Innexin Gap Junctions Research Articles

DEC-7/SUSD2, a sushi domain-containing protein, regulates an ultradian behavior mediated by intestinal epithelial Ca2+ oscillations in Caenorhabditis elegans.

In Caenorhabditis elegans, rhythmic posterior body wall muscle contractions mediate the highly regular defecation cycle. These contractions are regulated by inositol-1,4,5-trisphosphate (InsP3) receptor-dependent Ca2+ oscillations in intestinal epithelial cells. Here, we find that mutations in dec-7, which encodes the nematode ortholog of the human Sushi domain-containing 2 protein (SUSD2), lead to an increase in InsP3 receptor-dependent rhythmic posterior body wall muscle contractions. DEC-7 is highly expressed in the intestinal epithelia and localizes to the cell-cell junction. The increase in rhythmic activity caused by the loss of dec-7 is dependent on the innexin gap junction protein INX-16. Moreover, DEC-7 is required for the clustering of INX-16 to the cell-cell junction of the intestinal epithelia. We hypothesize that DEC-7/SUSD2 regulates INX-16 activity to mediate the rhythmic frequency of the defecation motor program. Thus, our data indicate a critical role of a phylogenetically conserved cell-cell junction protein in mediating an ultradian rhythm in the intestinal epithelia of C. elegans.NEW & NOTEWORTHY The conserved complement group protein DEC-7/SUSD2 acts at the apical cell-cell junction of C. elegans intestinal epithelia to mediate gap junction protein organization and function to facilitate a Ca2+ wave-regulated ultradian behavior.

Innexin-Mediated Adhesion between Glia Is Required for Axon Ensheathment in the Peripheral Nervous System.

Glia are essential to protecting and enabling nervous system function and a key glial function is the formation of the glial sheath around peripheral axons. Each peripheral nerve in the Drosophila larva is ensheathed by three glial layers, which structurally support and insulate the peripheral axons. How peripheral glia communicate with each other and between layers is not well established and we investigated the role of Innexins in mediating glial function in the Drosophila periphery. Of the eight Drosophila Innexins, we found two (Inx1 and Inx2) are important for peripheral glia development. In particular loss of Inx1 and Inx2 resulted in defects in the wrapping glia leading to disruption of the glia wrap. Of interest loss of Inx2 in the subperineurial glia also resulted in defects in the neighboring wrapping glia. Inx plaques were observed between the subperineurial glia and the wrapping glia suggesting that gap junctions link these two glial cell types. We found Inx2 is key to Ca2+ pulses in the peripheral subperineurial glia but not in the wrapping glia, and we found no evidence of gap junction communication between subperineurial and wrapping glia. Rather we have clear evidence that Inx2 plays an adhesive and channel-independent role between the subperineurial and wrapping glia to ensure the integrity of the glial wrap.SIGNIFICANCE STATEMENT Gap junctions are critical for glia communication and formation of myelin in myelinating glia. However, the role of gap junctions in non-myelinating glia is not well studied, yet non-myelinating glia are critical for peripheral nerve function. We found the Innexin gap junction proteins are present between different classes of peripheral glia in Drosophila. Here Innexins form junctions to facilitate adhesion between the different glia but do so in a channel-independent manner. Loss of adhesion leads to disruption of the glial wrap around axons and leads to fragmentation of the wrapping glia membranes. Our work points to an important role for gap junction proteins in mediating insulation by non-myelinating glia.

Anatomical distribution and functional roles of electrical synapses in Drosophila

Electrical synapses are present in almost all organisms that have a nervous system. However, their brain-wide expression patterns and the full range of contributions to neural function are unknown in most species. Here, we first provide a light-microscopic, immunohistochemistry-based anatomical map of all innexin gap junction proteins-the building blocks of electrical synapses-in the central nervous system of Drosophila melanogaster. Of those innexin types that are expressed in the nervous system, some localize to glial cells, whereas others are predominantly expressed in neurons, with shakB being the most widely expressed neuronal innexin. We then focus on the function of shakB in VS/HS cells-a class of visual projection neurons-thereby uncovering an unexpected role for electrical synapses. Removing shakB from these neurons leads to spontaneous, cell-autonomous voltage and calcium oscillations, demonstrating that electrical synapses are required for these cells' intrinsic stability. Furthermore, we investigate the role of shakB-type electrical synapses in early visual processing. We find that the loss of shakB from the visual circuits upstream of VS/HS cells differentially impairs ON and OFF visual motion processing pathways but is not required for the computation of direction selectivity per se. Taken together, our study demonstrates that electrical synapses are widespread across the Drosophila nervous system and that they play essential roles in neuronal function and visual information processing.

Molecular and morphological approach to study the innexin gap junctions in Rhynchosciara americana.

Gap junctions mediate communication between adjacent cells and are fundamental to the development and homeostasis in multicellular organisms. In invertebrates, gap junctions are formed by transmembrane proteins called innexins. Gap junctions allow the passage of small molecules through an intercellular channel, between a cell and another adjacent cell. The dipteran Rhynchosciara americana has contributed to studying the biology of invertebrates and the study of the interaction and regulation of genes during biological development. Therefore, this paper aimed to study the R. americana innexin-2 by molecular characterization, analysis of the expression profile and cellular localization. The molecular characterization results confirm that the message is from a gap junction protein and analysis of the expression and cellular localization profile shows that innexin-2 can participate in many physiological processes during the development of R. americana.

Formation of functional innexin hemichannels, as well as gap junctional channels, in an insect cell line, NIAs-AeAl-2, derived from Asian tiger mosquito Aedes albopictus (Diptera: Culicidae): A partial but significant contribution of innexin 2

In vertebrates, gap junctions and hemichannels consisting of connexins are important cell surface structures for communication with neighboring cells and for the regulation of various cell functions. To date, various gap-junction-related proteins have been found, including innexins in invertebrates and pannexins in vertebrates. Significant contributions of gap junctions by innexins and (hemi-)channels by pannexins to numerous functions have been reported. Verification of the presence and functional significance of innexin hemichannels, however, remains a gap in our knowledge in innexin physiology. In this study, we revealed the localization of an innexin protein (innexin 2) on the cell surface in mosquito tissues and cultured cells. Furthermore, we demonstrated the presence of functional hemichannels, as well as gap junctions, in mosquito cells using dye transfer assays. The inward uptake of fluorescent dye was inhibited by anti-innexin 2 antibody. These results suggest that innexin hemichannels are formed to function in cultured mosquito cells, in at least a partially innexin 2-dependent manner. Although only a few studies on insect hemichannels have been published, innexin-based hemichannels, as well as innexin gap junctions, could also significantly contribute to insect intercellular signal transduction.

Distinct roles for innexin gap junctions and hemichannels in mechanosensation.

Mechanosensation is central to a wide range of functions, including tactile and pain perception, hearing, proprioception, and control of blood pressure, but identifying the molecules underlying mechanotransduction has proved challenging. In Caenorhabditis elegans, the avoidance response to gentle body touch is mediated by six touch receptor neurons (TRNs), and is dependent on MEC-4, a DEG/ENaC channel. We show that hemichannels containing the innexin protein UNC-7 are also essential for gentle touch in the TRNs, as well as harsh touch in both the TRNs and the PVD nociceptors. UNC-7 and MEC-4 do not colocalize, suggesting that their roles in mechanosensory transduction are independent. Heterologous expression of unc-7 in touch-insensitive chemosensory neurons confers ectopic touch sensitivity, indicating a specific role for UNC-7 hemichannels in mechanosensation. The unc-7 touch defect can be rescued by the homologous mouse gene Panx1 gene, thus, innexin/pannexin proteins may play broadly conserved roles in neuronal mechanotransduction.

Structure of an innexin gap junction channel and cryo-EM sample preparation.

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.

Structure of the C. elegans Innexin-6 Gap Junction Channel

Structure of the <i>C. elegans</i> Innexin-6 Gap Junction Channel

Single-Particle Cryo-EM of an Innexin Gap Junction Channel at Atomic Resolution

Single-Particle Cryo-EM of an Innexin Gap Junction Channel at Atomic Resolution

Atomic structure of the innexin-6 gap junction channel determined by cryo-EM.

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.

Hexadecameric structure of an invertebrate gap junction channel

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.

Three-Dimensional Structure of Innexin GAP Junction Channels Studied by Electron Crystallography

Invertebrate-specific gap junction proteins, termed innexins, form a large family of four-transmembrane proteins. 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 vertebrate connexin gap junction channels, few structural studies of recombinant innexin channels have been published. Here we show a three-dimensional structure of two-dimensionally crystallized Caenorhabditis elegans innexin-6 (INX-6) gap junction channels. An N-terminal deletion INX-6 construct in which amino acids 2 through 19 were removed (INX-6-deltaN) was crystallized in lipid bilayers. The three-dimensional reconstruction based on cryo-electron crystallography revealed that the two-dimensional crystals of INX-6-deltaN comprise two lipid bilayers, including fully docked gap junction channels. A single INX-6-deltaN gap junction channel comprises 16 subunits, a hexa-decamer, in contrast to vertebrate connexin channels, which comprise 12 subunits. Two bulb densities were observed in each hemichannel, one in the pore and the other at the cytoplasmic side of the hemichannel in the channel pore pathway. The former is reminiscent of the plug observed in the connexin26 mutant structure we previously reported. A fluorescent dye transfer assay revealed that INX-6-deltaN junction channels were essentially impermeable. These findings imply the structural diversity of gap junction channels among multicellular organisms, and provide insight into the functional properties characteristic of invertebrates.

Electron microscopy and functional analysis of recombinant innexin gap junctions

Innexin is a molecular component of invertebrate gap junctions, which have an important role in neural and muscular electrical activity in invertebrates. Although the structure of vertebrate connexin26 was revealed by X-ray crystallography [1], the structure of innexin channels remains poorly understood. To study the structure of innexin gap junction channels, we expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels, and characterized their molecular dimensions and channel permeability using electron microscopy (EM) and a fluorescent dye transfer assay, respectively [2]. Negative-staining and thin-section EM of isolated INX-6 gap junction plaques revealed a loosely packed hexagonal lattice. We performed single particle analysis of purified INX-6 channels with negative-staining and cryo EM. Based on the negative-stain EM images, the class average of the junction form had a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class average of the hemichannels had diameters of up to 140 Å in the presence of detergent micelles. Cryo EM images revealed rotational peaks that could be related to the INX-6 subunits. Structural analysis of the reconstituted INX-6 channels with single particle analysis and electron tomography suggested that the oligomeric number of the INX-6 channel was distinct from that of the dodecameric connexin channel. Dye transfer experiments indicated that the INX-6-GFP-His channels were permeable to 3-kDa and 10-kDa dextran-conjugated tracers. These findings indicate that INX-6 channels have a characteristic oligomer component that differs from that in connexin gap junction channels.

Correction: Corrigendum: Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps

CORRIGENDUM: Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps

Innexin gap junctions in nerve cells coordinate spontaneous contractile behavior in Hydra polyps.

Nerve cells and spontaneous coordinated behavior first appeared near the base of animal evolution in the common ancestor of cnidarians and bilaterians. Experiments on the cnidarian Hydra have demonstrated that nerve cells are essential for this behavior, although nerve cells in Hydra are organized in a diffuse network and do not form ganglia. Here we show that the gap junction protein innexin-2 is expressed in a small group of nerve cells in the lower body column of Hydra and that an anti-innexin-2 antibody binds to gap junctions in the same region. Treatment of live animals with innexin-2 antibody eliminates gap junction staining and reduces spontaneous body column contractions. We conclude that a small subset of nerve cells, connected by gap junctions and capable of synchronous firing, act as a pacemaker to coordinate the contraction of the body column in the absence of ganglia.

2SBA-01 電子顕微鏡法により示された線虫イネキシン6ギャップ結合チャネルの特徴的なサブユニット構成(2SBA 日本顕微鏡学会合同シンポジウム:原子レベル分解能へ向かう生物電子顕微鏡技術,シンポジウム,第52回日本生物物理学会年会(2014年度))

2SBA-01 電子顕微鏡法により示された線虫イネキシン6ギャップ結合チャネルの特徴的なサブユニット構成(2SBA 日本顕微鏡学会合同シンポジウム:原子レベル分解能へ向かう生物電子顕微鏡技術,シンポジウム,第52回日本生物物理学会年会(2014年度))

Oligomeric Structure and Functional Characterization of Caenorhabditis elegans Innexin-6 Gap Junction Protein

Innexin is the molecular component of invertebrate gap junctions. Here we successfully expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels and characterized the molecular dimensions and channel permeability using electron microscopy (EM) and microinjection of fluorescent dye tracers, respectively. Negative staining and thin-section EM of isolated INX-6 gap junction membranes revealed a loosely packed hexagonal lattice and a greater cross-sectional width than that of connexin26 and connexin43 (Cx43)-GFP. In gel filtration analysis, the elution profile of purified INX-6 channels in dodecyl maltoside solution exhibited a peak at ∼400 kDa that was shifted to ∼800 kDa in octyl glucose neopentyl glycol. We also obtained the class averages of purified INX-6 channels from these peak fractions by single particle analysis. The class average from the ∼800-kDa fraction showed features of the junction form with a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class averages from the ∼400-kDa fraction showed diameters of up to 140 Å in the presence of detergent micelles. These findings indicate that the purified INX-6 channels are predominantly hemichannels in dodecyl maltoside and docked junction channels in octyl glucose neopentyl glycol. Dye transfer experiments revealed that the INX-6-GFP-His channels are permeable to 3- and 10-kDa tracers, whereas no significant amounts of these tracers passed through the Cx43-GFP channels. Based on these findings, INX-6 channels have a larger overall structure and greater permeability than connexin channels.

The MicroRNA mir-71 Inhibits Calcium Signaling by Targeting the TIR-1/Sarm1 Adaptor Protein to Control Stochastic L/R Neuronal Asymmetry in C. elegans

The Caenorhabditis elegans left and right AWC olfactory neurons communicate to establish stochastic asymmetric identities, AWCON and AWCOFF, by inhibiting a calcium-mediated signaling pathway in the future AWCON cell. NSY-4/claudin-like protein and NSY-5/innexin gap junction protein are the two parallel signals that antagonize the calcium signaling pathway to induce the AWCON fate. However, it is not known how the calcium signaling pathway is downregulated by nsy-4 and nsy-5 in the AWCON cell. Here we identify a microRNA, mir-71, that represses the TIR-1/Sarm1 adaptor protein in the calcium signaling pathway to promote the AWCON identity. Similar to tir-1 loss-of-function mutants, overexpression of mir-71 generates two AWCON neurons. tir-1 expression is downregulated through its 3′ UTR in AWCON, in which mir-71 is expressed at a higher level than in AWCOFF. In addition, mir-71 is sufficient to inhibit tir-1 expression in AWC through the mir-71 complementary site in the tir-1 3′ UTR. Our genetic studies suggest that mir-71 acts downstream of nsy-4 and nsy-5 to promote the AWCON identity in a cell autonomous manner. Furthermore, the stability of mature mir-71 is dependent on nsy-4 and nsy-5. Together, these results provide insight into the mechanism by which nsy-4 and nsy-5 inhibit calcium signaling to establish stochastic asymmetric AWC differentiation.

3F0924 線虫イネキシン6ギャップ結合チャネルの単離と機能特性(膜蛋白質,口頭発表)

3F0924 線虫イネキシン6ギャップ結合チャネルの単離と機能特性(膜蛋白質,口頭発表)

Electron tomographic analysis of gap junctions in lateral giant fibers of crayfish

Innexin-gap junctions in crayfish lateral giant fibers (LGFs) have an important role in escape behavior as a key component of rapid signal transduction. Knowledge of the structure and function of characteristic vesicles on the both sides of the gap junction, however, is limited. We used electron tomography to analyze the three-dimensional structure of crayfish gap junctions and gap junctional vesicles (GJVs). Tomographic analyses showed that some vesicles were anchored to innexons and almost all vesicles were connected by thin filaments. High densities inside the GJVs and projecting densities on the GJV membranes were observed in fixed and stained samples. Because the densities inside synaptic vesicles were dependent on the fixative conditions, different fixative conditions were used to elucidate the molecules included in the GJVs. The projecting densities on the GJVs were studied by immunoelectron microscopy with anti-vesicular monoamine transporter (anti-VMAT) and anti-vesicular nucleotide transporter (anti-VNUT) antibodies. Some of the projecting densities were labeled by anti-VNUT, but not anti-VMAT. Three-dimensional analyses of GJVs and excitatory chemical synaptic vesicles (CSVs) revealed clear differences in their sizes and central densities. Furthermore, the imaging data obtained under different fixative conditions and the immunolabeling results, in which GJVs were positively labeled for anti-VNUT but excitatory CSVs were not, support our model that GJVs contain nucleotides and excitatory CSVs do not. We propose a model in which characteristic GJVs containing nucleotides play an important role in the signal processing in gap junctions of crayfish LGFs.