A structural and functional comparison of gap junction channels composed of connexins and innexins

Abstract

Methods such as electron microscopy and electrophysiology led to the understanding that gap junctions were dense arrays of channels connecting the intracellular environments within almost all animal tissues. The characteristics of gap junctions were remarkably similar in preparations from phylogenetically diverse animals such as cnidarians and chordates. Although few studies directly compared them, minor differences were noted between gap junctions of vertebrates and invertebrates. For instance, a slightly wider gap was noted between cells of invertebrates and the spacing between invertebrate channels was generally greater. Connexins were identified as the structural component of vertebrate junctions in the 1980s and innexins as the structural component of pre-chordate junctions in the 1990s. Despite a lack of similarity in gene sequence, connexins and innexins are remarkably similar. Innexins and connexins have the same membrane topology and form intercellular channels that play a variety of tissue- and temporally specific roles. Both protein types oligomerize to form large aqueous channels that allow the passage of ions and small metabolites and are regulated by factors such as pH, calcium, and voltage. Much more is currently known about the structure, function, and structure-function relationships of connexins. However, the innexin field is expanding. Greater knowledge of innexin channels will permit more detailed comparisons with their connexin-based counterparts, and provide insight into the ubiquitous yet specific roles of gap junctions. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 522-547, 2017.

Keywords: connexin; function; gap junction; innexin; structure.

Figure 1

Comparison of gap junctions composed of connexins and innexins focusing on structure and sequence. Representative images are not adjusted to scale. Intercellular Gap: The gap between cells is slightly larger in invertebrate preparations. (Left) Section of mouse heart gap junction treated en bloc with lanthanum and stained with uranyl acetate. X 200,000. Intercellular gap ≈ 18 Å. Revel and Karnovsky, 1967. (Right) Section of a gap junction between muscle cells of Hydra treated en bloc with lanthanum and stained with lead citrate. X 144,000. Intercellular gap ≈ 30 Å. Hand and Gobel, 1972. Channel Spacing: Channels are spaced farther apart in invertebrate preparations. (Left) Electron micrograph of an isolated gap junction plaque from mouse liver. Center of connexons are marked. Caspar et al., 1977. (Right) Electron micrograph of gap junction plaque from Sf9 cells expressing c. elegans INX‐6 negatively stained with uranyl acetate. Oshima et al., 2013. Oligomerization: Connexin‐based channels are hexameric while innexin‐based channels are octameric. (Left) Sixfold rotationally filtered image of a connexon purified from rat liver (Stauffer et al., 1991). (Right) Projection map of a c. elegans INX‐6 deletion mutant expressed in Sf9 cells, solubilized, purified and negatively stained. Membrane Topology: The membrane topology of proteins that constitute gap junctions. Both connexins (left) and innexins (right) have four membrane‐spanning domains, two extracellular loops and cytoplasmic amino and carboxyl termini. Each of the two extracellular loop domains includes three conserved cysteines in connexins and two conserved cysteines in innexins. Gene Structure: Illustration summarizing gene structure of connexins (left) and innexins (right). Light grey denotes coding region bracketed by small sections of untranslated sequence (dark gray). Representative Introns are noted in cyan. Connexin genes do not contain introns within the coding region whereas innexin genes contain introns.

Figure 2

Comparison of gap junctions composed of connexins and innexins focusing on function. Permeability: Space‐filling models of a glycopeptide used to establish permeation‐limiting dimensions of gap junctions. Gap junctions of invertebrates were permeable to the larger version of the molecule (Left) while only the smaller version permeated mammalian junctions (Right) (Schwarzmann et al., 1981). Inhibited Long‐Chain Alcohols: Representative structure of 1‐octanol, a compound that inhibis gap junctions composed of connexins (Left) and innexins (Right). Long‐chain alcohols (Scemes et al., 2009), carbenoxlone (Bao et al., 2007) and arachidonic acid (Weingart and Bukauskas, 1998) also inhibit gap junctions from vertebrate and invertebrate tissue. Regulated by pH and Calcium: Gap junctions of vertebrates (Left) and invertebrates (Right) are known to be regulated by pH and calcium. Cytoplasmic acidification induces channel closure via conformational changes in cytoplasmic domains (Morley et al., 1996; Wang and Peracchia, 1998). Innexin‐based channels are also sensitive to pH (Giaume et al., 1980) but the mechanism is not understood. Coupling in vertebrate and invertebrate preparations is reduced by calcium ions (Lowenstein et al., 1967; Délèze and Loewenstein, 1976). Hemichannels: Some connexins (Left) and innexins (Right) function physiologically as half‐channels (hemichannels) mediating transport across the plasma membrane, a feature that does not seem to limit their ability to function as intercellular channels (reviewed by Ebihara, 2003; Bao et al., 2007). Vm‐Sensitivity: Intact gap junction channels may exhibit sensitivity to Vm as demonstrated for connexins (Left; Barrio et al., 1997)) and innexins (Right; DePriest et al., 2011). Vj‐Sensitivity: Under voltage clamp, junctional currents demonstrate unique properties in terms of time‐ and voltage‐dependence. Currents were recorded from oocytes expressing Cx32 [left top] and Cx26 [left bottom] (Oh et al., 1999) or Unc‐7L [right top] and Unc‐9 [right bottom] (Starich et al., 2009). Heterotypic and Heteromeric: Cartoon representing gap junction channels composed of different isoforms of connexins (Left) and Innexins (Right). Most native channels are likely to involve dynamic and complex interactions between protein isoforms (Koval et al., 2014). Rectification: Heterotypic combinations of Cx26/Cx32 (Left) and ShakB N + 16/ShakB L (Right) produce channels with properties of electrical rectification (Oh et al., 1999; Phelan et al., 2008).

Figure 3

Comparison of gap junction channels composed of connexins and innexins focusing on channel features and structure‐function analysis. Channel Features and Dimensions: Surface view structures of gap junction channels composed of Cx26 (Left) and INX‐6ΔN (Right). Scales alongside the channels indicate length of transmembrane (M), intracellular© and extracellular (G/gap) regions. The Cx26 channel is approximately 155Å in length with an outside diameter of 92Å (Maeda et al., 2009). The INX‐6 channel is approximately 240Å in length with an outside diameter of 115Å (Oshima et al., 2016). Images from Oshima et al., 2016. TM Domain Packing: Helical net plots showing residues where tryptophan substitution rendered channels nonfunctional during tryptophan scanning in Cx32 (Left) and ShakBL (Right). Cx32 TM1 was highly sensitive to tryptophan substitution indicative of tight packing (Brennan et al., 2015) whereas only a few sites were sensitive to tryptophan substitution in TM1 of ShakBL (DePriest et al., 2011). These results are consistent with structural data indicating that innexin‐based channels are larger and involve more subunits than their connexin‐based counterparts (Oshima et al., 2016). Amino Terminus: Membrane topology highlighting importance of the amino terminus (NT). In both connexin‐(Left) and innexin‐based channels (Right) the amino terminus is required for function and plays an important role in Vj‐dependent gating. In connexins, the NT is 22–23 amino acids in length including a short α‐helix. The NT likely folds into the pore, lining part of the conduction pathway, consistent with its involvement in permeability, conductance and Vj‐gating (reviewed by Beyer et al., 2012). Innexins also appear to require an NT which also plays a role in Vj‐gating and rectification (Marks and Skerrett, 2014). Coincidental Similarities: Two similarities were noted in structure‐function studies, aberrant hemichannel behavior (arrows showing transport across cell membrane) and a “reverse‐gating” phenotype. Currents recorded from “reverse‐gating” channels M34S in Cx32 (Left; Skerrett et al., 1999) and S39W in ShakBL (Right; DePriest et al., 2011) are shown. A characteristic of “reverse‐gating” mutants is that they form channels that remain predominantly closed (or in a low conductance state) at Vj = 0 mV but open with higher Vj. Currents are often only apparent in heterotypic pairings with wildtype.