The advent of multicellular organisms, some 800 million years ago, necessitated the development of mechanisms for cell-to-cell synchronization and for the spread of signals across increasingly large cell populations [168, 185]. Many structures and mechanisms have evolved to achieve such functions [4, 15]. Among these mechanisms, one which is prominent in both the invertebrate and the vertebrate world, across the entire phylogenetic scale, involves the transmembrane flux of large cytosolic and extracellular molecules [4, 15, 65, 66, 69–71, 121, 128, 129, 147, 154, 163]. These fluxes, in turn, are dependent on the formation of specific channels that in all animal classes are made by tetra-span integral membrane proteins [65, 66, 69–71, 121, 128, 129, 147, 154, 163] (Fig. 1). Fig. 1 Innexins, pannexins, and connexins form different types of gap junctional and “hemi-channels.” Invertebrates express many different innexins (purple; 25 isoforms in C. elegans) that may form either gap junction channels for cell-to-cell ... Keywords: Gap junctions, Connexons, Pannexons, Innexons, Membrane channels, Ca2+, ATP, Glutamate Three junctional protein families form membrane channels permeable to large molecules Early electrophysiological and electron microscopy studies converged in the realization that gap junctions, the membrane domains that concentrate intramembrane particles at sites of close membrane apposition, were the physical substrate of cell-to-cell communication in both invertebrate [59] and vertebrate tissues [139] (Fig. 1). The finding that similar drugs (the long-chain alcohols heptanol and octanol) and conditions (intracellular acidification) inhibited intercellular communication in both invertebrate and vertebrate systems [58, 85] was taken as further support that all gap junctions had a similar structure and function. Still, the different size of intramembrane particles, their different partition into the P- and the E-fracture faces of the cell membrane, and the different width of the gap space delineated by the two interacting membranes, suggested that the proteins making vertebrate gap junctions were different from those making the invertebrate structures [96]. These differences have functional consequences, as most elegantly demonstrated by co-culturing cell lines from different animal species. In these experiments, heterotypic coupling was shown between insect cells, as well as between different types of vertebrate cells, whereas virtually no coupling was observed between cells of phylogenetically distant species [48]. There is no longer any question that vertebrate gap junction channels are made by various combinations of different connexin proteins [66, 69–71]. The 20–21 isoforms of this family in rodents and man differ in size, but share a similar membrane topography. Thus, all connexins feature four transmembrane domains connected by two extracellular loops, each comprising three highly conserved Cys residues, a cytoplasmic loop, and both N and C termini in the cytosol (Fig. 2). The difference in size of the different connexin isoforms is essentially due to a different length of the cytoplasmic loop and/or the C terminus [39, 64, 65, 69–71, 109]. Current nomenclature (Table 1) designates connexin proteins as nCxZ, where n is the species (e.g., h, m, r for human, mouse, rat, respectively), and Z is the predicted molecular weight, in kDa. The genes encoding connexin proteins are named according to subgroups, in the order of discovery (e.g., Gjb1 encodes Cx32, Gjb2 encodes Cx26, Gja1 encodes Cx43, etc). Fig. 2 Membrane topography of the proteins forming gap junctions and “hemi-channels.” All invertebrate innexins, vertebrate ortholog pannexins, and nonhomologous vertebrate connexins feature four transmembrane domains connected by two extracellular ... Table 1 The family of human connexins Attempts to identify the proteins making invertebrate gap junctions initially revealed the proteins Ogre, Passover, Uncoordinated, and Shaking B in Drosophila and Caenorhabditis (hence the original OPUS acronym to name these proteins) with no primary sequence homology to connexins [128, 129, 195]. Since that time, more than 25 other junctional proteins revealing significant similarities have been identified in C. elegans, and it is clear that many other forms are expressed in other invertebrate species [75, 128, 129, 195] (Table 2). Collectively, these proteins have been named innexins to stress their invertebrate distribution and their role, analogous to that of connexins, in the formation of gap junctions (Fig. 1). Strikingly, innexins also share with connexins a similar structure and membrane topography (Fig. 2), even though the two sets of proteins have no homology in their primary amino acid sequence [75, 128, 129, 195]. Notably, connexins display three conserved Cys within each of the two extracellular loops, whereas innexins only carry two such residues. Thus, it is curious in retrospect that hydra development was reported to be blocked by an antibody prepared against Cx32 [53], that other antibodies have detected connexin-like proteins in anemone [112] and other marine invertebrates [5], and that junctional proteins isolated from hepatopancreas of crayfish and lobster were reported to have a sequence similar to that of rat liver connexins [51]. Table 2 A present view of the growing family of invertebrate innexins Sequencing of mammalian genomes has revealed a third family comprising only three genes that code for proteins with a primary sequence showing about 20% similarity to that of innexins [121, 154, 195]. On this basis, these proteins were thought to represent vertebrate homologs of the innexins, and were termed pannexins (Table 3) to encompass both invertebrate and vertebrate members [121, 154]. Like connexins and innexins, all three pannexins display N- and C-terminal domains within the cytoplasm, large extracellular and cytoplasmic loop domains, and four membrane spanning segments (Fig. 2). Like innexins but in contrast to connexins, pannexins contain two Cys residues in each extracellular loop [121, 154]. Furthermore, and in marked contrast with both innexins and connexins, pannexins display consensus sequences for glycosylation [13, 14, 125] (Fig. 2). The distribution of Pnx1 (Pnx1), the most studied form, is widespread and, in most types of cells and tissues, largely overlaps with that of connexins [17, 8]. While at least some phenotypes resulting from loss of specific connexin species are not compensated by pannexin changes [136, 146], suggesting a different function of the two protein families, in other cases, the effects of Pnx1 transfection mimicked that of Cx43, implicating a comparable/overlapping role of these two proteins [93]. Table 3 The family of human pannexins Connexins and innexins form cell-to-cell channels at gap junctional regions of the cell membrane Expression and deletion studies in a variety of systems have established that connexin hexamers, termed connexons, concentrate at gap junction domains of the cell membrane, where the intercellular space is reduced to a gap 2–3 nm wide. At these sites, the connexons of one cell align with, and strongly bind to the connexons of an adjacent cell, establishing a continuous intercellular hydrophilic pathway (Fig. 1) for the cell-to-cell exchange of multiple types of cytosolic molecules [64, 65, 69–71, 155]. The functional importance of this electrical and metabolic coupling is shown by a variety of striking and tissue-specific phenotypes that can be experimentally induced after overexpression or knock-out of individual connexin isoforms, as well as after the knock-in replacement of one isoform by another [92, 192]. It is further stressed by the identification of a number of diseases that are undoubtedly linked to connexin mutations [43, 57, 90, 94, 127, 143]. A variety of other diseases are thought to be due to altered amounts and/or function of these gap junction proteins [21, 108, 153]. Similarly, innexins oligomerize to form innexons that cluster at gap junctions of invertebrate cells (Fig. 1). Functional expression studies in paired Xenopus oocytes demonstrated that several, even though not all innexins also formed intercellular channels [8, 95, 166], and that at least some innexin mutations give rise to phenotypes expected for lack of gap junction-mediated intercellular communication [10, 25, 27, 37, 179]. In fact, it was on the basis of such dysfunctional phenotypes that the OPUS gene family was first identified. In contrast, and in spite of an initial report [18], pannexons appear unable to form sizable amounts of cell-to-cell channels under most conditions [76, 146, 147, 163] (Fig. 1). This lack of formation of functional gap junctions is likely due to the glycosylation of the extracellular loops of pannexins [13, 14, 125], which, as mentioned above, is not observed for either connexins or innexins. Still, forced expression of pannexins in paired Xenopus oocytes increased the conductance of the junctional cell membrane to current carrying ions in a way that can only be accounted for by the formation of pannexin cell-to-cell channels [13].