Jane A. Davies

Innexins: a family of invertebrate gap-junction proteins.

In vertebrates, intercellular communication via gap junctions is mediated by the connexin family of molecules, which is made up of at least 13 members (reviewed in Ref. 1). These proteins, which have four transmembrane domains and intracellular C- and N-termini, oligomerize to form hemichannels. Oligomers in the adjacent membranes of two closely apposed cells ‘dock’ to form intercellular channels, through which ions and small molecules move. Intercellular communication is a fundamental function of any multicellular organism and it is odd that no obvious homologues of the connexins have been found in any invertebrate. In view of the fact that over 90% of the genomic sequence of Caenorhabditis elegans is available for analysis, it is becoming increasingly unlikely that invertebrate connexins will be found. Conventional genetic dissection of C. elegans and Drosophila, however, has identified a gene family with some role in gap-junction communication. Although they bear no sequence similarity to the connexins, these genes are predicted to encode proteins with the same topology. In C. elegans, mutations in the unc-7 gene result in an uncoordinated phenotype, and the formation of ectopic electrical junctions between some interneurons and motoneurons (J.G. White, E. Southgate and J.N. Thomson, cited in Ref. 2). A worm with an eating disorder results from mutations in the eat-5 gene; here, some pharyngeal muscles fail to establish normal electrical connections with their neighbours2. In Drosophila, one of the transcripts from the shaking-B locus [shaking-B(neural); also known as Passover ] is required for electrical synapse function between neurons of the giant-fibre escape circuit3,4 and between embryonic somatic muscles (J.P. Bacon et al., 1996, Soc. Neurosci. Abstr. 22, 38). A second transcript from this Drosophila locus, known as shaking-B(lethal), the Drosophila gene optic ganglion reduced (ogre), and several other C. elegans genes share sequence similarity with this family5 but their mutant phenotypes have not yet been fully characterized. These worm and fly data did provide some circumstantial evidence that these loci encode gap-junction proteins. They were given the name OPUS (for ogre, passover, uncoordinated, shaking-B) in a previous letter to TIG (Ref. 6). We feel that the name OPUS is confusing because we now know that Passover and shaking-B are allelic and it has recently been brought to our attention that opus is the name of a Drosophila copia-like transposable element7. In addition, the recent determination of the role of one of these genes makes it timely to rename this family in a way that reflects function. Using heterologous expression in Xenopus oocyte pairs, it has been demonstrated unequivocally that Shaking-B(lethal) protein is sufficient to form homotypic gap junctions8. Interestingly, the closely related Shaking-B(neural) protein fails to form functional junctions in this system. We suspect that either Shaking-B(neural) forms gap junctions that are closed under the particular physiological conditions of Xenopus oocytes, or that it requires a partner to form hetero-oligomeric channels; a few connexins fail to form homotypic junctions in Xenopus oocytes (reviewed in Ref. 1). Despite these remaining uncertainties about the function of Shaking-B(neural) protein, Shaking-B (lethal) is the first invertebrate gap-junction protein to be identified. It means that this family of genes, and their proteins, can be given a functional name. We propose the name innexins (invertebrate analogues of the connexins) for invertebrate gap-junction proteins. We are anxious to avoid the gratuitous proliferation of names in an already overstuffed literature but we think it better to choose a name that reflects function rather than an acronym based on an incomplete set of mutant phenotypes. It will be important to examine the function of fly and worm Shaking-B(lethal)-like proteins, using heterologous expression systems, to determine whether they truly are innexins. Judging from the vertebrate connexin data, we anticipate that the innexin family will have many members that can either work alone or in concert to build a range of gap-junction types. Another task is to look for innexins in other invertebrate phyla; so far they have been described only in insects and nematodes. Given the recent proposal that moulting animals form a new phyletic clade, the Ecdysozoa9, it remains a real possibility that innexin proteins are a molecular marker for this clade, and will not be found outside it. Interestingly, our standard BLAST searches of the protein databases at the NCBI server have revealed no vertebrate proteins with sequence similarity to Shaking-B. Whether the similar (predicted) topology, without obvious sequence similarity, of innexins and connexins is a case of convergent evolution or one of extreme sequence divergence within a protein family, remains to be determined.

Drosophila Shaking-B protein forms gap junctions in paired Xenopus oocytes

In most multicellular organisms direct cell–cell communication is mediated by the intercellular channels of gap junctions. These channels allow the exchange of ions and molecules that are believed to be essential for cell signalling during development and in some differentiated tissues. Proteins called connexins, which are products of a multigene family, are the structural components of vertebrate gap junctions,. Surprisingly, molecular homologues of the connexins have not been described in any invertebrate. A separate gene family, which includes the Drosophila genes shaking-B and l(1)ogre, and the Caenorhabditis elegans genes unc-7 and eat-5, encodes transmembrane proteins with a predicted structure similar to that of the connexins. shaking-B and eat-5 are required for the formation of functional gap junctions,. To test directly whether Shaking-B is a channel protein, we expressed it in paired Xenopus oocytes. Here we show that Shaking-B localizes to the membrane, and that its presence induces the formation of functional intercellular channels. To our knowledge, this is the first structural component of an invertebrate gap junction to be characterized.

Gap junctions in Drosophila: developmental expression of the entire innexin gene family

Invertebrate gap junctions are composed of proteins called innexins and eight innexin encoding loci have been identified in the now complete genome sequence of Drosophila melanogaster. The intercellular channels formed by these proteins are multimeric and previous studies have shown that, in a heterologous expression system, homo- and hetero-oligomeric channels can form, each combination possessing different gating characteristics. Here we demonstrate that the innexins exhibit complex overlapping expression patterns during oogenesis, embryogenesis, imaginal wing disc development and central nervous system development and show that only certain combinations of innexin oligomerization are possible in vivo. This work forms an essential basis for future studies of innexin interactions in Drosophila and outlines the potential extent of gap-junction involvement in development.

Molecular Mechanism of Rectification at Identified Electrical Synapses in the Drosophila Giant Fiber System

Summary Electrical synapses are neuronal gap junctions that mediate fast transmission in many neural circuits [1–5]. The structural proteins of gap junctions are the products of two multigene families. Connexins are unique to chordates [3–5]; innexins/pannexins encode gap-junction proteins in prechordates and chordates [6–10]. A concentric array of six protein subunits constitutes a hemichannel; electrical synapses result from the docking of hemichannels in pre- and postsynaptic neurons. Some electrical synapses are bidirectional; others are rectifying junctions that preferentially transmit depolarizing current anterogradely [11, 12]. The phenomenon of rectification was first described five decades ago [1], but the molecular mechanism has not been elucidated. Here, we demonstrate that putative rectifying electrical synapses in the Drosophila Giant Fiber System [13] are assembled from two products of the innexin gene shaking-B. Shaking-B(Neural+16) [14] is required presynaptically in the Giant Fiber to couple this cell to its postsynaptic targets that express Shaking-B(Lethal) [15]. When expressed in vitro in neighboring cells, Shaking-B(Neural+16) and Shaking-B(Lethal) form heterotypic channels that are asymmetrically gated by voltage and exhibit classical rectification. These data provide the most definitive evidence to date that rectification is achieved by differential regulation of the pre- and postsynaptic elements of structurally asymmetric junctions.

Null mutation in shaking-B eliminates electrical, but not chemical, synapses in the Drosophila giant fiber system: A structural study

Mutations in the Drosophila shaking‐B gene perturb synaptic transmission and dye coupling in the giant fiber escape system. The GAL4 upstream activation sequence system was used to express a neuronal‐synaptobrevin‐green fluorescent protein (nsyb‐GFP) construct in the giant fibers (GFs); nsyb‐GFP was localized where the GFs contact the peripherally synapsing interneurons (PSIs) and the tergotrochanteral motorneurons (TTMns). Antibody to Shaking‐B protein stained plaquelike structures in the same regions of the GFs, although not all plaques colocalized with nsyb‐GFP. Electron microscopy showed that the GF‐TTMn and GF‐PSI contacts contained many chemical synaptic release sites. These sites were interposed with extensive regions of close membrane apposition (3.25 nm ± 0.12 separation), with faint cross striations and a single‐layered array of 41‐nm vesicles on the GF side of the apposition. These contacts appeared similar to rectifying electrical synapses in the crayfish and were eliminated in shaking‐B2 mutants. At mutant GF‐TTMn and GF‐PSI contacts, chemical synapses and small regions of close membrane apposition, more similar to vertebrate gap junctions, were not affected. Gap junctions with more vertebratelike separation of membranes (1.41 nm ± 0.08) were abundant between peripheral perineurial glial processes; these were unaffected in the mutants. J. Comp. Neurol. 404:449–458, 1999.

Evolution of gap junctions: the missing link?

Connexin molecules form gap-junction channels in vertebrates and there are at least 20 of them in humans [1]. Intuitively, one would imagine that cardinal features of the cellular machinery, such as gap-junctions, would be highly conserved. Paradoxically, however, Drosophila and Caenorhabditis elegans do not have connexin genes, but instead use innexins for gap-junctional communication, a protein family with the same 4-transmembrane topology but no sequence similarity to the connexins [2,3]. In this paper we show that the simple diploblastic organism Hydra appears to possess only innexins. We conclude that innexins are the primordial gap-junction molecules, while connexins evolved more recently in the deuterostomes. The major question is whether the connexin–innexin dichotomy is an extreme case of sequence divergence from a common ancestor or a convergent solution to the problem of intercellular communication. A critical experiment was to identify gap-junction proteins in diploblastic organisms, e.g cnidaria. These organisms have functional gap junctions [4] and represent an evolutionary grade before the deuterostome–protostome divergence. We focused on the hydrozoan Hydra because the cells that comprise its body have large gap-junction plaques and are electrically and dye coupled [4,5]. During a signal peptide screen, we recovered a fragment, which matched a set of 14 overlapping ESTs (contig number C_CD267995, available at http://mpc.uci.edu/ hampson/public_html/blast/jf), which we identified as a true innexin. The original EST collection had 3500 distinct sequences derived from 13,000 ESTs. The novel sequence has 396 amino acids and a predicted molecular weight of 44.9 kDa. A structural prediction using the Kyte-Doolittle algorithm showed that this molecule, named Hydra innexin-1 (Hv-inx1), has a typical innexin topology with 4-transmembrane (TM) domains and amino-and carboxy-terminal domains on the cytoplasmic face of the membrane. The extracellular loops contain pairs of invariant cysteine residues and the transmembrane domains (TM) contain signature residues Y, Q, W, P (second TM) and W, F (fourth TM) at conserved positions ([6]; Figure 1). This strongly supports the identification of Hv-inx1 as a true innexin despite low overall sequence identity. Expression of a Hv-inx1-GFP fusion protein in Hydra revealed a punctate pattern of GFP fluorescence along the basal lateral membrane of epithelial cells (Figure 2), corresponding to known sites of gap junctions [5]. revealed four more innexin homologs in addition to innexin-1. The enlarged EST collection was also searched for connexins using the BLAST algorithm but no statistically significant hits (e-value < 1) were recorded. This suggests that an earlier report of …

Molecular Basis of Gap Junctional Communication in the CNS of the Leech Hirudo medicinalis

Gap junctions are intercellular channels that allow the passage of ions and small molecules between cells. In the nervous system, gap junctions mediate electrical coupling between neurons. Despite sharing a common topology and similar physiology, two unrelated gap junction protein families exist in the animal kingdom. Vertebrate gap junctions are formed by members of the connexin family, whereas invertebrate gap junctions are composed of innexin proteins. Here we report the cloning of two innexins from the leech Hirudo medicinalis. These innexins show a differential expression in the leech CNS: Hm-inx1 is expressed by every neuron in the CNS but not in glia, whereas Hm-inx2 is expressed in glia but not neurons. Heterologous expression in the paired Xenopus oocyte system demonstrated that both innexins are able to form functional homotypic gap junctions. Hm-inx1 forms channels that are not strongly gated. In contrast, Hm-inx2 forms channels that are highly voltage-dependent; these channels demonstrate properties resembling those of a double rectifier. In addition, Hm-inx1 and Hm-inx2 are able to cooperate to form heterotypic gap junctions in Xenopus oocytes. The behavior of these channels is primarily that predicted from the properties of the constituent hemichannels but also demonstrates evidence of an interaction between the two. This work represents the first demonstration of a functional gap junction protein from a Lophotrochozoan animal and supports the hypothesis that connexin-based communication is restricted to the deuterostome clade.

Promotion of regeneration and axon growth following injury in an invertebrate nervous system by the use of three–dimensional collagen gels

We describe the application of three–dimensional collagen matrices to the study of nerve cord repair in the leech. Our experiments show that ganglia and connectives of the leech ventral nerve cord can be maintained for up to four weeks embedded in 3D gels constructed from mammalian type 1 collagen. Severed nerve cords embedded in the collagen gel reliably repaired within a few days of culture. The gel was penetrable by cells emigrating from the cut ends of nerves and connectives, and we consistently saw regenerative outgrowth of severed peripheral and central axons into the gel matrix. Thus, 3D gels provide an in vitro system in which we can reliably obtain repair of severed nerve cords in the dish, and visualize cell behaviour underlying regenerative growth at the damage site; and which offers the possibility of manipulating the regenerating cells and their extracellular environment in various ways at stages during repair. Using this system it should be possible to test the effect on the repair process of altering expression of selected genes in identified nerve cells.

A subtractive cDNA library from an identified regenerating neuron is enriched in sequences up-regulated during nerve regeneration

We have constructed a subtractive cDNA library from regenerating Retzius cells of the leech,Hirudo medicinalis. It is highly enriched in sequences up-regulated during nerve regeneration. Sequence analysis of selected recombinants has identified both novel sequences and sequences homologous to molecules characterised in other species. Homologies include α-tubulin, a calmodulin-like protein, CAAT/enhancer-binding protein (C/EBP), protein 4.1 and synapsin. These types of proteins are exactly those predicted to be associated with axonal growth and their identification confirms the quality of the library. Most interesting, however, is the isolation of 5 previously uncharacterised cDNAs which appear to be up-regulated during regeneration. Their analysis is likely to provide new information on the molecular mechanisms of neuronal regeneration.

Innexins in the lobster stomatogastric nervous system: cloning, phylogenetic analysis, developmental changes and expression within adult identified dye and electrically coupled neurons

Gap junctions play a key role in the operation of neuronal networks by enabling direct electrical and metabolic communication between neurons. Suitable models to investigate their role in network operation and plasticity are invertebrate motor networks, which are built of comparatively few identified neurons, and can be examined throughout development; an excellent example is the lobster stomatogastric nervous system. In invertebrates, gap junctions are formed by proteins that belong to the innexin family. Here, we report the first molecular characterization of two crustacean innexins: the lobster Homarus gammarus innexin 1 (Hg‐inx1) and 2 (Hg‐inx2). Phylogenetic analysis reveals that innexin gene duplication occurred within the arthropod clade before the separation of insect and crustacean lineages. Using in situ hybridization, we find that each innexin is expressed within the adult and developing lobster stomatogastric nervous system and undergoes a marked down‐regulation throughout development within the stomatogastric ganglion (STG).The number of innexin expressing neurons is significantly higher in the embryo than in the adult. By combining in situ hybridization, dye and electrical coupling experiments on identified neurons, we demonstrate that adult neurons that express at least one innexin are dye and electrically coupled with at least one other STG neuron. Finally, two STG neurons display no detectable amount of either innexin mRNAs but may express weak electrical coupling with other STG neurons, suggesting the existence of other forms of innexins. Altogether, we provide evidence that innexins are expressed within small neuronal networks built of dye and electrically coupled neurons and may be developmentally regulated.