Neuronal Gap Junctions Research Articles

Assembly mechanisms of the neuronal gap junction channel connexin 36 elucidated by Cryo-EM

Electrical synapses are essential components of neural circuits. Neuronal signal transduction across electrical synapses is primarily mediated by gap junction channels composed of Connexin36 (Cx36), the lack of which causes impaired electrical coupling between certain neurons including cortical interneurons and thalamic reticular nucleus (TRN) neurons. However, the structural basis underlying Cx36 function and assembly remains elusive. Recently, Lee et al. reported cryo-EM structures of Cx36, thus provided first insights of its gating mechanism. Here, we report a consistent cryo-EM structure of Cx36 determined in parallel, and describe unique interactions underpinning its assembly mechanism in complementary to the competing work. In particular, we found non-canonical electrostatic interactions between protomers from opposing hemichannels and a steric complementary site between adjacent protomers within a hemichannel, which together provide a structural explanation for the assembly specificity in homomeric and heteromeric gap junction channels.

Transmissible Spongiform Encephalopathies Prion Proteins: A Systematic Review

Prion proteins (PrPc) have been implicated as the causative agent of “Transmissible Spongiform Encephalopathies” (TSE). Apart from this devilish role, prions also have a bright facet of their own and their identity holds much more than just being a pathogenic entity. Role of prions as scaffolding proteins for ligand binding and signal transduction has been reported by several researchers. Role of prions in nerve impulse transmission at neuronal junctions, glyapse and gap junctions have been reported. Prion mediated regulation of calcium ion flux and redox status in turn regulates many major cellular functions. In this review we have focussed mainly on the physiological aspects of prion function apart from its pathological role in TSE. Role of prions in mediation of neuropathic pain, neuroinflammatory diseases and chronic headache has been reported by few researchers. In this review we have tried to correlate such effects of prions and also discuss various therapeutic targets for various diseases influenced by prions.

Antiepileptogenic and neuroprotective effect of mefloquine after experimental status epilepticus

Acquired temporal lobe epilepsy (TLE) characterized by spontaneous recurrent seizures (SRS) and hippocampal inhibitory neuron dysfunction is often refractory to current therapies. Gap junctional or electrical coupling between inhibitory neurons has been proposed to facilitate network synchrony and intercellular molecular exchange suggesting a role in both seizures and neurodegeneration. While gap junction blockers can limit acute seizures, whether blocking neuronal gap junctions can modify development of chronic epilepsy has not been examined. This study examined whether mefloquine, a selective blocker of Connexin 36 gap junctions which are well characterized in inhibitory neurons, can limit epileptogenesis and related cellular and behavioral pathology in a model of acquired TLE. A single, systemic dose of mefloquine administered early after pilocarpine-induced status epilepticus (SE) in rat reduced both development of SRS and behavioral co-morbidities. Immunostaining for interneuron subtypes identified that mefloquine treatment likely reduced delayed inhibitory neuronal loss after SE. Uniquely, parvalbumin expressing neurons in the hippocampal dentate gyrus appeared relatively resistant to early cell loss after SE. Functionally, whole cell patch clamp recordings revealed that mefloquine treatment preserved inhibitory synaptic drive to projection neurons one week and one month after SE. These results demonstrate that mefloquine, a drug already approved for malaria prophylaxis, is potentially antiepileptogenic and can protect against progressive interneuron loss and behavioral co-morbidities of epilepsy.

Neurobeachin controls the asymmetric subcellular distribution of electrical synapse proteins

The subcellular positioning of synapses and their specialized molecular compositions form the fundamental basis of neural circuits. Like chemical synapses, electrical synapses are constructed from an assortment of adhesion, scaffolding, and regulatory molecules, yet little is known about how these molecules localize to specific neuronal compartments. Here, we investigate the relationship between the autism- and epilepsy-associated gene Neurobeachin, the neuronal gap junction channel-forming Connexins, and the electrical synapse scaffold ZO1. Using the zebrafish Mauthner circuit, we find Neurobeachin localizes to the electrical synapse independently of ZO1 and Connexins. By contrast, we show Neurobeachin is required postsynaptically for the robust localization of ZO1 and Connexins. We demonstrate that Neurobeachin binds ZO1 but not Connexins. Finally, we find Neurobeachin is required to restrict electrical postsynaptic proteins to dendrites, but not electrical presynaptic proteins to axons. Together, the results reveal an expanded understanding of electrical synapse molecular complexity and the hierarchical interactions required to build neuronal gap junctions. Further, these findings provide novel insight into the mechanisms by which neurons compartmentalize the localization of electrical synapse proteins and provide a cell biological mechanism for the subcellular specificity of electrical synapse formation and function.

The Effect of Pronase on Mollusk, Leech, and Frog Nerve Ganglia Causes the Formation of Neuron–Neuronal Gap Junctions

The Effect of Pronase on Mollusk, Leech, and Frog Nerve Ganglia Causes the Formation of Neuron–Neuronal Gap Junctions

Cryo-EM structures of human Cx36/GJD2 neuronal gap junction channel

Connexin 36 (Cx36) is responsible for signal transmission in electrical synapses by forming interneuronal gap junctions. Despite the critical role of Cx36 in normal brain function, the molecular architecture of the Cx36 gap junction channel (GJC) is unknown. Here, we determine cryo-electron microscopy structures of Cx36 GJC at 2.2–3.6 Å resolutions, revealing a dynamic equilibrium between its closed and open states. In the closed state, channel pores are obstructed by lipids, while N-terminal helices (NTHs) are excluded from the pore. In the open state with pore-lining NTHs, the pore is more acidic than those in Cx26 and Cx46/50 GJCs, explaining its strong cation selectivity. The conformational change during channel opening also includes the α-to-π-helix transition of the first transmembrane helix, which weakens the protomer-protomer interaction. Our structural analyses provide high resolution information on the conformational flexibility of Cx36 GJC and suggest a potential role of lipids in the channel gating.

Cryo-EM structures of human Cx36/GJD2 neuronal gap junction channel

Cryo-EM structures of human Cx36/GJD2 neuronal gap junction channel

Electrical synaptic transmission requires a postsynaptic scaffolding protein.

Electrical synaptic transmission relies on neuronal gap junctions containing channels constructed by Connexins. While at chemical synapses neurotransmitter-gated ion channels are critically supported by scaffolding proteins, it is unknown if channels at electrical synapses require similar scaffold support. Here, we investigated the functional relationship between neuronal Connexins and Zonula Occludens 1 (ZO1), an intracellular scaffolding protein localized to electrical synapses. Using model electrical synapses in zebrafish Mauthner cells, we demonstrated that ZO1 is required for robust synaptic Connexin localization, but Connexins are dispensable for ZO1 localization. Disrupting this hierarchical ZO1/Connexin relationship abolishes electrical transmission and disrupts Mauthner cell-initiated escape responses. We found that ZO1 is asymmetrically localized exclusively postsynaptically at neuronal contacts where it functions to assemble intercellular channels. Thus, forming functional neuronal gap junctions requires a postsynaptic scaffolding protein. The critical function of a scaffolding molecule reveals an unanticipated complexity of molecular and functional organization at electrical synapses.

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.

Understanding the Molecular and Cell Biological Mechanisms of Electrical Synapse Formation

In this review article, we will describe the recent advances made towards understanding the molecular and cell biological mechanisms of electrical synapse formation. New evidence indicates that electrical synapses, which are gap junctions between neurons, can have complex molecular compositions including protein asymmetries across joined cells, diverse morphological arrangements, and overlooked similarities with other junctions, all of which indicate new potential roles in neurodevelopmental disease. Aquatic organisms, and in particular the vertebrate zebrafish, have proven to be excellent models for elucidating the molecular mechanisms of electrical synapse formation. Zebrafish will serve as our main exemplar throughout this review and will be compared with other model organisms. We highlight the known cell biological processes that build neuronal gap junctions and compare these with the assemblies of adherens junctions, tight junctions, non-neuronal gap junctions, and chemical synapses to explore the unknown frontiers remaining in our understanding of the critical and ubiquitous electrical synapse.

Gap junctions and expression of Cx36, Cx43 and Cx45 in the posterodorsal medial amygdala of adult rats.

The posterodorsal medial amygdala (MePD) has an adapted synaptic organization that dynamically modulates reproduction and other social behaviors in rats. Discrete gap junctions between glial cells were previously reported in the MePD neuropil. Connexins (Cx) are components of gap junctions and indicative of cellular electrical coupling. Here, we report the ultrastructural occurrence of gap junctions between neurons in the MePD and demonstrate the expression and immunofluorescent labeling of Cx36, Cx43 and Cx45 in this subcortical area of adult male rats. Few neuronal gap junctions were found in the MePD and, when identified, occurred between dendrites. On the other hand, there is a diffuse presence and distribution of punctate labelling for the tested Cxs. Puncta were visualized isolated or forming clusters in the same focal plane of cell bodies or along the MePD neuropil. The Cx36 puncta were found in neurons, Cx43 in astrocytes and Cx45 in both neurons and astrocytes. Our data indicate the presence of few gap junctions and different Cxs composition in the MePD. Because Cxs can assemble, form hemichannel units and/or serve as transcriptional regulator, it is likely that additional modulation of intercellular communication can occur besides the chemical transmission in the MePD of adult rats.

Purinergic modulation of neuronal gap junction circuits in the CNS of the leech.

Gap junctions (GJs) are widely distributed in brains across the animal kingdom. To visualize the GJ- coupled networks of two major mechanosensory neurons in the ganglia of medicinal leeches, we injected these cells with the GJ-permeable tracer Neurobiotin. When diffusion time was limited to only 30min, tracer coupling was highly variable for both cells, suggesting a possible modulation of GJ permeability. In invertebrates the innexins (homologs of vertebrate pannexins) form the GJs. Because extracellular adenosine triphosphate (ATP) modulates pannexin and leech innexin hemichannel permeability and is released by leech glial cells following injury, we tested the effects of bath application of ATP after the injection of Neurobiotin and observed a significant increase in the number of neurons tracer coupled to the sensory neurons. This effect required the elevation of intracellular Ca2+ and could be produced by bath application of caffeine. Conversely, scavenging endogenous extracellular ATP with the ATPase apyrase decreased the number of coupled cells. ATP also increased electrical conductance and tracer permeability between the bilateral Retzius neurons. This modulatory effect of ATP on GJ coupling was blocked by siRNA knockdown of a P1-like adenosine receptor. Finally, exposure of leech ganglia to extracellular ATP induced a characteristic low frequency (<0.3Hz) rhythmic bursting activity that was roughly synchronous among multiple neurons, a behavior that was significantly attenuated by the GJ blocker octanol. These findings highlight the mediation by ATP of a robust physiological mechanism for modifying neuronal circuits by rapidly recruiting neurons into active networks and entraining synchronized bursting activity.

Localization of Retinal Ca2+/Calmodulin-Dependent Kinase II-β (CaMKII-β) at Bipolar Cell Gap Junctions and Cross-Reactivity of a Monoclonal Anti-CaMKII-β Antibody With Connexin36.

Neuronal gap junctions formed by connexin36 (Cx36) and chemical synapses share striking similarities in terms of plasticity. Ca2+/calmodulin-dependent protein kinase II (CaMKII), an enzyme known to induce memory formation at chemical synapses, has recently been described to potentiate electrical coupling in the retina and several other brain areas via phosphorylation of Cx36. The contribution of individual CaMKII isoforms to this process, however, remains unknown. We recently identified CaMKII-β at electrical synapses in the mouse retina. Now, we set out to identify cell types containing Cx36 gap junctions that also express CaMKII-β. To ensure precise description, we first tested the specificity of two commercially available antibodies on CaMKII-β-deficient retinas. We found that a polyclonal antibody was highly specific for CaMKII-β. However, a monoclonal antibody (CB-β-1) recognized CaMKII-β but also cross-reacted with the C-terminal tail of Cx36, making localization analyses with this antibody inaccurate. Using the polyclonal antibody, we identified strong CaMKII-β expression in bipolar cell terminals that were secretagogin- and HCN1-positive and thus represent terminals of type 5 bipolar cells. In these terminals, a small fraction of CaMKII-β also colocalized with Cx36. A similar pattern was observed in putative type 6 bipolar cells although there, CaMKII expression seemed less pronounced. Next, we tested whether CaMKII-β influenced the Cx36 expression in bipolar cell terminals by quantifying the number and size of Cx36-immunoreactive puncta in CaMKII-β-deficient retinas. However, we found no significant differences between the genotypes, indicating that CaMKII-β is not necessary for the formation and maintenance of Cx36-containing gap junctions in the retina. In addition, in wild-type retinas, we observed frequent association of Cx36 and CaMKII-β with synaptic ribbons, i.e., chemical synapses, in bipolar cell terminals. This arrangement resembled the composition of mixed synapses found for example in Mauthner cells, in which electrical coupling is regulated by glutamatergic activity. Taken together, our data imply that CaMKII-β may fulfill several functions in bipolar cell terminals, regulating both Cx36-containing gap junctions and ribbon synapses and potentially also mediating cross-talk between these two types of bipolar cell outputs.

Domain-specific distribution of gap junctions defines cellular coupling to establish a vascular relay in the retina.

In the retina, diverse functions of neuronal gap junctions (GJs) have been established. However, the distribution and function of vascular GJs are less clear. Here in the mouse retina whole mounts, we combined structural immunohistochemical analysis and a functional assessment of cellular coupling with a GJ-permeable tracer Neurobiotin to determine distribution patterns of three major vascular connexins. We found that Cx43 was expressed in punctate fashion on astroglia, surrounding all types of blood vessels and in continuous string-like structures along endothelial cell contacts in specialized regions of the vascular tree. Specifically, these Cx43-positive strings originated at the finest capillaries and extended toward the feeding artery. As this structural arrangement promoted strong and exclusive coupling of pericytes and endothelial cells along the corresponding branch, we termed this region a "vascular relay." Cx40 expression was found predominantly along the endothelial cell contacts of the primary arteries and did not overlap with Cx43-positive strings. At their occupied territories, Cx43 and Cx40 clustered with tight junctions and, to a lesser extent, with adhesion contacts, both key elements of the blood-retina barrier. Finally, Cx37 puncta were associated with the entire surface of both mural and endothelial cells across all regions of the vascular tree. This combinatorial analysis of vascular connexins and identification of the vascular relay region will serve as a structural foundation for future studies of neurovascular signaling in health and disease.

Vgsc-interacting proteins are genetically associated with pyrethroid resistance in Aedes aegypti.

Association mapping of factors that condition pyrethroid resistance in Aedes aegypti has consistently identified genes in multiple functional groups. Toward better understanding of the mechanisms involved, we examined high throughput sequencing data (HTS) from two Aedes aegypti aegypti collections from Merida, Yucatan, Mexico treated with either permethrin or deltamethrin. Exome capture enrichment for coding regions and the AaegL5 annotation were used to identify genes statistically associated with resistance. The frequencies of single nucleotide polymorphisms (SNPs) were compared between resistant and susceptible mosquito pools using a contingency χ2 analysis. The -log10(χ2 p value) was calculated at each SNP site, with a weighted average determined from all sites in each gene. Genes with -log10(χ2 p value) ≥ 4.0 and present among all 3 treatment groups were subjected to gene set enrichment analysis (GSEA). We found that several functional groups were enriched compared to all coding genes. These categories were transport, signal transduction and metabolism, in order from highest to lowest statistical significance. Strikingly, 21 genes with demonstrated association to synaptic function were identified. In the high association group (n = 1,053 genes), several genes were identified that also genetically or physically interact with the voltage-gated sodium channel (VGSC). These genes were eg., CHARLATAN (CHL), a transcriptional regulator, several ankyrin-domain proteins, PUMILIO (PUM), a translational repressor, and NEDD4 (E3 ubiquitin-protein ligase). There were 13 genes that ranked among the top 10%: these included VGSC; CINGULIN, a predicted neuronal gap junction protein, and the aedine ortholog of NERVY (NVY), a transcriptional regulator. Silencing of CHL and NVY followed by standard permethrin bottle bioassays validated their association with permethrin resistance. Importantly, VGSC levels were also reduced about 50% in chl- or nvy-dsRNA treated mosquitoes. These results are consistent with the contribution of a variety of neuronal pathways to pyrethroid resistance in Ae. aegypti.

E3 ubiquitin ligases LNX1 and LNX2 localize at neuronal gap junctions formed by connexin36 in rodent brain and molecularly interact with connexin36

Electrical synapses in the mammalian central nervous system (CNS) are increasingly recognized as highly complex structures for mediation of neuronal communication, both with respect to their capacity for dynamic short- and long-term modification in efficacy of synaptic transmission and their multimolecular regulatory and structural components. These two characteristics are inextricably linked, such that understanding of mechanisms that contribute to electrical synaptic plasticity requires knowledge of the molecular composition of electrical synapses and the functions of proteins associated with these synapses. Here, we provide evidence that the key component of gap junctions that form the majority of electrical synapses in the mammalian CNS, namely connexin36 (Cx36), directly interacts with the related E3 ubiquitin ligase proteins Ligand of NUMB protein X1 (LNX1) and Ligand of NUMB protein X2 (LNX2). This is based on immunofluorescence colocalization of LNX1 and LNX2 with Cx36-containing gap junctions in adult mouse brain versus lack of such coassociation in LNX null mice, coimmunoprecipitation of LNX proteins with Cx36, and pull-down of Cx36 with the second PDZ domain of LNX1 and LNX2. Furthermore, cotransfection of cultured cells with Cx36 and E3 ubiquitin ligase-competent LNX1 and LNX2 isoforms led to loss of Cx36-containing gap junctions between cells, whereas these junctions persisted following transfection with isoforms of these proteins that lack ligase activity. Our results suggest that a LNX protein mediates ubiquitination of Cx36 at neuronal gap junctions, with consequent Cx36 internalization, and may thereby contribute to intracellular mechanisms that govern the recently identified modifiability of synaptic transmission at electrical synapses.

Structural and Intermolecular Associations Between Connexin36 and Protein Components of the Adherens Junction–Neuronal Gap Junction Complex

Intimate structural and functional relationships between gap junctions and adherens junctions have been demonstrated in peripheral tissues, but have not been thoroughly examined in the central nervous system, where adherens junctions are often found in close proximity to neuronal gap junctions. Here, we used immunofluorescence approaches to document the localization of various protein components of adherens junctions in relation to those that we have previously reported to occur at electrical synapses formed by neuronal gap junctions composed of connexin36 (Cx36). The adherens junction constituents N-cadherin and nectin-1 were frequently found to localize near or overlap with Cx36-containing gap junctions in several brain regions examined. This was also true of the adherens junction-associated proteins α-catenin and β-catenin, as well as the proteins zonula occludens-1 and AF6 (aka, afadin) that were reported constituents of both adherens junctions and gap junctions. The deployment of the protein constituents of these junctions was especially striking at somatic contacts between primary afferent neurons in the mesencephalic trigeminal nucleus (MesV), where the structural components of adherens junctions appeared to be maintained in connexin36 null mice. These results support emerging views concerning the multi-molecular composition of electrical synapses and raise possibilities for various structural and functional protein–protein interactions at what now can be considered the adherens junction–neuronal gap junction complex. Further, the results point to intracellular signaling pathways that could potentially contribute to the assembly, maintenance and turnover of this complex, as well as to the dynamic nature of neuronal communication at electrical synapses.

Electrical Synapses are Involved in Orofacial Neuropathic Pain

Accumulated evidences suggest important roles of glial GAP-junctions in pain. However, only a few studies have explored the role of neuronal GAP-junctions or electrical synapses in neuropathic pain (NP). Therefore, the present study explores the role of connexin 36 (Cx36) in NP using the chronic constriction injury of the infraorbital nerve (CCI-IoN) model in rat. A significant increase in Cx36 labeling was observed in the medullary dorsal horn (MDH) of CCI-IoN-lesioned compared to sham rats. The expression of Cx36 in CCI-IoN-lesioned rats revealed a rostroventral gradient of punctuate labeling within lamina IIo of the MDH. Cx36-positive somata and processes were also observed in MDH laminae IIi and III–V. These somata were mostly of the Gamma aminobutyric acid (GABA) and occasionally Glycine transporter 2 (GlyT2) cell subtypes. Moreover the GABA cell subtypes are highly coupled in lamina IIo as revealed by the intense Cx36 staining in this lamina. Pharmacological Cx36 blockade by intracisternal administration of mefloquine decreased significantly the mechanical allodynia observed in CCI-IoN-lesioned rats. Altogether, our findings demonstrated that Cx36 play an important role in mechanical allodynia by coupling GABA cells. Increasing cell coupling by enhancing Cx36 expression favors neuropathic pain while disrupting this coupling alleviates it. This mechanism may constitute a novel target for the treatment of orofacial mechanical allodynia.

Neuronal Gap Junctions in Cortical Columns and Nuclei of the Ventral Thalamus of Rats

Functional cortical columns and nuclei of the ventral thalamus play a key role in processing of sensory information; therefore, detailed studies on formation of neuron-to-neuron gap junctions in these areas are of great theoretical and practical importance. In the present study, we applied electron-microscopy methods to examine the structure and specific distribution of interneuronal gap junctions in the cortical layer IV and thalamic nuclei, including VPM, RTN, Pom, and VPL. In the cortex, we found more interneuronal gap junctions than in thalamic nuclei. In all structures studied we revealed and described axo-dendritic, dendrodendritic, and “mixed” synapses. We report on the axo-dendritic gap junctions for the first time. It is suggested that this type of contacts plays some functional role in local synchronization of neuronal activity within one ensemble on the presynaptic level.

Electrical synapses mediate synergism between pheromone and food odors in Drosophila melanogaster

In Drosophila melanogaster, the sex pheromone produced by males, cis-vaccenyl acetate (cVA), evokes a stereotypic gender-specific behavior in both males and females. As Drosophila adults feed, mate, and oviposit on food, they perceive the pheromone as a blend against a background of food odors. Previous studies have reported that food odors enhance flies' behavioral response to cVA, specifically in virgin females. However, how and where the different olfactory inputs interact has so far remained unknown. In this study, we elucidated the neuronal mechanism underlying the response at an anatomical, functional, and behavioral level. Our data show that in virgin females cVA and the complex food odor vinegar evoke a synergistic response in the cVA-responsive glomerulus DA1. This synergism, however, does not appear at the input level of the glomerulus, but is restricted to the projection neuron level only. Notably, it is abolished by a mutation in gap junctions in projection neurons and is found to be mediated by electrical synapses between excitatory local interneurons and projection neurons. As a behavioral consequence, we demonstrate that virgin females in the presence of vinegar become receptive more rapidly to courting males, while male courtship is not affected. Altogether, our results suggest that lateral excitation via gap junctions modulates odor tuning in the antennal lobe and drives synergistic interactions between two ecologically relevant odors, representing food and sex.