1.A.25 The Gap Junction-forming Innexin (Innexin) Family

Innexins comprise a large family of proteins that form intercellular gap junctional channels in invertebrates, but only a few have been functionally characterized. These junctions allow electrical coupling as well as the free flow of small molecules between cells. The C. elegans INX-3, but not a paralogue, EAT-5, induced electrical coupling between Xenopus oocyte pairs (Landesman et al., 1999). Voltage and pH gating of INX-3 channels is functionally similar to that of vertebrate connexin channels (TC# 1.A.24). Many paralogues of the innexin family are found in both C. elegans and D. melanogaster as well as other invertebrates, and these proteins are subject to differential developmental control in various body tissues. Innexins exhibit a 4 TMS topology. Homologues, called pannexins, have been identified in vertebrates (Hua et al., 2003; Yen and Saier, 2007). The LRRC8 family (TC# 1.A.25.3) is a member of the Pfam pannexin-like superfamily. The structures of LRRC8 proteins have been determined, and they resemble connexins (Deneka et al. 2018). C. elegans unpiared innexins form gap junctions that transport ions including K⁺ and small molecules such as ATP (Sangaletti et al. 2014).

Gap junctions are widespread in immature neuronal circuits. A transient network formed by the innexin gap-junction protein NSY-5 coordinates left-right asymmetry in the developing nervous system of C. elegans. NSY-5 forms hemichannels and intercellular gap-junction channels, consistent with a combination of cell-intrinsic and network functions (Chuang et al., 2007). In addition to making gap junctions, innexins also form non-junctional membrane channels with properties similar to those of pannexons (Bao et al., 2007).  N-terminal- elongated innexins can act as a plug to manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction from the intracellular to the extracellular compartment (Chen et al. 2016). The physiology of hemichannels and gap junctions, including ion blockage of hemichannels, voltage gating of gap junctions, and asymmetry and delay of electrical synaptic transmission have been discussed (Wang and Liu 2021).

Pannexins in vertebrates have been studied in some detail (Shestopalov and Panchin, 2008; Boyce et al. 2013). They can form nonjunctional transmembrane 'hemichannels' for transport of molecules of less than 1000 Da, or intercellular gap junctions. They transport Ca²⁺, ATP, inositol triphosphate, and other small molecules. They can be present in plasma, ER and golgi membranes. Pannexin1 can form homooligomeric channels and heterooligomeric channels with Pannexin2. They form hemichannels with greater ease than connexin subunits (Shestopalov and Panchin, 2008). Scemes (2011) summarized the published data on hemichannel formation by junctional proteins. Silverman et al. 2008 have showed that probenecid inhibited currents mediated by pannexin 1 channels in the same concentration range as observed for inhibition of transport processes. Probenecid did not affect channels formed by connexins. Thus, probenecid allows for discrimination between channels formed by connexins and pannexins. A large protein, Nucleoside-diphosphate-kinase of P. gingivalis is secreted from epithelial cells In the absence of a leader sequence through a Pannexin-1 interactome (Atanasova et al. 2016). Pannexin 1 regulates skeletal muscle regeneration by promoting bleb-based myoblast migration and fusion through a lipid based signaling mechanism (Suarez-Berumen et al. 2021).

The volume-reglated Anion Channel, VRAC, consists of the leucine-rich repeat-containing protein 8A with N-terminal pannexin-like domain, LRRC8A, together with other LRRC8 subunits (B, C, D and E). The first two TMSs of the 4 TMS LRRC8 proteins appear as DUF3733 in CDD (Abascal and Zardoya, 2012). The C-terminal soluble domain shows sequence similarity to the heme-binding protein Shv (9.A.63.1.1) and pollen-specific leucine-rich repeat extension-like proteins (3.A.20.1.1).  The volume-regulated anion channel, VRAC, has LRRC8A as a VRAC component. It forms heteromers with other LRRC8 membrane proteins (Voss et al. 2014). Genomic disruption of LRRC8A ablated VRAC currents. Cells with disruption of all five LRRC8 genes required LRRC8A cotransfection with other LRRC8 isoforms to reconstitute VRAC currents. The isoform combination determined the VRAC inactivation kinetics. Taurine flux and regulatory volume decrease also depended on LRRC8 proteins. Thus, VRAC defines a class of anion channels, suggests that VRAC is identical to the volume-sensitive organic osmolyte/anion channel VSOAC, and explains the heterogeneity of native VRAC currents (Voss et al. 2014).

Connexins participate in the generation of intercellular calcium waves, in which calcium-mediated signaling responses spread to contiguous cells through gap junction to transmit calcium signaling throughout the airway epithelium. Pannexins in the nasal mucosa contribute not only to ciliary beat modulation via ATP release, but also regulation of mucus blanket components via H₂O efflux. The synchronized roles of pannexin and connexin may allow effective mucociliary clearance in nasal mucosa (Ohbuchi and Suzuki 2018).

Using cryo-electron microscopy and X-ray crystallography, Deneka et al. 2018 determined the structure of a homomeric channel of the obligatory subunit LRRC8A (TC# 1.A.25.3.1). This protein conducts ions and has properties in common with endogenous heteromeric channels. Its modular structure consists of a transmembrane pore domain followed by a cytoplasmic leucine-rich repeat domain. The transmembrane domain, which is structurally related to connexins, is wide towards the cytoplasm but constricted on the outside by a structural unit that acts as a selectivity filter. An excess of basic residues in the filter and throughout the pore attracts anions by electrostatic interaction (Deneka et al. 2018).

The transport reaction catalyzed by innexin gap junctions is:

Small molecules (cell 1 cytoplasm)   Small molecules (cell 2 cytoplasm)

or for hemichannels:

Small molecules (cell cytoplasm)  Small molecules (out)