Animals from humans to comb jellies use gap junction channels to couple adjacent cells and thus enable direct intercellular communication. Interestingly, gap junction channels are formed by two unrelated integral membrane proteins: innexins and connexins. The innexins are the primordial gap junction proteins that have been identified in all eumetazoans except sponges, placozoa, and echinoderms (Slivko-Koltchik et al., 2019). The connexins arose de novo during the early chordate evolution and constitute the gap junction channels of all living chordates except lancelets (Abascal and Zardoya, 2013; Mikalsen et al., 2021; Slivko-Koltchik et al., 2019). Both types of intercellular gap junction channels are formed by the docking of two hemichannels of two neighboring cells. Each hemichannel is composed of six connexins or eight innexins, respectively (Skerrett and Williams, 2017). As connexins and innexins are encoded by large gene families (Mikalsen et al., 2021; Yen and Saier, 2007), different connexin- and innexin-based gap junction channels can be formed to fulfill unique functions. Moreover, a diversity of innexins (or connexins) co-expressed in the same cell can dramatically increase the functional diversity in both types of gap junction channels. This is because each hemichannel can freely combine the N different co-expressed innexins or connexins so that theoretically up to N⁶ (connexins) or N⁸ (innexins) heteromeric hemichannels would be possible.

Despite the lack of sequence homology (Alexopoulos et al., 2004), the topology (Maeda et al., 2009; Michalski et al., 2020; Oshima et al., 2016; Figure 1A and B) and function (Pereda and Macagno, 2017; Skerrett and Williams, 2017) of connexin- and innexin-based gap junction channels are remarkably similar. Nevertheless, it is thought that chordates have completely replaced the innexin-based gap junctions with the novel connexin-based gap junctions. Vertebrates still express innexin-homologs (Baranova et al., 2004; Panchin et al., 2000), called pannexins, but it is supposed that these stopped forming gap junctions and since then only function as non-junctional membrane channels (Dahl and Muller, 2014; Esseltine and Laird, 2016; Sosinsky et al., 2011). This hypothesis is based on the discovery that the three pannexins of humans and mice are glycoproteins. Each of the pannexins contains an identified consensus motif (Asn-X-Ser/Thr) for asparagine (N)-linked glycosylation within either the first or the second extracellular loop (EL; Penuela et al., 2007; Penuela et al., 2014a; Ruan et al., 2020; Sanchez-Pupo et al., 2018). This enables the posttranslational attachment of sugar moieties at the asparagine residue within the consensus sequence which hinders two pannexin channels of adjacent cells to form intercellular channels (Ruan et al., 2020; Figure 1C). Based on these findings, it has been assumed that each vertebrate pannexin is equipped with an N-linked glycosylation site (NGS) and thus lost its gap junction function (Sosinsky et al., 2011). However, it remains unclear whether really all vertebrate pannexins are glycosylated and thus presumably only function as single membrane channels. It is also unknown whether glycosylation is indeed a novel modification gained by chordates to prevent their innexin-homologs from forming gap junctions. Specifically, previous studies have shown that at least two non-chordate species, Aedes aegypti (Calkins et al., 2015) and Caenorhabditis elegans (Kaji et al., 2007), possess an innexin protein with an extracellular NGS that is glycosylated. Additionally, putative NGS have also been described in a terrestrial slug (Limax valentianus) (Sadamoto et al., 2021). These findings raise the intriguing possibility that N-glycosylation might actually be rather common in both chordate and non-chordate innexins, and that N-glycosylation might have played an important role in the evolution of gap junction proteins.

Figure 1 with 1 supplement see all

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Figure 1—source data 1

A list of the identified N-glycosylation sites (NGSs) in the extracellular loops of innexins in non-chordate species.

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Figure 1—source data 2

Multiple sequence alignment of all non-chordate innexins.

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Figure 1—source data 3

Extracellular N-glycosylation sites (NGSs) in non-chordate innexins with confirmed gap junction function.

The identified NGSs have a high potential score (see Materials and methods) and the amino acid distribution around the N-glycosylation motif shows no indications for an unoccupancy of the predicted NGSs (Petrescu, 2003).

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Figure 1—source data 4

Protein sequences of the 1521 non-chordate innexins identified in this study.

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Since the experimental identification of N-glycosylated proteins is technically demanding, time-consuming, and expensive, accurate computational methods are commonly used to identify NGSs in primary amino acid sequences (Gupta and Brunak, 2002; Pitti et al., 2019). In this study, we used the wealth of data that is now available in several public protein, genome, and transcriptome databases to analyze the occurrence of NGSs in non-chordate and chordate innexins in silico. Based on our findings, we suggest a new evolutionary scenario in which a loss in innexin diversity could explain why connexins arose de novo during the early chordate evolution and why connexins have completely replaced the innexins that so successfully serve diverse functions in the nervous systems of invertebrates.

We first screened for innexin proteins across multiple non-chordate taxa by using innexin proteins as sequence queries in BLAST searches. Only hits that fulfilled defined criteria were included in our study (for more details, see Materials and methods). In total, we collected the amino acid sequences of 1521 non-chordate innexins from 174 species across nine higher-level taxonomic groups (ctenophores, cnidarians, molluscs, annelids, platyhelminthes, nematodes, arthropods, xenacoelomorphs, and echinoderms). We subsequently searched in each of the sequences for the consensus motif for N-glycosylation (Asn-X-Ser/Thr). As the extracellular glycosylation of pannexins hinders the gap junction formation (Ruan et al., 2020), we only included NGSs that are located in the ELs of the innexins in our study. Surprisingly, we found that innexins with extracellular NGSs are widespread among the examined non-chordate phyla, comprising 67% of the innexins in the ctenophores (Figure 1D and E, Figure 1—source data 1). The position of the NGSs within the ELs as well as the residues around the N-glycosylation consensus motifs are not conserved between the phyla (Figure 1F). Within the single phyla, we found some innexin orthologs that have highly conserved NGSs and ELs (Figure 1—figure supplement 1). However, we did not find any extracellular NGS that was conserved in all species within a phylum. This finding is presumably based on the phylum-specific diversification of innexins. As shown in previous studies (Abascal and Zardoya, 2013; Hasegawa and Turnbull, 2014; Moroz et al., 2014), and demonstrated in Figure 1D, innexins originated early in metazoan evolution and have undergone diversification within the different non-chordate phyla. Thus, innexins with extracellular NGSs evolved independently numerous times within the single phyla.

Another point of interest is that connexins were clearly absent in all species. But there is one apparent exception, the red sea urchin (Mesocentrotus franciscanus). Here, a connexin (GenBank accession number GHJZ01010392.1) appears evident in the transcriptome (Wong et al., 2019) but is absent in the genomic data (Sergiev et al., 2016). Moreover, the putative connexin is clearly absent in any of the 41 available transcriptomes of other echinoderm species, including close relatives (Strongylocentrotus purpuratus and Hemicentrotus pulcherrimus). If it could be confirmed, it would be an interesting case of an aberrant and remarkably singular occurrence of a connexin.

Notably, our survey also solved an old paradox in the ambulacrarians that form gap junctions (as identified by electron microscopy and voltage-clamp experiments) (Andreuccetti et al., 1987; Slivko-Koltchik et al., 2019; Yazaki et al., 1999) but were assumed to lack both innexins and connexins (Abascal and Zardoya, 2013; Burke et al., 2006; Slivko-Koltchik et al., 2019), causing speculations on the nature of their junctions. Our identification of innexins in ambulacrarian transcriptomes (Figure 1D–F and Figure 1—source data 1) provides a simple solution of this puzzle.

The wide occurrence of innexins with NGSs in all non-chordate phyla (Figure 1D and E) as well as the experimentally confirmed NGSs (Calkins et al., 2015; Kaji et al., 2007) strongly suggest that a large fraction of non-chordate innexins are glycoproteins. These glycosylated innexin channels might then also not be able to form gap junction channels but rather function as non-junctional channels. It is interesting to note that seven of the innexins with identified NGSs have previously been shown to be parts of functional gap junction channels (Figure 1F, Figure 1—source data 3). The NGS were not conserved across these seven innexins (Figure 1F, Figure 1—source data 3) and we found no of the indications (Petrescu, 2003) that would suggest that these NGS might not be glycosylated.

Our findings demonstrate that non-chordate animals possess a vast diversity of innexins, with and without NGSs, and thus presumably function either as non-junctional membrane channels or as intercellular gap junction channels. This finding is in sharp contrast to the situation in chordates, where all innexins are assumed to be glycosylated and unable to form gap junction channels (Dahl and Muller, 2014; Esseltine and Laird, 2016; Sosinsky et al., 2011). But is there really not a single chordate species that uses pannexin-based gap junctions? Up to now, extracellular NGSs were only identified in human (Ruan et al., 2020), mouse (Penuela et al., 2007), rat (Boassa et al., 2007), and zebrafish (Kurtenbach et al., 2013; Prochnow et al., 2009) pannexins. To clarify the prevalence of extracellular NGSs in chordates, we again used public databases to screen for innexin proteins across multiple chordate taxa. In total, we collected the amino acid sequences of 867 chordate innexins from 276 species across nine higher-level taxonomic groups (lancelets, tunicates, lampreys, cartilaginous fish, bony fish, amphibians, reptiles, birds, and mammals) and then searched, as described above, in the ELs of each of the sequences for the consensus motif for N-glycosylation (Asn-X-Ser/Thr).

Our results clearly show that each single innexin in every chordate species has at least one NGS in its ELs (Figure 2A and B, Figure 2—source data 1). The NGSs are highly conserved in lancelets, lampreys and across all vertebrate taxa. NGS were, however, not conserved across the tunicates (Figure 2C–E), which may be consistent with their unusually high amino acid evolutionary rates (Berná and Alvarez-Valin, 2014). Across the different vertebrate taxa, the sequences of the ELs as well as the positions of the glycosylation motifs are highly conserved (Figure 2F–H). Among the three pannexins, conservation is particularly high in the ELs of Pannexi 2. Interestingly, conservation is still seen even after the whole-genome duplication in the common ancestor of teleost fishes (Glasauer and Neuhauss, 2014), an event that generally provides a source of genetic raw material for evolutionary innovation and functional divergence. Still, each single species retained their pannexins with NGSs (Figure 2—source data 1). This is remarkable because a single mutation in the N-glycosylation motif might be sufficient to recover the ability of pannexins to form gap junction channels (Ruan et al., 2020). The surprisingly high conservation of the location and the surrounding sequences of NGSs strongly suggests that at least the vertebrate innexins serve essential roles, with correspondingly high stabilizing selective pressures (Abascal and Zardoya, 2013).

Figure 2

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Figure 2—source data 1

A list of the identified N-glycosylation sites (NGS) in the extracellular loops of innexins in chordate species.

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Figure 2—source data 2

Multiple sequence alignment of all chordate innexins.

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Figure 2—source data 3

Protein sequences of the 867 chordate innexins identified in this study.

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In summary, we show that N-glycosylation is present in both non-chordate and chordate species. Already simple organisms at the beginning of the metazoan evolution attached sugar moieties to some of their innexins to presumably prevent them from forming gap junction channels. In consequence, the vertebrate pannexins did not diverge and change their function driven by the appearance of the connexins but rather originate from an innexin already equipped with NGS. This would be consistent with findings that single membrane channels formed by pannexins and innexins have the same physiological functions and are similar in their biophysical and pharmacological properties (Dahl and Muller, 2014).

If it is typical for invertebrates to use a great diversity of glycosylated and non-glycosylated innexins and to even form gap junctions from both (Figure 1—source data 3), then the situation in the vertebrates becomes even more puzzling: Why do all vertebrates exclusively retain glycosylated innexins? Why do they not form gap junctions from them (Ruan et al., 2020) and instead evolved and exclusively use the new connexins for functions that could equally be fulfilled by an innexin? We suggest that looking at the early chordate evolution may solve this puzzle. Chordates are comprised of three subphyla: the lancelets, the tunicates, and the vertebrates. The lancelets represent the most basal chordate lineage that diverged before the split between tunicates and vertebrates (Putnam et al., 2008). The vertebrates split into the jawless fish (lampreys), the most ancient vertebrate group (Smith et al., 2018), and the jawed vertebrates (Figure 3A). As our previous analysis revealed, the lancelets and the lampreys as well as most of the tunicates have only one innexin. In the jawed vertebrates, we found three innexins (called pannexins) in each single species. This is expected from the two whole-genome duplications at the early vertebrate lineage leading first to Pannexin-2 and afterward to Pannexin-1 and Pannexin-3 (Abascal and Zardoya, 2013; Fushiki et al., 2010). Only teleost fishes have a fourth pannexin generated by a whole-genome duplication event during the teleost evolution (Bond et al., 2012; Glasauer and Neuhauss, 2014). The limited genetic diversity is thus in strong contrast to the rich innexin diversity within the non-chordate phyla (Figures 1D and 3A; Abascal and Zardoya, 2013; Hasegawa and Turnbull, 2014; Moroz et al., 2014). Here, the large gene families and the co-expression of several innexins allow impressive numbers of combinations of innexins to form heteromeric and heterotypic gap junction channels leading thus to a rich diversity of functionally unique channels (Hall, 2017). With the loss of innexin diversity, this aspect of functional complexity is clearly missing in chordates. Furthermore, we found that each of the innexins of lancelets, tunicates, and lampreys has an extracellular NGS (Figures 2A–E ,–3C). Specifically, in lancelets the only innexin available contains an NGS in its EL1. Hence, lancelets, not only lost the diversity of innexins but retained one that might not even be capable of forming gap junctions. Our work and earlier studies Mikalsen et al., 2021; Slivko-Koltchik et al., 2019 found no connexin-like sequences in lancelets genomes and transcriptomes (Figure 3C). With connexins absent, lancelets could thus either have no gap junctions at all (see Baatrup, 1981; Lane et al., 1987; Soledad Ruiz and Anadón, 1989) or one formed from an innexin with an NGS. Both options, either the complete absence of regular gap junctions or the loss of all functional diversity among them are highly relevant for the evolution of connexins. These arose de novo just at this time of chordate evolution (Figure 3C) and rapidly developed into diverse gene families (Figure 3B) with up to 22 connexins in mammals and 46 connexins in bony fish (Mikalsen et al., 2021) that again enabled a huge diversity of functionally unique gap junctions.

Figure 3

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Based on our findings, we thus propose that a bottleneck effect at the origin of chordates might have been crucial for the evolution of the novel connexins (Figure 3D). In this evolutionary scenario, innexins were recruited as gap junction proteins in the last common ancestor of eumetazoa. While the innexins functionally diverge in ctenophores, cnidarians, and protostomes, the last common ancestor of the deuterostomes had lost all innexin diversity and thus the capacity to form functionally diverse gap junctions (Figure 3C). The strikingly high conservation of NGSs in lancelet, lamprey, and jawed vertebrate innexins (Figure 2C–H), their expression in every organ (Penuela et al., 2014a) and their association with a variety of diseases (Esseltine and Laird, 2016; Penuela et al., 2014b) suggest that the non-junctional innexin channels already served essential physiological functions in the last common ancestor of chordates and could not be converted into gap junctions. The loss of diversity in gap junctions is thus extreme, with just one, if any at all, functional gap junction. This finding and that of the strict conservation of the one remaining innexin could thus explain rather simply why the connexin family arose de novo and why it became the exclusive gap junction protein in all chordates although innexin-based gap junctions would have been fully capable to serve all functions (Baker and Macagno, 2014; Bao et al., 2007; Bhattacharya et al., 2019; Lane et al., 2018; Liu et al., 2016; Phelan and Starich, 2001; Skerrett and Williams, 2017; Welzel and Schuster, 2018; Yaksi and Wilson, 2010) as they do so successfully in the sophisticated nervous systems of invertebrates (Calabrese et al., 2016; Hall, 2017; Kristan et al., 2005; Marder et al., 2005; Otopalik et al., 2019).

All data generated or analysed during this study are included in the manuscript and in the supporting files. Source Data files have been provided for Figure 1 and 2.

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Author details

1. Georg Welzel

   Department of Animal Physiology, University of Bayreuth, Bayreuth, Germany

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft

   For correspondence

   georg.welzel@uni-bayreuth.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7017-604X

2. Stefan Schuster

   Department of Animal Physiology, University of Bayreuth, Bayreuth, Germany

   Contribution

   Conceptualization, Resources, Supervision, Validation, Writing - review and editing

   For correspondence

   stefan.schuster@uni-bayreuth.de

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0873-8996

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