1.A.24 The Gap Junction-forming Connexin (Connexin) Family

Gap junctions, found in the plasma membranes of vertebrate animal cells, consist of clusters of closely packed pairs of transmembrane channels, the connexons, through which small molecules diffuse between neighboring cells (Zhou and Jiang 2014). The connexons consist of homo- or heterohexameric arrays of connexins, and the connexon in one plasma membrane docks end-to-end with a connexon in the membrane of a closely opposed cell. The hemichannel is made of six connexin subunits (Kar et al., 2012). The properties and possible functions of unpaired connexin and pannexin hemichannels and the implications this has for a variety of events, such as cell death, glutamate release, oxidative stress, cortical spreading depression, that occur during an ischemic insult and may affect its outcome, have been reviewed (Bargiotas et al. 2009). The two connexons are docked by interdigitated, anti-parallel beta strands across the extracellular gap. The second extracellular loop guides selectivity in docking between connexons formed by different isoforms (Kovacs et al. 2007). There is considerably more sequence variability of the N-terminal portion of E2; possibly this region dictates connexon coupling.  Structure/function relationships for connexins have been reviewed (Beyer and Berthoud 2017). The roles of connexin hemichannels in normal cochlear function and in promoting hearing loss have been reviewed (Verselis 2017).

Over 15 connexin subunit isoforms are known. They vary in size between about 25 kDa and 60 kDa. They have four putative transmembrane α-helical spanners, and direct experimental evidence favors the α-helical folding of at least two of these TMSs. Connexins are similar in sequence and are designated connexins α1-8 and β1-6. Low resolution structural data are available for a gap junction membrane channel. A dodecameric channel is formed by the end-to-end docking of two hexamers, each displaying 24 TMSs (4 α-helical TMSs per connexin subunit) (Bosco et al., 2011). Gap junctional channels are parts of multiprotein complexes (Hervé et al., 2011).  Regulation of cardiovascular connexins have been reviewed (Meens et al. 2013). The proteins interacting with Cx43, the most prevalent connexin (TC# 1.A.24.1.1; the rat and human orthologs are 98 % identical), include: c-Src (TC#1.A.23.1.12; P12931), ZO-1 (8.A.24.1.9; Q07157), drebrin (TC#; DBN1; Q16643), CIP85 (TC# 8.A.87.1.5; Q96HU1) and CCN3 (8.A.87.1.6; P48745), as well as feedback between gap junctions, adherens junctions (N-cadherin and catenins) and the cytoskeleton (microtubules and actin) (Giepmans 2006).

Connexin channels have been reconstituted in unilamellar phospholipid vesicles from purified rat liver connexin 43. The vesicles were shown to be permeable to sucrose and the dye, lucifer yellow, and channel activity was reversibly inhibited by phosphorylation of connexin 43 by mitogen-activated protein (MAP) kinase. Other kinases may also effect inhibition. Gating of connexin 43 channels may therefore be regulated by phosphorylation of the connexin subunit in vivo. However, the cytoplasmic tails differ considerably in the size and amino acid sequence for different connexins and are predicted to be involved in the channel open and closed conformations. A ball and chain model for hemichannel conformational changes has been proposed for some connexins (e.g., Cx43) with large cytoplasmic tails (Liu et al., 2006). The tail folds into a ball or 'gating particle' and binds to the cytoplasmic loop domain, leading to channel closure (Liu et al., 2006).

Different connexins may exhibit differing specificities for solutes. For example, adenosine passed about 12-fold better through channels formed by Cx32 while AMP and ADP passed about 8-fold better, and ATP greater than 300-fold better, through channels formed by Cx43. Thus, addition of phosphate to adenosine appears to shift its relative permeability from channels formed by Cx32 to channels formed by Cx43. This may have functional consequence because the energy status of a cell could be controlled via connexin expression and channel formation (Goldberg et al., 2002).

There are about 20 isoforms of connexin proteins, each forming channels with distinct channel properties (Ayad et al., 2006). Moreover, connexins can form both homomeric and heteromeric connexin channels. Two homomeric channels may have different permeability properties that differ from those of the heteromeric channels including both proteins (see 1.A.24.1.3; Ayad et al., 2006). Connexin23 has only 4 conserved cysteines in the extracellular domain, but they still form hemichannels (Iovine et al., 2008)  A robust and updated classification of the human 4 TMS protein complement has appeared (Attwood et al. 2016). The connexin gene family is under extensive regulation at the transcriptional and post-transcriptional levels, and they undergoes numerous modifications at the protein level, including phosphorylation, which ultimately affects their trafficking, stability, and function (Aasen et al. 2018).

Deletion or mutation of the various connexin isoforms produces distinctive phenotypes and pathologies. This observation reflects (1) the different molecular specificities, (2) the different relative magnitudes of transport rates of various compounds via these channels, and (3) the regulatory properties via these dissimilar channels.  Genetic diseases indicate that the normal function of CNS myelin depends on connexin32 (Cx32) and Cx47, gap junction (GJ) proteins expressed by oligodendrocytes. GJs couple oligodendrocytes to themselves (O/O channels), astrocytes to themselves (A/A channels), and oligodendrocytes to astrocytes (O/A channels). Astrocytes and oligodendrocytes express different connexins. Cx47/Cx43 and Cx32/Cx30 efficiently form functional channels, but neither Cx47 nor Cx43 formed channels with Cx30 or Cx32 (Orthoann-Murphy et al., 2007). Cx47/Cx43 and Cx32/Cx30 channels have distinct properties and permeabilities. Cx47 mutants that cause Pelizaeus-Merzbacher-like disease do not efficiently form functional channels with Cx43, indicating that disrupted Cx47/Cx43 channels cause this disease.  The mutations in connexins that give rise to disease have been summarized and discussed (Pfenniger et al. 2011).  While mutations in Cx43 are mostly linked to oculodentodigital dysplasia, Cx47 mutations are associated with Pelizaeus-Merzbacher-like disease and lymphedema. Cx40 mutations are principally linked to atrial fibrillation. Mutations in Cx37 have not yet been described, but polymorphisms in the Cx37 gene have been implicated in the development of arterial disease (Molica et al. 2014).

Maeda et al. (2009) have reported the crystal structure of the gap junction channel formed by human connexin 26 (Cx26, also known as GJB2) at 3.5 Å resolution. The density map showed the two membrane-spanning hemichannels and the arrangement of the four transmembrane helices of the six protomers forming each hemichannel. The hemichannels feature a postively charged cytoplasmic entrance, a funnel, a negatively charged transmembrane pathway, and an extracellular cavity. The pore is narrowed at the funnel, which is formed by the six amino-terminal helices lining the wall of the channel, which thus determines the molecular size restriction at the channel entrance. The structure of the Cx26 gap junction channel also has implications for the gating of the channel by the transjunctional voltage (Nakagawa et al., 2010). The N-terminal half of connexin 46 appears to contain the core elements of the pore and voltage gates (Kronengold et al., 2012). 

Research has revealed a multilevel platform via which connexins (Cxs) and pannexins (Panxs) can influence the following cellular functions within a tissue: (1) Cx gap junctional channels (GJCs) enable direct cell-cell communication of small molecules, (2) Cx hemichannels and Panx channels can contribute to autocrine/paracrine signaling pathways, and (3) different structural domains of these proteins allow for channel-independent functions, such as cell-cell adhesion, interactions with the cytoskeleton, and the activation of intracellular signaling pathways. Decrock et al. 2015 discuss their multifaceted contributions to brain development and specific processes in the NGVU, including synaptic transmission and plasticity, glial signaling, vasomotor control, and blood-brain barrier integrity in the mature CNS. Connectosomes, cell-derived lipid vesicles that contain functional gap junction channels and encapsulate molecular cargos, have been used to deliver cargos such as drugs into the cytoplasm of a cell (Gadok et al. 2016).

Connexins (Cx) contain both highly ordered domains (i.e., 4 transmembrane domains) and primarily unstructured regions (i.e., N- and C-terminal domains). The C-terminal domains vary in length and amino acid composition from the shortest on Cx26 to the longest on Cx43. With the exception of Cx26, the C-terminal domains contain multiple sites for posttranslational modification (PTM) including serines (S), threonines (T), and tyrosines (Y) for phosphorylation as well as cysteines (C) for S-nitrosylation. These PTMs are critical for regulating cellular localization, protein-protein interactions, and channel functionality (Lohman et al. 2016).

Fatty acids (FAs) have effects on connexin- and pannexin-based channels. FAs regulate diverse cellular functions, including the activities of connexin (Cx) and Panx channels which form hexameric hemichannels (HCs), which assemble into dodecameric gap junction channels (GJCs).  It has been shown that FAs decrease GJC-mediated cell-cell communication. Changes in GJCs mediated by FAs have been associated with post-translational modifications (e.g., phosphorylation), and seem to be directly related to chemical properties of FAs (Puebla et al. 2017). 

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).

Gadok et al. 2016 have developed 'connectosomes', cell-derived lipid vesicles that contain functional gap junction channels and encapsulate molecular cargos. They showed that these vesicles form gap junctions with cells, opening a direct and efficient route for the delivery of molecular cargo to the cellular cytoplasm. Specifically, they demonstrated that using gap junctions to deliver doxorubicin reduces the therapeutically effective dose of the drug by more than an order of magnitude (Gadok et al. 2016).  Single-domain antibodies on connectosomes allows gap junction-mediated drug targetting to specific cell types (Gadok et al. 2018).

The transport reaction catalyzed by connexin gap junctions is:

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

Small molecules include small proteins, cyclic nucleotides, chemotherapeutics and small RNAs.



This family belongs to the Tetraspan Junctional Complex Protein (4JC) Superfamily.

 

References:

Aasen, T., S. Johnstone, L. Vidal-Brime, K.S. Lynn, and M. Koval. (2018). Connexins: Synthesis, Post-Translational Modifications, and Trafficking in Health and Disease. Int J Mol Sci 19:.

Alstrom JS., Hansen DB., Nielsen MS. and MacAulay N. (2015). Isoform-specific phosphorylation-dependent regulation of connexin hemichannels. J Neurophysiol. 114(5):3014-22.

Attwood, M.M., A. Krishnan, V. Pivotti, S. Yazdi, M.S. Almén, and H.B. Schiöth. (2016). Topology based identification and comprehensive classification of four-transmembrane helix containing proteins (4TMs) in the human genome. BMC Genomics 17: 268.

Ayad, W.A., D. Locke, I.V. Koreen, and A.L. Harris. (2006). Heteromeric, but not homomeric, connexin channels are selectively permeable to inositol phosphates. J. Biol. Chem. 281: 16727-16739.

Banerjee, D., S. Das, S.A. Molina, D. Madgwick, M.R. Katz, S. Jena, L.K. Bossmann, D. Pal, and D.J. Takemoto. (2011). Investigation of the reciprocal relationship between the expression of two gap junction connexin proteins, connexin46 and connexin43. J. Biol. Chem. 286: 24519-24533.

Bargiotas, P., H. Monyer, and M. Schwaninger. (2009). Hemichannels in cerebral ischemia. Curr Mol Med 9: 186-194.

Bevans, C.G., M. Kordel, S.K. Rhee, and A.L. Harris. (1998). Isoform composition of connexin channels determines selectivity among second messengers and uncharged molecules. J. Biol. Chem. 273: 2808-2816.

Beyer, E.C. and V.M. Berthoud. (2017). Gap junction structure: unraveled, but not fully revealed. F1000Res 6: 568.

Beyer, E.C., D.L. Paul, and D.A. Goodenough. (1987). Connexin43: a protein from rat heart homologous to a gap junction protein from liver. J. Cell Biol. 105: 2621-2629.

Bosco, D., J.A. Haefliger, and P. Meda. (2011). Connexins: key mediators of endocrine function. Physiol. Rev. 91: 1393-1445.

Brennan MJ., Karcz J., Vaughn NR., Woolwine-Cunningham Y., DePriest AD., Escalona Y., Perez-Acle T. and Skerrett IM. (2015). Tryptophan Scanning Reveals Dense Packing of Connexin Transmembrane Domains in Gap Junction Channels Composed of Connexin32. J Biol Chem. 290(28):17074-84.

Cascella, R., C. Strafella, S. Gambardella, G. Longo, P. Borgiani, F. Sangiuolo, G. Novelli, and E. Giardina. (2016). Two molecular assays for the rapid and inexpensive detection of GJB2 and GJB6 mutations. Electrophoresis 37: 860-864.

Coelho, J.P.L., M. Stahl, N. Bloemeke, K. Meighen-Berger, C.P. Alvira, Z.R. Zhang, S.A. Sieber, and M.J. Feige. (2019). A network of chaperones prevents and detects failures in membrane protein lipid bilayer integration. Nat Commun 10: 672.

Da, Y., W. Wang, Z. Liu, H. Chen, L. Di, L. Previch, and Z. Chen. (2016). Aberrant trafficking of a Leu89Pro connexin32 mutant associated with X-linked dominant Charcot-Marie-Tooth disease. Neurol Res 38: 897-902.

Decrock, E., M. De Bock, N. Wang, G. Bultynck, C. Giaume, C.C. Naus, C.R. Green, and L. Leybaert. (2015). Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology? Cell Mol Life Sci 72: 2823-2851.

Derosa, A.M., C.H. Xia, X. Gong, and T.W. White. (2007). The cataract-inducing S50P mutation in Cx50 dominantly alters the channel gating of wild-type lens connexins. J. Cell. Sci. 120:4107-4116.

Ek Vitorín, J.F., T.K. Pontifex, and J.M. Burt. (2016). Determinants of Cx43 Channel Gating and Permeation: The Amino Terminus. Biophys. J. 110: 127-140.

Gabriel, L.A., R. Sachdeva, A. Marcotty, E.J. Rockwood, and E.I. Traboulsi. (2011). Oculodentodigital dysplasia: new ocular findings and a novel connexin 43 mutation. Arch Ophthalmol 129: 781-784.

Gadok, A.K., C. Zhao, A.I. Meriwether, S. Ferrati, T.G. Rowley, J. Zoldan, H.D.C. Smyth, and J.C. Stachowiak. (2018). The Display of Single-Domain Antibodies on the Surfaces of Connectosomes Enables Gap Junction-Mediated Drug Delivery to Specific Cell Populations. Biochemistry 57: 81-90.

Gadok, A.K., D.J. Busch, S. Ferrati, B. Li, H.D. Smyth, and J.C. Stachowiak. (2016). Connectosomes for Direct Molecular Delivery to the Cellular Cytoplasm. J. Am. Chem. Soc. 138: 12833-12840.

Giepmans, B.N. (2006). Role of connexin43-interacting proteins at gap junctions. Adv Cardiol 42: 41-56.

Goldberg, G.S., A.P. Moreno, and P.D. Lampe. (2002). Gap junctions between cells expressing connexon 43 or 32 show inverse permselectivity to adenosine and ATP. J. Biol. Chem. 277: 36725-36730.

Grek, C.L., J.M. Rhett, J.S. Bruce, G.S. Ghatnekar, and E.S. Yeh. (2016). Connexin 43, breast cancer tumor suppressor: Missed connections? Cancer Lett 374: 117-126.

Hervé, J.C., M. Derangeon, D. Sarrouilhe, B.N. Giepmans, and N. Bourmeyster. (2012). Gap junctional channels are parts of multiprotein complexes. Biochim. Biophys. Acta. 1818: 1844-1865.

Hervé, J.C., P. Phelan, R. Bruzzone, and T.W. White. (2005). Connexins, innexins and pannexins: bridging the communication gap. Biochim. Biophys. Acta. 1719: 3-5.

Hong, H.M., J.J. Yang, C.C. Su, J.Y. Chang, T.C. Li, and S.Y. Li. (2010). A novel mutation in the connexin 29 gene may contribute to nonsyndromic hearing loss. Hum Genet 127: 191-199.

Hua, V.B., A.B. Chang, J.H. Tchieu, P.A. Nielsen, and M.H. Saier, Jr. (2003). Sequence and phylogenetic analysis of 4 TMS junctional proteins: Connexins, innexins, claudins and occludins. J. Mem. Biol. 194: 59-76.

Iossa, S., E. Marciano, and A. Franzé. (2011). GJB2 Gene Mutations in Syndromic Skin Diseases with Sensorineural Hearing Loss. Curr Genomics 12: 475-785.

Iovine, M.K., A.M. Gumpert, M.M. Falk, and T.C. Mendelson. (2008). Cx23, a connexin with only four extracellular-loop cysteines, forms functional gap junction channels and hemichannels. FEBS Lett. 582: 165-170.

Jara O., Acuna R., Garcia IE., Maripillan J., Figueroa V., Saez JC., Araya-Secchi R., Lagos CF., Perez-Acle T., Berthoud VM., Beyer EC. and Martinez AD. (2012). Critical role of the first transmembrane domain of Cx26 in regulating oligomerization and function. Mol Biol Cell. 23(17):3299-311.

Kang, J., N. Kang, D. Lovatt, A. Torres, Z. Zhao, J. Lin, and M. Nedergaard. (2008). Connexin 43 hemichannels are permeable to ATP. J. Neurosci. 28: 4702-4711.

Kar, R., N. Batra, M.A. Riquelme, and J.X. Jiang. (2012). Biological role of connexin intercellular channels and hemichannels. Arch Biochem Biophys 524: 2-15.

Katoch, P., S. Mitra, A. Ray, L. Kelsey, B.J. Roberts, J.K. Wahl, 3rd, K.R. Johnson, and P.P. Mehta. (2015). The carboxyl tail of connexin32 regulates gap junction assembly in human prostate and pancreatic cancer cells. J. Biol. Chem. 290: 4647-4662.

Kim, D.Y., Y. Kam, S.K. Koo, and C.O. Joe. (1998). Gating connexin 43 channels reconstituted in lipid vesicles by mitogen-activated protein kinase phosphorylation. J. Biol. Chem. 274: 5581-5587.

Kim, I.S., P. Ganesan, and D.K. Choi. (2016). Cx43 Mediates Resistance against MPP⁺-Induced Apoptosis in SH-SY5Y Neuroblastoma Cells via Modulating the Mitochondrial Apoptosis Pathway. Int J Mol Sci 17:.

Kopanic, J.L., B. Schlingmann, M. Koval, A.F. Lau, P.L. Sorgen, and V.F. Su. (2015). Degradation of gap junction connexins is regulated by the interaction with Cx43-interacting protein of 75 kDa (CIP75). Biochem. J. 466: 571-585.

Kovacs, J.A., K.A. Baker, G.A. Altenberg, R. Abagyan, and M. Yeager. (2007). Molecular modeling and mutagenesis of gap junction channels. Prog Biophys Mol Biol 94: 15-28.

Kronengold, J., M. Srinivas, and V.K. Verselis. (2012). The N-terminal half of the connexin protein contains the core elements of the pore and voltage gates. J. Membr. Biol. 245: 453-463.

Kuo, D.S., J.T. Sokol, P.J. Minogue, V.M. Berthoud, A.M. Slavotinek, E.C. Beyer, and D.B. Gould. (2017). Characterization of a variant of gap junction protein α8 identified in a family with hereditary cataract. PLoS One 12: e0183438.

Kyle JW., Berthoud VM., Kurutz J., Minogue PJ., Greenspan M., Hanck DA. and Beyer EC. (2009). The N terminus of connexin37 contains an alpha-helix that is required for channel function. J Biol Chem. 284(30):20418-27.

Leithe, E. and E. Rivedal. (2007). Ubiquitination of gap junction proteins. J. Membr. Biol. 217: 43-51.

Liang, W.G., C.C. Su, J.H. Nian, A.S. Chiang, S.Y. Li, and J.J. Yang. (2011). Human connexin30.2/31.3 (GJC3) does not form functional gap junction channels but causes enhanced ATP release in HeLa cells. Cell Biochem Biophys 61: 189-197.

Liu, F., F.T. Arce, S. Ramachandran, and R. Lal. (2006). Nanmechanics of hemichannel conformations. Connexin flexibility underlying channel opening and closing. J. Biol. Chem. 281: 23207-23217.

Lohman, A.W., A.C. Straub, and S.R. Johnstone. (2016). Identification of Connexin43 Phosphorylation and S-Nitrosylation in Cultured Primary Vascular Cells. Methods Mol Biol 1437: 97-111.

Lukashkina, V.A., S. Levic, A.N. Lukashkin, N. Strenzke, and I.J. Russell. (2017). A connexin30 mutation rescues hearing and reveals roles for gap junctions in cochlear amplification and micromechanics. Nat Commun 8: 14530.

Maeda, S., S. Nakagawa, M. Suga, E. Yamashita, A. Oshima, Y. Fujiyoshi, and T. Tsukihara. (2009). Structure of the connexin 26 gap junction channel at 3.5 Å resolution. Nature 458: 597-602.

Meens MJ., Pfenniger A., Kwak BR. and Delmar M. (2013). Regulation of cardiovascular connexins by mechanical forces and junctions. Cardiovasc Res. 99(2):304-14.

Misu, A., H. Yamanaka, T. Aramaki, S. Kondo, I.M. Skerrett, M.K. Iovine, and M. Watanabe. (2016). Two Different Functions of Connexin43 Confer Two Different Bone Phenotypes in Zebrafish. J. Biol. Chem. 291: 12601-12611.

Molica, F., M.J. Meens, S. Morel, and B.R. Kwak. (2014). Mutations in cardiovascular connexin genes. Biol Cell 106: 269-293.

Nakagawa, S., S. Maeda, and T. Tsukihara. (2010). Structural and functional studies of gap junction channels. Curr. Opin. Struct. Biol. 20: 423-430.

Ohbuchi, T. and H. Suzuki. (2018). Synchronized roles of pannexin and connexin in nasal mucosal epithelia. Eur Arch Otorhinolaryngol. [Epub: Ahead of Print]

Orthmann-Murphy, J.L., M. Freidin, E. Fischer, S.S. Scherer, and C.K. Abrams. (2007). Two distinct heterotypic channels mediate gap junction coupling between astrocyte and oligodendrocyte connexins. J. Neurosci. 27: 13949-13957.

Pfenniger, A., A. Wohlwend, and B.R. Kwak. (2011). Mutations in connexin genes and disease. Eur J Clin Invest 41: 103-116.

Pinto, B.I., I.E. García, A. Pupo, M.A. Retamal, A.D. Martínez, R. Latorre, and C. González. (2016). Charged residues at the first transmembrane region contribute to the voltage dependence of connexins slow gate. J. Biol. Chem. [Epub: Ahead of Print]

Press, E.R., Q. Shao, J.J. Kelly, K. Chin, A. Alaga, and D.W. Laird. (2017). Induction of cell death and gain-of-function properties of connexin26 mutants predict severity of skin disorders and hearing loss. J. Biol. Chem. [Epub: Ahead of Print]

Puebla, C., B.A. Cisterna, D.P. Salas, F. Delgado-López, P.D. Lampe, and J.C. Sáez. (2016). Linoleic acid permeabilizes gastric epithelial cells by increasing connexin 43 levels in the cell membrane via a GPR40- and Akt-dependent mechanism. Biochim. Biophys. Acta. 1861: 439-448.

Puebla, C., M.A. Retamal, R. Acuña, and J.C. Sáez. (2017). Regulation of Connexin-Based Channels by Fatty Acids. Front Physiol 8: 11.

Puk, O., J. Löster, C. Dalke, D. Soewarto, H. Fuchs, B. Budde, P. Nürnberg, E. Wolf, M.H. de Angelis, and J. Graw. (2008). Mutation in a novel connexin-like gene (Gjf1) in the mouse affects early lens development and causes a variable small-eye phenotype. Invest Ophthalmol Vis Sci 49: 1525-1532.

Rash, J.E., K.G. Vanderpool, T. Yasumura, J. Hickman, J.T. Beatty, and J.I. Nagy. (2016). KV1 channels identified in rodent myelinated axons, linked to Cx29 in innermost myelin: support for electrically active myelin in mammalian saltatory conduction. J Neurophysiol 115: 1836-1859.

Ribeiro-Rodrigues, T.M., T. Martins-Marques, S. Morel, B.R. Kwak, and H. Girão. (2017). Role of connexin 43 in different forms of intercellular communication - gap junctions, extracellular vesicles and tunnelling nanotubes. J Cell Sci 130: 3619-3630.

Sanchez, H.A., N. Slavi, M. Srinivas, and V.K. Verselis. (2016). Syndromic deafness mutations at Asn 14 differentially alter the open stability of Cx26 hemichannels. J Gen Physiol 148: 25-42.

Šeda, O., D. Křenová, O. Oliyarnyk, L. Šedová, M. Krupková, F. Liška, B. Chylíková, L. Kazdová, and V. Křen. (2016). Heterozygous connexin 50 mutation affects metabolic syndrome attributes in spontaneously hypertensive rat. Lipids Health Dis 15: 199.

Shin, D.J., A.L. Germann, A.D. Johnson, S.A. Forman, J.H. Steinbach, and G. Akk. (2018). Propofol Is an Allosteric Agonist with Multiple Binding Sites on Concatemeric Ternary GABA Receptors. Mol Pharmacol 93: 178-189.

Slavi, N., A.H. Toychiev, S. Kosmidis, J. Ackert, S.A. Bloomfield, H. Wulff, S. Viswanathan, P.D. Lampe, and M. Srinivas. (2018). Suppression of connexin 43 phosphorylation promotes astrocyte survival and vascular regeneration in proliferative retinopathy. Proc. Natl. Acad. Sci. USA 115: E5934-E5943.

Stridh, M.H., M. Tranberg, S.G. Weber, F. Blomstrand, and M. Sandberg. (2008). Stimulated efflux of amino acids and glutathione from cultured hippocampal slices by omission of extracellular calcium: likely involvement of connexin hemichannels. J. Biol. Chem. 283(16): 10347-10356.

Su, C.C., S.Y. Li, Y.C. Yen, J.H. Nian, W.G. Liang, and J.J. Yang. (2013). Mechanism of two novel human GJC3 missense mutations in causing non-syndromic hearing loss. Cell Biochem Biophys 66: 277-286.

Sugiura, K., M. Arima, K. Matsunaga, and M. Akiyama. (2015). The novel GJB3 mutation p.Thr202Asn in the M4 transmembrane domain underlies erythrokeratodermia variabilis. Br J Dermatol 173: 309-311.

Tarzemany, R., G. Jiang, H. Larjava, and L. Häkkinen. (2015). Expression and function of connexin 43 in human gingival wound healing and fibroblasts. PLoS One 10: e0115524.

Teubner B., B. Odermatt, M. Guldenagel, G. Sohl, J. Degen, F. Bukauskas, J. Kronengold, V.K. Verselis, Y.T. Jung, C.A. Kozak, K. Schilling, K. Willecke. (2001). Functional expression of the new gap junction gene connexin47 transcribed in mouse brain and spinal cord neurons. J. Neurosci. 21: 1117-1126.

Unger, V.M., N.M. Kumar, N.B. Gilula, and M. Yeager. (1999). Three-dimensional structure of a recombinant gap junction membrane channel. Science 283: 1176-1180.

Valdez Capuccino, J.M., P. Chatterjee, I.E. García, W.M. Botello-Smith, H. Zhang, A.L. Harris, Y. Luo, and J.E. Contreras. (2018). The connexin26 human mutation N14K disrupts cytosolic intersubunit interactions and promotes channel opening. J Gen Physiol. [Epub: Ahead of Print]

Valiunas V., R. Mui, E. McLachlan, G. Valdimarsson, P.R. Brink, T.W. White. (2004). Biophysical characterization of zebrafish connexin35 hemichannels. Am J Physiol. Cell Physiol. 287: C1596-1604

Verselis, V.K. (2017). Connexin hemichannels and cochlear function. Neurosci Lett. [Epub: Ahead of Print]

Wang, C.H., A.W. Duster, B.O. Aydintug, M.G. Zarecki, and H. Lin. (2018). Chloride Ion Transport by theCLC Cl/HAntiporter: A Combined Quantum-Mechanical and Molecular-Mechanical Study. Front Chem 6: 62.

Wang, J., Z.Y. Yang, Y.F. Guo, J.Y. Kuang, X.W. Bian, and S.C. Yu. (2017). Targeting different domains of gap junction protein to control malignant glioma. Neuro Oncol. [Epub: Ahead of Print]

Wang, K.J. and S.Q. Zhu. (2012). A novel p.F206I mutation in Cx46 associated with autosomal dominant congenital cataract. Mol Vis 18: 968-973.

White, T.W. and D.L. Paul. (1999). Genetic diseases and gene knockouts reveal diverse connexin functions. Annu. Rev. Physiol. 61: 283-310.

White, T.W., H. Wang, R. Mui, J. Litteral, and P.R. Brink. (2004). Cloning and functional expression of invertebrate connexins from Halocynthia pyriformis. FEBS Lett. 577: 42-48.

Wong, S.H., W.H. Wang, P.H. Chen, S.Y. Li, and J.J. Yang. (2017). Functional analysis of a nonsyndromic hearing loss-associated mutation in the transmembrane II domain of the GJC3 gene. Int J Med Sci 14: 246-256.

Yeager, M. and N.B. Gilula. (1992). Membrane topology and quaternary structure of cardiac gap junction ion channels. J. Mol. Biol. 223: 929-948.

Yeager, M., V.M. Unger, and M.M. Falk. (1998). Synthesis, assembly and structure of gap junction intercellular channels. Curr. Opin. Struct. Biol. 8: 517-524.

Zhang, D., C. Zhou, Q. Wang, L. Cai, W. Du, X. Li, X. Zhou, and J. Xie. (2018). Extracellular Matrix Elasticity Regulates Osteocyte Gap Junction Elongation: Involvement of Paxillin in Intracellular Signal Transduction. Cell Physiol Biochem 51: 1013-1026.

Zhang, X., T. Zou, Y. Liu, and Y. Qi. (2006). The gating effect of calmodulin and calcium on the connexin50 hemichannel. Biol Chem 387: 595-601.

Zhang, X.H., J. Da Wang, H.Y. Jia, J.S. Zhang, Y. Li, Y. Xiong, J. Li, X.X. Li, Y. Huang, G.Y. Zhu, S.S. Rong, M. Wormstone, and X.H. Wan. (2018). Mutation profiles of congenital cataract genes in 21 northern Chinese families. Mol Vis 24: 471-477.

Zhou JZ. and Jiang JX. (2014). Gap junction and hemichannel-independent actions of connexins on cell and tissue functions--an update. FEBS Lett. 588(8):1186-92.

Zonta, F., D. Buratto, G. Crispino, A. Carrer, F. Bruno, G. Yang, F. Mammano, and S. Pantano. (2018). Cues to Opening Mechanisms From Electric Field Excitation of Cx26 Hemichannel and Mutagenesis Studies in HeLa Transfectans. Front Mol Neurosci 11: 170.

Examples:

TC#NameOrganismal TypeExample
1.A.24.1.1

Connexin 43 (gap junction α-1 protein), CX43 (transports ATP, ADP and AMP better than CX32 does; Goldberg et al., 2002). Hemichannels mediate efflux of glutathione, glutamate and other amino acids as well as ATP (Stridh et al., 2008; Kang et al., 2008). CX43 has a half life of ~3 h due to ubiquitination and lysosomal and proteasomal degradation (Leithe and Rivedal, 2007). Cx43 and Cx46 regulate each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells (Banerjee et al., 2011). A mutant form of Connexin 43 causes Oculodentodigital dysplasia (Gabriel et al., 2011).  Suppressing the function of Cx43 promotes expression of wound healing-associated genes and hibitits scarring (Tarzemany et al. 2015).  Channel conductance and size selectivity are largely determined by pore diameter, whereas charge selectivity results from the amino-terminal domains; transitions between fully open and (multiple) closed states involves global changes in structure of the pore-forming domains (Ek Vitorín et al. 2016). The human Cx43 orthologue is almost identical to the rat protein.  It may mediate resistance against the parkinsonian toxin, 1-methyl-4-phenylpyridine (MPP+) which induces apoptosis in neuroblastoma cells by modulating mitochondrial apoptosis (Kim et al. 2016).  Dopamine neurons may be the target of MPP+ and play a role in Parkinson's disease. In humans, Cx43 plays roles in the development of the central nervous system and in the progression of glioma (Wang et al. 2017).  It interacts with and is regulated by many proteins including NOV (CCN3, IGFBP9; P48745) (Giepmans 2006). Cx43 plays roles in intercellular communication mediated by extracellular vesicles, tunnelling nanotubes and gap junctions (Ribeiro-Rodrigues et al. 2017). Phosphorylation of Cx43 leads to astrocytic coupling and apoptosis, and ultimately, to vascular regeneration in retinal ischemia. Paxillin (Pxn; 591 aas; P49023), a cytoskeletal protein involved in focal adhesion, leads to changes in connexin 43 by direct protein-protein binding, thereby influencing osteocyte gap junction elongation (Zhang et al. 2018).

Animals

CX43 of Rattus norvegicus

 
1.A.24.1.10

 

Connexin31, Cx31 of 270 aas and 4 TMSs.  Also called the gap junction β-3 protein. Mutation Thr202Asn in TMS4 gives rise to erythrokeratodermia (Sugiura et al. 2015).

Cx31 of Homo sapiens

 
1.A.24.1.11

Gap junction α-1 protein, GJα-1, Cx43, shf, sof, of 281 aas and 4 TMSs.  Can function both as a gap junction and a hemichannel and plays critical diverse roles in zebrafish bone growth (Misu et al. 2016).

Cx43 of Danio rerio (Zebrafish) (Brachydanio rerio)

 
1.A.24.1.12

Connexin 29 (Cx29, Gjc3, Gje1) of 269 aas and 4 TMSs.  The Cx29E269D mutant has a dominant negative effect on the formation and function of gap junctions, explaining the role Cx29 in the development of hearing loss (Hong et al. 2010).  Direct axon-to-myelin linkage by abundant KV1 (TC# 1.A.1.2.10 and 12)/Cx29 channel interactions in rodent axons supports the idea of an electrically active role for myelin in increasing both the saltatory conduction velocity and the maximal propagation frequency in mammalian myelinated axons (Rash et al. 2016).

Cx29 of Mus musculus

 
1.A.24.1.13

Connexin36, connexin delta2, Cxδ2, GJD2, of 321 aas and 4 TMSs.

Cx36 of Homo sapiens

 
1.A.24.1.2

Connexin 32 (gap junction β1-protein), CX32 (transports adenosine better than CX43 does; Goldberg et al., 2002).  The carboxyl tail regulates gap junction assembly (Katoch et al. 2015).  The modeled channel pore-facing regions of TMSs 1 and 2 were highly sensitive to tryptophan substitution while lipid-facing regions of TMSs 3 and 4 were variably tolerant. Residues facing a putative intracellular water pocket (the IC pocket) were also sensitive.  Interactions important for voltage gating occurred mainly in the mid-region of the channel in TMS 1. TMS 1 of Cx43 was scanned revealing similar but not identical sensitivities (Brennan et al. 2015). Single point mutations in Cx32, which cause Charcot-Marie-Tooth disease, causes failure in membrane integration, transport defects and rapid degradation. Multiple chaperones detect and remedy this aberrant behavior including the ER-membrane complex (EMC) which helps insert low-hydrophobicity TMSs (Coelho et al. 2019). If they fail to integrate, they are recognized by the ER-lumenal chaperone BiP. Ultimately, the E3 ligase gp78 ubiquitinates Cx32, targeting it for degradation. Thus, cells use a coordinated system of chaperones for membrane protein biogenesis.

Animals

CX32 of Rattus norvegicus

 
1.A.24.1.3

Heteromeric connexin (Cx)32/Cx26) (transports cAMP, cGMP and all inositol phosphates with 1-4 esterified phosphate groups (homomeric Cx26(β2) or homomeric Cx32 do not transport the inositol phosphates as well) (Ayad et al., 2006). The GJB2 gene encodes connexin 26, the protein involved in cell-cell attachment in many tissues. GJB2 mutations cause autosomal recessive (DFNB1) and sometimes dominant (DFNA3) non-syndromic sensorineural hearing loss as well as various skin disease phenotypes (Iossa et al., 2011). TMS1 regulates oligomerization and function (Jara et al., 2012).  The carboxyl tail pg Cx32 regulates gap junction assembly (Katoch et al. 2015).  In Cx46, neutralization of negative charges or addition of positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50 the addition of a glutamate in the same region decreased the voltage dependence and the neutralization of a negative charge increased it. Thus, the charges at the end of TMS1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity (Pinto et al. 2016).  Syndromic deafness mutations at Asn14 alter the open stability of Cx26 hemichannels (Sanchez et al. 2016). The Leu89Pro substitution in the second TMS of CX32 disrupts the trafficking of the protein, inhibiting the assembly of CX32 gap junctions, which in turn may result in peripheral neuropathy (Da et al. 2016).  Cx26 mutants that promote cell death or exert transdominant effects on other connexins in keratinocytes lead to skin diseases and hearing loss, whereas mutants having reduced channel function without aberrant effects on coexpressed connexins cause only hearing loss (Press et al. 2017). When challenged by a field of 0.06 V/nm, the Cx26 hemichannel relaxed toward a novel configuration characterized by a widened pore and an increased bending of the second TMS at the level of the conserved Pro87. A point mutation that inhibited such a transition impeded hemichannel opening in electrophysiology and dye uptake experiments.  Thus, the Cx26 hemichannel uses a global degree of freedom to transit between different configuration states, which may be shared among all connexins (Zonta et al. 2018). A group of human mutations within the N-terminal (NT) domain of connexin 26 hemichannels produce aberrant channel activity, which gives rise to deafness and skin disorders, including keratitis-ichthyosis-deafness (KID) syndrome. Structural and functional studies indicate that the NT domain of connexin hemichannels is folded into the pore, where it plays important roles in permeability and gating. The mutation, N14K disrupts cytosolic intersubunit interactions and promotes channel opening (Valdez Capuccino et al. 2018).

Animals

Cx26/Cx32 of Homo sapiens
Cx26 (P29033)
Cx32 (P08034)

 
1.A.24.1.4Connexin 35 hemichannels (activated by depolarization; deactivated by hyperpolarization; expressed in retina and brain (Valiunas et al., 2004).AnimalsConnexin 35 of Danio rerio (Zebrafish)
(Q8JFD6)
 
1.A.24.1.5

Heteromeric (or homomeric) Connexin46/Connexin50 junction (Cx46/Cx50)  Mutations in CX46 or Cx50 cause cataracts) (Derosa et al., 2007; Wang and Zhu 2012), and mutations in Cx46 can cause breast cancer (Grek et al. 2016). Cx43 and Cx46 regulate each other's expression and turnover in a reciprocal manner in addition to their conventional roles as gap junction proteins in lens cells (Banerjee et al., 2011).  The N-terminal half of connexin 46 appears to contain the core elements of the pore and voltage gates (Kronengold et al. 2012).  In Cx46, neutralization of negative charges or addition of positive charge in the Cx26 equivalent region reduced the slow gate voltage dependence. In Cx50 the addition of a glutamate in the same region decreased the voltage dependence and the neutralization of a negative charge increased it. Thus, the charges at the end of TMS1 are part of the slow gate voltage sensor in Cxs. The fact that Cx42, which has no charge in this region, still presents voltage dependent slow gating suggests that charges still unidentified also contribute to the slow gate voltage sensitivity (Pinto et al. 2016).  Cx43 is regulated by phosphorylation of ser-373 (Puebla et al. 2016). A connexin50 mutation in the heterozygous state affects the lipid profile and the oxidative stress parameters in a spontaneously hypertensive rat strain (Šeda et al. 2016). Mutations in Cx50 (N220D and V44M) are responsible for congenital cataracts (Kuo et al. 2017; Zhang et al. 2018). Cx50 is important for eye lens transparency, and calmoduin and Ca2+ cooperate in the gating control of Cx50 hemichannels (Zhang et al. 2006).

Animals

Cx46/Cx50 of Homo sapiens:
Cx46 (Q9Y6H8)
Cx50 (P48165)

 
1.A.24.1.6Connexin37 (Cx37). The N-terminus contains an α-helix that is required for channel function (Kyle et al., 2009).

Animals

Connexin37 of Homo sapiens (P35212)

 
1.A.24.1.7

Connexin 30 complex (connexin30.2/connexin31.3 (CX30.2/CX31.3)). Also called connexinΥ3/GJC3/GJε1; 279 aas, encoded by the GJB6 (13q12) gene (Cascella et al. 2016)). ATP is released from cells that stably expressed CX30.2 in a medium with low calcium, suggesting a hemichannel-based function. Liang et al. (2011) suggested that it shares functional properties with pannexin hemichannels rather than gap junction channels.  Defects cause nonsyndromic hypoacusia (hearing loss) due to partial loss of channel activity (Su et al. 2012; Su et al. 2013;  Cascella et al. 2016).  Cx30, but not Cx43, hemichannels close upon protein kinase C activation, showing that connexin hemichannels display not only isoform-specific permeability profiles but also isoform-specific regulation by PKC (Alstrom et al. 2015). The W77S mutant has a dominant negative effect on the formation and function of the gap junction and is probably responsible for hearing loss (Wong et al. 2017). Mutations in Cs30 rescue hearing and reveal roles for gap junctions in cochlear amplification (Lukashkina et al. 2017).

and micromechanics

Animals

 

Cx30.2 of Homo sapiens (Q8NFK1)

 
1.A.24.1.8

Connexin40 (Cx40; Gap Junction Protein δ4; GJδ4) of 370 aas and 4 TM (Kopanic et al. 2015).

Animals

Cx40 of Homo sapiens

 
1.A.24.1.9

Gap junction epsilon-1 protein, Gjf1 of 205 aas and 4 TMSs.  Mutations result in variable small eyes and affect lens development (Puk et al. 2008).

Gjf1 of Mus musculus

 
Examples:

TC#NameOrganismal TypeExample
1.A.24.2.1Connexin 47 gap junction (catalyzes intercellular diffusion of neurobiotin, Lucifer yellow and 4',6-diamidino-2-phenylindole; expressed in brain and spinal cord neurons) (Teubner et al., 2001). Possesses sequences between TMSs 2 and 3 and following TMS 4 that differ from these regions in most other connexins.AnimalsConnexin 47 of Mus musculus
(Q8BQU6)
 
1.A.24.2.2

Invertebrate cordate Connexin 47 (White et al., 2004).

Tunicates

Connexin 47 of Halocynthia pyriformis (Q6U1M0)

 
1.A.24.2.3

Inverebrate cordate Connexin (Hervé et al., 2005).

Tunicates

Connexin of Oikopleura dioica (E4YIP4)

 
1.A.24.2.4

Connexin45 (cx45; Gap Junction protein γ1; GJγ1; CxG1) of 396 aas and 4 TMSs (Kopanic et al. 2015).

Animals

Cx45 of Homo sapiens