains (E1 and E2) have been shown to be important for the docking, but the molecular mechanisms behind the docking and formation of GJ channels are not clear. Recent developments in atomic GJ structure and functional studies on a series of connexin mutants revealed that E1 and E2 are likely to play different roles in the docking. Noncovalent interactions at the docking interface, including hydrogen bonds, are predicted to form between interdocked extracellular domains. Protein sequence alignment analysis on the docking compatible/incompatible connexins indicate that the E1 domain is important for the formation of the GJ channel and the E2 domain is important in the docking compatibility in heterotypic channels. Interestingly, the hydrogen-bond forming or equivalent residues in both E1 and E2 domains are mutational hot spots for connexinlinked human diseases. Understanding themolecularmechanisms of GJ docking can assist us to develop novel strategies in rescuing the disease-linked connexin mutants. [ABSTRACT FROM AUTHOR]

Connexin 37 (Cx37) suppresses cell proliferation when expressed in rat insulinoma (Rin) cells, an effect also manifest in vivo during vascular development and in response to tissue injury. Mutant forms of Cx37 with nonfunctional channels but normally localized, wild-type carboxy termini are not growth suppressive. Here we determined whether the carboxy-terminal (CT) domain is required for Cx37-mediated growth suppression and whether the Cx37 pore-forming domain can be replaced with the Cx43 pore-forming domain and still retain growth-suppressive properties. We show that despite forming functional gap junction channels and hemichannels, Cx37 with residues subsequent to 273 replaced with a V5-epitope tag (Cx37-273tr*V5) had no effect on the proliferation of Rin cells, did not facilitate G1-cell cycle arrest with serum deprivation, and did not prolong cell cycle time comparably to the wild-type protein. The chimera Cx43*CT37, comprising the poreforming domain of Cx43 and CT of Cx37, also did not suppress proliferation, despite forming functional gap junctions with a permselective profile similar to wild-type Cx37. Differences in channel behavior of both Cx37-273tr*V5 and Cx43*CT37 relative to their wild-type counterparts and failure of the Cx37-CT to interact as the Cx43-CT does with the Cx43 cytoplasmic loop suggest that the Cx37-CT and pore-forming domains are both essential to growth suppression by Cx37. [ABSTRACT FROM AUTHOR]

Gap junctions are unique intercellular channels formed by the proper docking of two hemichannels from adjacent cells. Each hemichannel is a hexamer of connexins (Cxs) - the gap junction subunits, which are encoded by 21 homologous genes in the human genome. The docking of two hemichannels to form a functional gap junction channel is only possible between compatible Cxs, but the underlying molecular mechanism is unclear. On the basis of the crystal structure of the Cx26 gap junction, we developed homology models for homotypic and heterotypic channels from Cx32 and/or Cx26; these models predict six hydrogen bonds at the docking interface of each pair of the second extracellular domain (E2). A Cx32 mutation N175H and a human-disease-linked mutant N175D were predicted to lose the majority of the hydrogen bonds at the E2 docking-interface; experimentally both mutations failed to form morphological and functional gap junctions. To restore the lost hydrogen bonds, two complementary Cx26 mutants - K168V and K168A were designed to pair with the Cx32 mutants. When docked with Cx26K168V or K168A, the Cx32N175H mutant was successfully rescued morphologically and functionally in forming gap junction channels, but not Cx32 mutant N175Y. By testing more homotypic and heterotypic Cx32 and/or Cx26 mutant combinations, it is revealed that a minimum of four hydrogen bonds at each E2- docking interface are required for proper docking and functional channel formation between Cx26 and Cx32 hemichannels. Interestingly, the disease-linked Cx32N175D could be rescued by Cx26D179N, which restored five hydrogen bonds at the E2-docking interface. Our findings not only provide a mechanism for gap junction docking for Cx26 and Cx32 hemichannels, but also a potential therapeutic strategy for gap junction channelopathies. [ABSTRACT FROM AUTHOR]

Abstract: Connexin mimetic peptides corresponding to short conserved extracellular loop sequences of connexins have been used widely as reversible inhibitors of gap junctional intercellular communication. These peptides also block movement of ATP and Ca2+ across connexin hemichannels, i.e. hexameric channels yet to dock with partners in aligned cells and to generate the gap junction cell–cell conduit. By means of electrophysiology, we compared the effects of Gap26, a mimetic peptide corresponding to a short linear sequence in the first extracellular loop of connexin43, on connexin channel function in HeLa cells expressing connexin43. We demonstrate that Gap26 inhibited electrical coupling in cell pairs mediated by gap junctions after exposure for 30min. In contrast, Gap26 applied to single cells, inhibited hemichannel currents evoked in low Ca2+ solution with a response time of less than 5min. The results further support the view that the likely primary and direct inhibitory effect of Gap26 is on connexin hemichannels, with gap junctions becoming inhibited later. The mechanism of action of Gap26 in blocking hemichannels and gap junction channels is discussed in the context of their different functions and locations. [Copyright &y& Elsevier]