Connexons Coupling to Gap Junction Channel: Potential Role for Extracellular Protein Stabilization Centers

Connexin (Cx) proteins establish intercellular gap junction channels (Cx GJCs) through coupling of two apposed hexameric Cx hemichannels (Cx HCs, connexons). Pre- and post-GJ interfaces consist of extracellular EL1 and EL2 loops, each with three conserved cysteines. Previously, we reported that known peptide inhibitors, mimicking a variety of Cx43 sequences, appear non-selective when binding to homomeric Cx43 vs. Cx36 GJC homology model subtypes. In pursuit of finding potentially Cx subtype-specific inhibitors of connexon-connexon coupling, we aimed at to understand better how the GJ interface is formed. Here we report on the discovery of Cx GJC subtype-specific protein stabilization centers (SCs) featuring GJ interface architecture. First, the Cx43 GJC homology model, embedded in two opposed membrane bilayers, has been devised. Next, we endorsed the fluctuation dynamics of SCs of the interface domain of Cx43 GJC by applying standard molecular dynamics under open and closed cystine disulfide bond (CS-SC) preconditions. The simulations confirmed the major role of of the unique trans-GJ SC pattern comprising conserved (55N, 56T) and non-conserved (57Q) residues of the apposed EL1 loops in the stabilization of the GJC complex. Importantly, clusters of SC patterns residing close to the GJ interface domain appear to orient the interface formation via the numerous SCs between EL1 and EL2. These include central 54CS-S198C or 61CS-S192C contacts with residues 53R, 54C, 55N, 197D, 199F or 64V, 191P, respectively. In addition, we revealed that GJC interface formation is favoured when the psi dihedral angle of the nearby 193P residue is stable around 180° and the interface SCs disappear when this angle moves to the 0° to −45° range. The potential of the association of non-conserved residues with SC motifs in connexon-connexon coupling makes the development of Cx subtype-specific inhibitors viable.

Calmodulin-Connexin Partnership in Gap Junction Channel Regulation-Calmodulin-Cork Gating Model

In the past four decades numerous findings have indicated that gap junction channel gating is mediated by intracellular calcium concentrations ([Ca2+i]) in the high nanomolar range via calmodulin (CaM). We have proposed a CaM-based gating model based on evidence for a direct CaM role in gating. This model is based on the following: CaM inhibitors and the inhibition of CaM expression to prevent chemical gating. A CaM mutant with higher Ca2+ sensitivity greatly increases gating sensitivity. CaM co-localizes with connexins. Connexins have high-affinity CaM-binding sites. Connexin mutants paired to wild type connexins have a higher gating sensitivity, which is eliminated by the inhibition of CaM expression. Repeated trans-junctional voltage (Vj) pulses progressively close channels by the chemical/slow gate (CaM’s N-lobe). At the single channel level, the gate closes and opens slowly with on-off fluctuations. Internally perfused crayfish axons lose gating competency but recover it by the addition of Ca-CaM to the internal perfusion solution. X-ray diffraction data demonstrate that isolated gap junctions are gated at the cytoplasmic end by a particle of the size of a CaM lobe. We have proposed two types of CaM-driven gating: “Ca-CaM-Cork” and “CaM-Cork”. In the first, the gating involves Ca2+-induced CaM activation. In the second, the gating occurs without a [Ca2+]i rise.

Remodeling of Cardiac Gap Junctional Cell–Cell Coupling

The heart works as a functional syncytium, which is realized via cell-cell coupling maintained by gap junction channels. These channels connect two adjacent cells, so that action potentials can be transferred. Each cell contributes a hexameric hemichannel (=connexon), formed by protein subuntis named connexins. These hemichannels dock to each other and form the gap junction channel. This channel works as a low ohmic resistor also allowing the passage of small molecules up to 1000 Dalton. Connexins are a protein family comprising of 21 isoforms in humans. In the heart, the main isoforms are Cx43 (the 43 kDa connexin; ubiquitous), Cx40 (mostly in atrium and specific conduction system), and Cx45 (in early developmental states, in the conduction system, and between fibroblasts and cardiomyocytes). These gap junction channels are mainly located at the polar region of the cardiomyocytes and thus contribute to the anisotropic pattern of cardiac electrical conductivity. While in the beginning the cell–cell coupling was considered to be static, similar to an anatomically defined structure, we have learned in the past decades that gap junctions are also subject to cardiac remodeling processes in cardiac disease such as atrial fibrillation, myocardial infarction, or cardiomyopathy. The underlying remodeling processes include the modulation of connexin expression by e.g., angiotensin, endothelin, or catecholamines, as well as the modulation of the localization of the gap junctions e.g., by the direction and strength of local mechanical forces. A reduction in connexin expression can result in a reduced conduction velocity. The alteration of gap junction localization has been shown to result in altered pathways of conduction and altered anisotropy. In particular, it can produce or contribute to non-uniformity of anisotropy, and thereby can pre-form an arrhythmogenic substrate. Interestingly, these remodeling processes seem to be susceptible to certain pharmacological treatment.

Gap Junction Channelopathies and Calmodulinopathies. Do Disease-Causing Calmodulin Mutants Affect Direct Cell–Cell Communication?

The cloning of connexins cDNA opened the way to the field of gap junction channelopathies. Thus far, at least 35 genetic diseases, resulting from mutations of 11 different connexin genes, are known to cause numerous structural and functional defects in the central and peripheral nervous system as well as in the heart, skin, eyes, teeth, ears, bone, hair, nails and lymphatic system. While all of these diseases are due to connexin mutations, minimal attention has been paid to the potential diseases of cell–cell communication caused by mutations of Cx-associated molecules. An important Cx accessory protein is calmodulin (CaM), which is the major regulator of gap junction channel gating and a molecule relevant to gap junction formation. Recently, diseases caused by CaM mutations (calmodulinopathies) have been identified, but thus far calmodulinopathy studies have not considered the potential effect of CaM mutations on gap junction function. The major goal of this review is to raise awareness on the likely role of CaM mutations in defects of gap junction mediated cell communication. Our studies have demonstrated that certain CaM mutants affect gap junction channel gating or expression, so it would not be surprising to learn that CaM mutations known to cause diseases also affect cell communication mediated by gap junction channels.

Structural insights into the gating mechanism of human Cx43/GJA1 gap junction channel

Connexin family proteins assemble into hexameric hemichannels in a cell membrane, which dock together between two adjacent membranes to form gap junction intercellular channels (GJIChs). The most ubiquitously expressed connexin Cx43 plays important roles in numerous biological processes. Here we report cryo-EM structures of Cx43 GJIChs at 3.1–3.6 Å resolutions, which show dynamic conformational changes of N-terminal helices (NTHs) caused by pH change or C-terminal truncation. Cx43 GJIChs in a channel-closing condition contain 12 protomers in gate-covering NTH (GCN) conformation, while those in opening conditions have varying compositions of GCNs and pore-lining NTHs (PLNs) resulting in various pore dimensions and electrostatic surface potentials. GCN-to-PLN transition accompanies π-helix formation in the first transmembrane helix (TM1), movement of TM2-4 that creates a side opening to the membrane, and structural stabilization of the cytoplasmic loop. Our study provides structural insights into the intercellular ion/metabolite transfer and the lateral lipid transport through Cx43 GJICh.

Dual Role for Astroglial Copper-Assisted Polyamine Metabolism during Intense Network Activity

Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na+ symport. GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 μM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.

The potential antiepileptogenic effect of neuronal Cx36 gap junction channel blockage

Epilepsy is one of the most prevalent neurological disorders and can result in neuronal injury and degeneration. Consequently, research into new antiepileptic drugs capable of providing protection against neuronal injury and degeneration is extremely important. Neuronal Cx36 gap junction channels have been found to play an important role in epilepsy; thus, pharmacological interference using Cx36 gap junction channel blockers may be a promising strategy for disrupting the synchronization of neurons during seizure activity and protecting neurons. Based on these promising findings, several in vivo and in vitro studies are ongoing and the first encouraging results have been published. The results bring hope that neurons can be protected from injury and degeneration in patients with epilepsy, which is currently impossible.

Oligodendrocyte Progenitor Cells Increase Chi3l1 Secretion in Exosome to Activate Myh9 and Promote Proliferation through Connexin47 Connected to Astrocytes

Background: Neurodegenerative diseases, caused by the loss of neurons or myelin sheath, are some of the most important neurological diseases that threaten the health of the elderly. In the CNS, oligodendrocytes (OLs) are the only cells that can form myelin. Astrocytes (ASTs) play a generally beneficial role in remyelination, including the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) to OLs. However, the specific downstream mechanism is unclear.Methods: This study investigated the proliferation of OPCs in OPCs mono-culture, OPCs culture with ASTs supernatant, and ASTs-OPCs co-culture. Gene Ontology (GO) analysis were used to analyze the differentially expressed genes after transcriptome sequencing of these OPCs. Electron microscope, Nanoparticle Tracking Analysis (NTA), Fluorescence tracing of exosomes and Western blot were used to evaluate the effects of exsomes. Pull-down, co-immunoprecipitation (Co-IP) and mass spectrometry analys were conducted to find the downstream signal proliferation which is transmitted information into OPCs.Reasults: Direct contact co-culture of ASTs and OPCs promotes the proliferation of OPCs. After Cx47 siRNA interference under ASTs-OPCs co-culture, Chi3l1 secretion in exosome reveals associated decrease, and OPCs proliferation decreased. The cell proliferation induced by Chi3l1 was inhibited after siRNA interfered with Myh9, and the expression of cyclin D1 was also decreased.Conclusions: These results suggest that ASTs transmit information to OPCs by increasing gap junction channel Cx47, thereby promoting the secretion of Chi3l1 in exosome of OPCs. The secretory form of Chi3l1 in exosome might be easier to enter the target cell than in extracellular supernatant, which is beneficial to the activation of Myh9 to promote OPCs proliferation. This may be a potential target for drugs rescuing neurodegeneration diseases related to remyelination.

Oligodendrocyte Progenitor Cells Increase Chi3l1 Secretion in exosome to Activate Myh9 and Promote Proliferation through Connexin47 Connected to Astrocytes

Background: Neurodegenerative diseases, caused by the loss of neurons or myelin sheath, are some of the most important neurological diseases that threaten the health of the elderly. In the CNS, oligodendrocytes (OLs) are the only cells that can form myelin. Astrocytes (ASTs) play a generally beneficial role in remyelination, including the proliferation and differentiation of oligodendrocyte precursor cells (OPCs) to OLs. However, the specific downstream mechanism is unclear.Methods: This study investigated the proliferation of OPCs in OPCs mono-culture, OPCs culture with ASTs supernatant, and ASTs-OPCs co-culture. Gene Ontology (GO) analysis were used to analyze the differentially expressed genes after transcriptome sequencing of these OPCs. Electron microscope, Nanoparticle Tracking Analysis (NTA), Fluorescence tracing of exosomes and Western blot were used to evaluate the effects of exsomes. Pull-down, co-immunoprecipitation (Co-IP) and mass spectrometry analys were conducted to find the downstream signal proliferation which is transmitted information into OPCs.Reasults: Direct contact co-culture of ASTs and OPCs promotes the proliferation of OPCs. After Cx47 siRNA interference under ASTs-OPCs co-culture, Chi3l1 secretion in exosome reveals associated decrease, and OPCs proliferation decreased. The cell proliferation induced by Chi3l1 was inhibited after siRNA interfered with Myh9, and the expression of cyclin D1 was also decreased.Conclusions: These results suggest that ASTs transmit information to OPCs by increasing gap junction channel Cx47, thereby promoting the secretion of Chi3l1 in exosome of OPCs. The secretory form of Chi3l1 in exosome might be easier to enter the target cell than in extracellular supernatant, which is beneficial to the activation of Myh9 to promote OPCs proliferation. This may be a potential target for drugs rescuing neurodegeneration diseases related to remyelination.