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Cardiac connexin-43 (Cx43) creates gap junction channels (GJCs) at intercellular contacts and hemi-channels (HCs) at the peri-junctional plasma membrane and sarcolemmal caveolae/rafts compartments. GJCs are fundamental for the direct cardiac cell-to-cell transmission of electrical and molecular signals which ensures synchronous myocardial contraction. The HCs and structurally similar pannexin1 (Panx1) channels are active in stressful conditions. These channels are essential for paracrine and autocrine communication through the release of ions and signaling molecules to the extracellular environment, or for uptake from it. The HCs and Panx1 channel-opening profoundly affects intracellular ionic homeostasis and redox status and facilitates via purinergic signaling pro-inflammatory and pro-fibrotic processes. These conditions promote cardiac arrhythmogenesis due to the impairment of the GJCs and selective ion channel function. Crosstalk between GJCs and HCs/Panx1 channels could be crucial in the development of arrhythmogenic substrates, including fibrosis. Despite the knowledge gap in the regulation of these channels, current evidence indicates that HCs and Panx1 channel activation can enhance the risk of cardiac arrhythmias. It is extremely challenging to target HCs and Panx1 channels by inhibitory agents to hamper development of cardiac rhythm disorders. Progress in this field may contribute to novel therapeutic approaches for patients prone to develop atrial or ventricular fibrillation.

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation 1 , apoptotic cell clearance 2 and human oocyte development 3 . Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels. Cryo-electron microscopy structures of the ATP-permeable channel pannexin 1 reveal a gating mechanism involving multiple distinct ion-conducting pathways.

Gap junctions establish direct pathways for cell-to-cell communication through the assembly of twelve connexin subunits that form intercellular channels connecting neighbouring cells. Co-assembly of different connexin isoforms produces channels with unique properties and enables communication across cell types. Here we used single-particle cryo-electron microscopy to investigate the structural basis of connexin co-assembly in native lens gap junction channels composed of connexin 46 and connexin 50 (Cx46/50). We provide the first comparative analysis to connexin 26 (Cx26), which—together with computational studies—elucidates key energetic features governing gap junction permselectivity. Cx46/50 adopts an open-state conformation that is distinct from the Cx26 crystal structure, yet it appears to be stabilized by a conserved set of hydrophobic anchoring residues. ‘Hot spots’ of genetic mutations linked to hereditary cataract formation map to the core structural–functional elements identified in Cx46/50, suggesting explanations for many of the disease-causing effects. Cryo-electron microscopy structures of connexin channels composed of connexin 46 and connexin 50 in an open-state reveal features that govern permselectivity and the location of mutated residues linked to herediatry cataracts.

Pannexins are large-pore forming channels responsible for ATP release under a variety of physiological and pathological conditions. Although predicted to share similar membrane topology with other large-pore forming proteins such as connexins, innexins, and LRRC8, pannexins have minimal sequence similarity to these protein families. Here, we present the cryo-EM structure of a frog pannexin 1 (Panx1) channel at 3.0 Å. We find that Panx1 protomers harbor four transmembrane helices similar in arrangement to other large-pore forming proteins but assemble as a heptameric channel with a unique constriction formed by Trp74 in the first extracellular loop. Mutating Trp74 or the nearby Arg75 disrupt ion selectivity, whereas altering residues in the hydrophobic groove formed by the two extracellular loops abrogates channel inhibition by carbenoxolone. Our structural and functional study establishes the extracellular loops as important structural motifs for ion selectivity and channel inhibition in Panx1.

The plasma membrane adenosine triphosphate (ATP) release channel pannexin 1 (PANX1) has been implicated in many physiological and pathophysiological processes associated with purinergic signaling, including cancer progression, apoptotic cell clearance, inflammation, blood pressure regulation, oocyte development, epilepsy and neuropathic pain. Here we present near-atomic-resolution structures of human and frog PANX1 determined by cryo-electron microscopy that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the size of permeable substrates. The amino and carboxyl termini, not resolved in the density map, appeared to be structurally dynamic and might contribute to narrowing of the pore during channel gating. In combination with functional characterization, this work elucidates the previously unknown architecture of pannexin channels and establishes a foundation for understanding their unique channel properties.Cryo-EM structures of plasma membrane ATP release channel pannexin 1 reveal heptameric architecture, wide pore and a constriction potentially restricting the size of permeable substrates. Combined with functional assays, they offer insights into channel gating.

Innexins, a large protein family comprising invertebrate gap junction channels, play an essential role in nervous system development and electrical synapse formation. Here we report the cryo-electron microscopy structures of Caenorhabditis elegans innexin-6 (INX-6) gap junction channels at atomic resolution. We find that the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomeric structure are highly similar to those of connexin-26 (Cx26), despite the lack of significant sequence similarity. The INX-6 gap junction channel comprises hexadecameric subunits but reveals the N-terminal pore funnel, consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated one-by-one through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore. These observations suggest that the INX-6 cytoplasmic domains are cooperatively associated with the N-terminal funnel conformation, and an essential linkage of the N-terminal with channel activity is presumably preserved across gap junction families. Gap junctions have critical roles in maintaining homeostasis in multicellular organisms. Here the authors present cryo-EM structures of the C. elegans innexin-6 gap junction channel, revealing high structural similarity to human connexin 26 despite a different oligomeric number and lack of sequence similarity.

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

Connexin molecules form intercellular membrane channels facilitating electronic coupling and the passage of small molecules between adjoining cells. Connexin26 (Cx26) is the second smallest member of the gap junction protein family, and mutations in Cx26 cause certain hereditary human diseases such as skin disorders and hearing loss. Here, we report the electron crystallographic structure of a human Cx26 mutant (M34A). Although crystallization trials used hemichannel preparations, the density map revealed that two hemichannels redocked at their extracellular surfaces into full intercellular channels. These orthorhombic crystals contained two sets of symmetry-related intercellular channels within three lipid bilayers. The 3D map shows a prominent density in the pore of each hemichannel. This density contacts the innermost helices of the surrounding connexin subunits at the bottom of the vestibule. The density map suggests that physical blocking may play an important role that underlies gap junction channel regulation. Our structure allows us to suggest that the two docked hemichannels can be independent and may regulate their activity autonomously with a plug in the vestibule.

Key Points Gap junctions are aggregates of intercellular channels that join adjacent cells. Intercellular channels are composed of a pair of connexons, each of which is a hexamer of connexin proteins. As precursors to the intercellular channels, connexons can be found in the plasma membranes of cells. In some cellular systems, however, connexons might function independently of gap junctions. Although there are data implicating gap-junctional intercellular channels in the cell–cell transmission of Ca 2+ waves, it is also clear that these waves can spread by paracrine signalling owing to the extracellular release of ATP that binds to purinergic receptors. Evidence is accumulating that the connexon might provide a regulated channel that is responsible for the ATP release. The bisphosphonate alendronate has been shown to have an anti-apoptotic effect on bone cells. Recent data indicate that connexons might be the receptor for alendronate, transducing a survival signal through the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway. A long-standing puzzle has been the interaction of retinal horizontal cells with cones as part of centre-surround antagonism. The interaction between these cells might involve connexons. The voltage-induced opening of connexons in the horizontal-cell membrane might function to create a negative extracellular potential in the invaginating synapses of the cone pedicles, opening Ca 2+ channels and stimulating glutamate release. Gap junctions consist of intercellular channels that connect the cytoplasm of adjacent cells directly and allow the exchange of small molecules. These channels are unique in that they span two plasma membranes — the more orthodox ion or ligand-gated channels span only one. Each cell contributes half of the intercellular channel, and each half is known as a connexon or hemichannel. Recent studies indicate that connexons are also active in single plasma membranes and that they might be essential in intercellular signalling beyond their incorporation into gap junctions.

The coxsackievirus and adenovirus receptor (CAR) is a transmembrane protein that belongs to the family of adhesion molecules. In the postnatal heart, it is localized predominantly at the intercalated disc, where its function is not known. Here, we demonstrate that a first degree or complete block of atrioventricular (AV) conduction developed in the absence of CAR in the adult mouse heart and that prolongation of AV conduction occurred in the embryonic heart of the global CAR-KO mouse. In the cardiac-specific CAR-KO (CAR-cKO) mouse, we observed the loss of connexin 45 localization to the cell-cell junctions of the AV node but preservation of connexin 40 and 43 in contracting myocardial cells and connexin 30.2 in the AV node. There was also a marked decrease in beta-catenin and zonula occludens-1 (ZO-1) localization to the intercalated discs of CAR-cKO mouse hearts at 8 weeks before the mice developed cardiomyopathy at 21 weeks of age. We also found that CAR formed a complex with connexin 45 via its PSD-95/DigA/ZO-1-binding (PDZ-binding) motifs. We conclude that CAR expression is required for normal AV-node conduction and cardiac function. Furthermore, localization of connexin 45 at the AV-node cell-cell junction and of beta-catenin and ZO-1 at the ventricular intercalated disc are dependent on CAR.