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)



This family belongs to the Leucine-rich Repeat-containing Domain (LRRD) Superfamily.

 

References:

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Examples:

TC#NameOrganismal TypeExample
1.A.25.1.1Invertebrate innexin, (gap junction protein), INX3 Invertebrates INX3 of C. elegans
 
1.A.25.1.10

Leech innexin, Inx2 (Kandarian et al. 2012; Firme et al. 2012)

Animals

Inx2 of Hirudo verbana

 
1.A.25.1.11

Duplicated innexin of 801 aas and 8 TMSs.

Innexin of Ascaris suum

 
1.A.25.1.12

Duplicated innexin protein of 813 aas and 8 TMSs.

Duplicated innexin of Trichinella spiralis (Trichina worm)

 
1.A.25.1.13

Innexin2, Inx2 of 359 aas and 4 TMSs. N-terminally elongated domains in innexins may act to plug or manipulate hemichannel closure and provide a mechanism connecting the effect of hemichannel closure directly to apoptotic signaling transduction (Chen et al. 2016).

Inx2 of Spodoptera litura (Asian cotton leafworm)

 
1.A.25.1.14

Innexin 2, Inx2; Prp33, of 367 aas and 4 TMSs. It is a structural components of gap junctions, and is involved in gap junctional communication between germline and somatic cells which is essential for normal oogenesis (Bohrmann and Zimmermann 2008). In embryonic epidermis, it is required for epithelial morphogenesis as well as for keyhole formation during early stages of proventriculus development in response to wg signaling (Bauer et al. 2004). In follicle cells, it promotes the formation of egg chambers, in part through regulation of shg and baz at the boundary between germ cells and follicle cells. In inner germarial sheath cells, it is required for survival of early germ cells and for cyst formation (Mukai et al. 2011). 

 

Inx2 or Anon-37B-2 of Drosophila melanogaster (Fruit fly)

 
1.A.25.1.15

Innexin-2 of 358 aas and 6 TMSs. Innexin-2 can participate in many physiological processes during the development of R. americana (Neves et al. 2021).

Innexin-2 of Rhynchosciara americana

 
1.A.25.1.2Invertebrate innexin, UNC-7 InvertebratesUNC-7 of C. elegans
 
1.A.25.1.3Invertebrate innexin, Ogre InvertebratesOgre of Drosophila melanogaster
 
1.A.25.1.4Invertebrate innexin, passover protein (shaking B locus) InvertebratesPassover protein of Drosophila melanogaster
 
1.A.25.1.5Invertebrate innexin, NSY-5 (INX-19) (Chuang et al., 2007) (establishes left-right neuronal asymmetry) (Oviedo and Levin, 2007)InvertebratesNSY-5 (INX-19) of Caenorhabditis elegans (NP_490983)
 
1.A.25.1.6

Innexin-14 (Protein Opu-14)

Worm

Inx-14 of Caenorhabditis elegans

 
1.A.25.1.7

Innexin-6 protein, Inx-6 or Opu-6, of 389 aas and 4 TMSs. A single INX-6 gap junction channel consists of 16 subunits, a hexadecamer, in contrast to chordate connexin channels, which consist of 12 subunits. The channel pore diameters at the cytoplasmic entrance and extracellular gap region are larger than those of connexin26 (Oshima et al. 2016). Nevertheless, the arrangements of the transmembrane helices and extracellular loops of the INX-6 monomer are highly similar to those of connexin-26 (Cx26). The INX-6 gap junction channel comprises hexadecameric subunits but reveals an N-terminal pore funnel consistent with Cx26. The helix-rich cytoplasmic loop and C-terminus are intercalated through an octameric hemichannel, forming a dome-like entrance that interacts with N-terminal loops in the pore (Oshima et al. 2016).

Worm

Inx-6 of Caenorhabditis elegans

 
1.A.25.1.8

Innexin Inx4 (Innexin-4) (Protein zero population growth)

Animals

Zpg of Drosophila melanogaster

 
1.A.25.1.9

Leech innexin, Inx6 (Kandarian et al. 2012; Firme et al. 2012)

Animals

Inx6 of Hirudo verbana

 
Examples:

TC#NameOrganismal TypeExample
1.A.25.2.1

Pannexin-1 (PANX1) has been reported to form functional, single membrane, cell surface channels (Penuela et al., 2007). Pannexin1 is part of the pore forming unit of the P2X(7) receptor death complex (Locovei et al., 2007). It can catalyze ATP release from cells (Huang and Roper, 2010) and promote ATP signalling in mice (Suadicani et al. 2012). It also promotes acetaminophen liver toxicity by allowing it to enter the cell (Maes et al. 2016).  Pannexin1 and pannexin2 channels show quaternary similarities to connexons but different oligomerization numbers (Ambrosi et al., 2010). Pannexin 1 constitutes the large conductance cation channel of cardiac myocytes (Kienitz et al., 2011). Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) underlie channel function in neurons and contribute to ischemic brain damage (Bargiotas et al., 2011). Single cysteines in the extracellular and transmembrane regions modulate pannexin 1 channel function (Bunse et al., 2011).  Spreading depression triggers migraine headaches by activating neuronal pannexin1 (panx1) channels  (Karatas et al. 2013).  The channel in the mouse orthologue opens upon apoptosis (Spagnol et al. 2014). Transports ATP out of the cell since L-carbenoxolone (a Panx1 channel blocker) inhibits ATP release from the nasal mucosa, but flufenamic acid (a connexin channel blocker) and gadolinium (a stretch-activated channel blocker) do not (Ohbuchi et al. 2014). CALHM1 (TC#1.N.1.1.1) and PANX1 both play roles in ATP release and downstream ciliary beat frequency modulation following a mechanical stimulus in airway epithelial cells (Workman et al. 2017). Pannexin1 may play a role in the pathogenesis of liver disease (Willebrords et al. 2018). Inhibition of pannexin1 channel opening may provide a novel approach for the treatment of drug (acetaminophen-induced)-induced hepatotoxicity (Maes et al. 2017). Pannexin-1 is necessary for capillary tube formation on Matrigel and for VEGF-C-induced invasion. It is highly expressed in HDLECs and is required for in vitro lymphangiogenesis (Boucher et al. 2018). cryo-EM structure of a pannexin 1 reveals unique motifs for ion selection and inhibition. The cryo-EM structure of a pannexin 1 revealed unique motifs for ion selection and inhibition (Michalski et al. 2020). In another study, Deng et al. 2020 obtained near-atomic-resolution structures of human and frog PANX1 determined by cryo-EM that revealed a heptameric channel architecture. Compatible with ATP permeation, the transmembrane pore and cytoplasmic vestibule were exceptionally wide. An extracellular tryptophan ring located at the outer pore created a constriction site, potentially functioning as a molecular sieve that restricts the sizes of permeable substrates. Pannexin 1 channels in renin-expressing cells influence renin secretion and homeostasis (DeLalio et al. 2020). Structures of human pannexin 1 have revealed ion pathways and mechanism of gating (Ruan et al. 2020). PANX1 is critical for functions such as blood pressure regulation, apoptotic cell clearance and human oocyte development. Ruan et al. 2020 presented several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 Å, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. A gating mechanism was revealed that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. A carbenoxolone (a channel blocker)-binding site is embraced by W74 in the extracellular entrance. A gap-junction-like structure was observed as expected (Yen and Saier 2007; Chou et al. 2017). Navis et al. 2020 provided a review of the literature on Panx1 structural biology and known pharmacological agents that target it. The R217H mutation perturbs the conformational flexibility of the C-terminus, leading to channel dysfunction (Purohit and Bera 2021). Panx1 plays decisive roles in multiple physiological and pathological settings, including oxygen delivery to tissues, mucociliary clearance in airways, sepsis, neuropathic pain, and epilepsy. It exerts some of these roles in the context of purinergic signaling by providing a transmembrane pathway for ATP, but Panx1 can also act as a highly selective membrane channel for chloride ions without ATP permeability (Mim et al. 2021). 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).  Pannexin-1 activation by phosphorylation is crucial for platelet aggregation and thrombus formation (Metz and Elvers 2022). Data suggest that in response to hypotonic stress, the intact rat lens is capable of releasing ATP. This seems to be mediated via the opening of pannexin channels in a specific zone of the outer cortex of the lens (Suzuki-Kerr et al. 2022).

Vertebrates

Pannexin-1 of Homo sapiens

 
1.A.25.2.2

Pannexin1 and pannexin2 channels show quaternary similarities to connexons but different oligomerization numbers (Ambrosi et al., 2010). Pannexin 1 (Px1, Panx1) and pannexin 2 (Px2, Panx2) underlie channel function in neurons and contribute to ischemic brain damage (Bargiotas et al., 2011).

Vertebrates

Pannexin-2 of Homo sapiens (Q96RD6)

 
1.A.25.2.3

Pannexin-3, PANX3, of 392 aas and 5 TMSs, is reported to form functional, single membrane, cell surface channels (Penuela et al., 2007)). It functions as an ER Ca2+ channel, hemichannel, and gap junction to promote osteoblast differentiation (Ishikawa et al., 2011). However, Panx3 expression in osteoblasts is not required for postnatal bone remodeling (Yorgan et al. 2019).

Vertebrates

Pannexin-3 of Homo sapiens (gi16418453)

 
1.A.25.2.4

Pannexin 1a, Panx1a, of 417 aas and 4 TMSs, an ATP channel.  Aromatic-aromatic interaction involving Trp123 and Tyr205 in TMSs 2 and 3, respectively are important for the assembly and trafficking of the Zebrafish Panx1a membrane channel (Timonina et al. 2020).

Panx1a of Danio rerio (Zebrafish) (Brachydanio rerio)

 
Examples:

TC#NameOrganismal TypeExample
1.A.25.3.1

The volume-regulated Anion Channel, VRAC, or volume-sensitive outward rectifying anion channel, VSOR. It is also called the SWELL1 protein. It consists of the leucine-rich repeat-containing protein 8A, with an 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, 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, suggesting 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).  Point mutations in two amino-acyl residues (Lys98 and Asp100 in LRRC8A and equivalent residues in LRRC8C and -E) upon charge reversal, alter the kinetics and voltage-dependence of inactivation (Ullrich et al. 2016). Using cryo-electron microscopy and X-ray crystallography, Deneka et al. 2018 and Kasuya et al. 2018  determined the structures of a homomeric channel of the obligatory subunit LRRC8A. 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 structure shows a hexameric assembly, and the transmembrane region features a topology similar to gap junction channels. The LRR region, with 15 leucine-rich repeats, forms a long, twisted arc. The channel pore is located along the central axis and constricted on the extracellular side, where highly conserved polar and charged residues at the tip of the extracellular helix contribute to the permeability to anions and other osmolytes. Two structural populations were identified, corresponding to compact and relaxed conformations. Comparing the two conformations suggests that the LRR region is flexible and mobile with rigid-body motions, which might be implicated in structural transitions on pore opening (Kasuya et al. 2018). VRAC is inhibited by Tamoxifen and Mefloquine (Lee et al. 2017). The intracellular loop connecting TMSs 2 and 3 of LRRC8A and the first extracellular loop connecting transmembrane domains 1 and 2 of LRRC8C, LRRC8D, or LRRC8E are essential for VRAC activity (Yamada and Strange 2018). The N termini of the LRRC8 subunits may line the cytoplasmic portion of the VRAC pore, possibly by folding back into the ion permeation pathway (Zhou et al. 2018).  On the adipocyte plasma membrane, the SWELL1-/LRRC8 channel complex activates in response to increases in adipocyte volume in the context of obesity. SWELL1 is required for insulin-PI3K-AKT2 signalling to regulate adipocyte growth and systemic glycaemia (Gunasekar et al. 2019).

Animals

The VRAC channel consisting of LRRC8A together with one or two of the subunits, LRRC8B, LRRC8C, LRRC8D and/or LRRC8E of Homo sapiens (Q8IWT6)

 
1.A.25.3.2

The LRRC8B homologue of 480 aas.  Its cytoplasmic domains are regulators of channel activity by allosteric mechanisms (Deneka et al. 2021).

Animals

LRRC8B of Ciona intestinalis (Transparent sea squirt) (Ascidia intestinalis)

 
1.A.25.3.3

Uncharacterized protein of 467 aas

Animals

UP of Branchiostoma floridae (Florida lancelet) (Amphioxus)

 
1.A.25.3.4

Uncharacterized ADP-binding protein of 1311 aas and 2 TMSs.  May be involved in defense responses.

Plants

UP of Oryza sativa

 
1.A.25.3.5

Volume-regulated anion channel subunit LRRC8B-like protein of 666 aas and 4 TMSs.

LRRC8B of Mizuhopecten yessoensis

 
1.A.25.3.6

Uncharacterized protein of 610 aas and 4 TMSs.  It is of the Pannexin-like Superfamily.

UP of Thelohanellus kitauei

 
1.A.25.3.7

Leucine-rich repeat-containing protein 59, LRRC59, of 307 aas and 1 C-terminal TMS. It is a tail-anchored protein  that localizes to the ER and the nuclear envelope and is required for nuclear import of FGF1. It might regulate nuclear import by facilitating interaction with the nuclear import machinery and by transporting cytosolic FGF1 to, and possibly through, the nuclear pore (TC# 1.I.1) (Zhen et al. 2012). LRRC59 is post-translationally inserted into ER-derived membranes, possibly by diffusion (Blenski and Kehlenbach 2019).


LRRC59 of Homo sapiens