Diversity in connexin biology
2023; Elsevier BV; Volume: 299; Issue: 11 Linguagem: Inglês
10.1016/j.jbc.2023.105263
ISSN1083-351X
AutoresSergiu A. Lucaciu, Stephanie E. Leighton, Alexandra Hauser, Ryan Yee, Dale W. Laird,
Tópico(s)Yersinia bacterium, plague, ectoparasites research
ResumoOver 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semicrystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies. Acceptance of connexins as having bifunctional channel properties was initially met with some resistance, which has given way in recent years to the premise that connexins have multifunctional properties. Depending on the connexin isoform and cell of origin, connexins have wide-ranging half-lives that vary from a couple of hours to the life expectancy of the cell. Diversity in connexin channel characteristics and molecular properties were further revealed by X-ray crystallography and single-particle cryo-EM. New avenues have seen connexins or connexin fragments playing roles in cell adhesion, tunneling nanotubes, extracellular vesicles, mitochondrial membranes, transcription regulation, and in other emerging cellular functions. These discoveries were largely linked to Cx43, which is prominent in most human organs. Here, we will review the evolution of knowledge on connexin expression in human adults and more recent evidence linking connexins to a highly diverse array of cellular functions. Over 35 years ago the cell biology community was introduced to connexins as the subunit employed to assemble semicrystalline clusters of intercellular channels that had been well described morphologically as gap junctions. The decade that followed would see knowledge of the unexpectedly large 21-member human connexin family grow to reflect unique and overlapping expression patterns in all organ systems. While connexin biology initially focused on their role in constructing highly regulated intercellular channels, this was destined to change as discoveries revealed that connexin hemichannels at the cell surface had novel roles in many cell types, especially when considering connexin pathologies. Acceptance of connexins as having bifunctional channel properties was initially met with some resistance, which has given way in recent years to the premise that connexins have multifunctional properties. Depending on the connexin isoform and cell of origin, connexins have wide-ranging half-lives that vary from a couple of hours to the life expectancy of the cell. Diversity in connexin channel characteristics and molecular properties were further revealed by X-ray crystallography and single-particle cryo-EM. New avenues have seen connexins or connexin fragments playing roles in cell adhesion, tunneling nanotubes, extracellular vesicles, mitochondrial membranes, transcription regulation, and in other emerging cellular functions. These discoveries were largely linked to Cx43, which is prominent in most human organs. Here, we will review the evolution of knowledge on connexin expression in human adults and more recent evidence linking connexins to a highly diverse array of cellular functions. As understood from the human genome project, identification of the connexin family of gap junction (GJ) proteins is likely complete with 21 members in humans (1Sohl G. Willecke K. An update on connexin genes and their nomenclature in mouse and man.Cell Commun. Adhes. 2003; 10: 173-180Crossref PubMed Google Scholar) (Table 1). Connexin proteins have acquired a nomenclature that reflects their predicted molecular weight (e.g., Cx43 = 43 kD) (2Beyer E.C. Paul D.L. Goodenough D.A. Connexin43: a protein from rat heart homologous to a gap junction protein from liver.J. Cell Biol. 1987; 105: 2621-2629Crossref PubMed Google Scholar). Connexin genes are found on seven chromosomes and follow a more conventional gene nomenclature system, with GJ, followed by a Greek letter representing the subfamily and a number reflecting the order of discovery (e.g., GJA1, gap junction alpha 1, which encodes Cx43 and was the first discovered member of the alpha subfamily) (3Sohl G. Willecke K. Gap junctions and the connexin protein family.Cardiovasc Res. 2004; 62: 228-232Crossref PubMed Scopus (805) Google Scholar). Human connexin genes have now been classified into five subfamilies based on sequence homology and are denoted as GJ-A, GJ-B, GJ-C, GJ-D, or GJ-E, reflecting alpha, beta, gamma, delta, and epsilon subtypes (4Kirichenko E.Y. Skatchkov S.N. Ermakov A.M. Structure and functions of gap junctions and their constituent connexins in the mammalian CNS.Biochem. (Mosc) Suppl. Ser. A. Membr. Cell Biol. 2021; 15: 107-119PubMed Google Scholar, 5Mikalsen S.O. S I.K. Tausen M. Connexins during 500 Million years-from Cyclostomes to Mammals.Int. J. Mol. Sci. 2021; 22: 1584Crossref PubMed Scopus (5) Google Scholar)(Table 1). Since 1990, there has been a virtual explosion of articles that focus on GJs, connexins, and/or Cx43 (Fig. 1). The depth of knowledge and understanding of connexin family members is variable with over 50% of published reports since 1995 focusing on Cx43 (Fig. 1). Novel discoveries revealed through interrogation of Cx43 have commonly been extrapolated to other connexin family members. Furthermore, insights into nonhuman connexins are frequently used to deduce information about their putative human orthologs. While this approach continues to provide systemic insights into the entire human connexin family, caution needs to be exercised as each connexin has many unique properties that distinguishes it from its family members. In fact, unexpected diversity even within individual connexin family members (e.g., Cx43 versus N-terminal truncated Cx43) continues to reveal that the "same" connexins can have remarkably different functions with distinct modes of regulation.Table 1Connexin diversity and links to inherited diseasesConnexinGeneOrgans expressed# Of diseases associated with gene variantsHigh-resolution structure solvedCx43GJA1576108, 109Cx46GJA371101, 102Cx37GJA4181∗—Cx40GJA5273∗—Cx50GJA831101, 102Cx59GJA950—Cx62GJA10100—Cx32GJB1351—Cx26GJB239898–100, 103–106Cx31GJB3103—Cx30.3GJB491—Cx31.1GJB580—Cx30GJB6184—Cx25GJB7100—Cx45GJC1221∗—Cx47GJC253—Cx30.2 (Cx31.3)GJC370107Cx36GJD290110Cx31.9GJD3100—Cx40.1GJD460—Cx23GJE100—A list of the 21 connexin encoding genes and corresponding protein names. Connexin genes are typically divided into five subtypes denoted by A, B, C, D, and E, reflective of sequence homologies. The number of adult human organs where these connexins have been detected are noted as revealed by a systematic analysis of articles available on PubMed. Seventy-five organs were assessed, with 62 organs exhibiting evidence for expression of at least one connexin. Note that Cx43, Cx26, and Cx32 are the three most widely distributed connexin isoforms being found in numerous human organs. The vast majority of the 33 human diseases attributed to mutations/variants within the genes encoding twelve connexin isoforms are annotated within the Online Mendelian Inheritance in Man database, while others have been reported elsewhere. Asterisks indicate that one or more of the diseases included was identified from the Genetic Testing Registry or a PubMed search of disease-associated connexin gene variants that require further patient studies to confirm linkage. Connexin gene polymorphisms associated with disease were not included. The high-resolution structure of six connexins found within gap junctions and/or hemichannels are listed along with corresponding references. Open table in a new tab A list of the 21 connexin encoding genes and corresponding protein names. Connexin genes are typically divided into five subtypes denoted by A, B, C, D, and E, reflective of sequence homologies. The number of adult human organs where these connexins have been detected are noted as revealed by a systematic analysis of articles available on PubMed. Seventy-five organs were assessed, with 62 organs exhibiting evidence for expression of at least one connexin. Note that Cx43, Cx26, and Cx32 are the three most widely distributed connexin isoforms being found in numerous human organs. The vast majority of the 33 human diseases attributed to mutations/variants within the genes encoding twelve connexin isoforms are annotated within the Online Mendelian Inheritance in Man database, while others have been reported elsewhere. Asterisks indicate that one or more of the diseases included was identified from the Genetic Testing Registry or a PubMed search of disease-associated connexin gene variants that require further patient studies to confirm linkage. Connexin gene polymorphisms associated with disease were not included. The high-resolution structure of six connexins found within gap junctions and/or hemichannels are listed along with corresponding references. Canonically, in their capacity as GJ forming integral membrane proteins that span the lipid bilayer four times, connexins oligomerize into connexons (commonly referred to as hemichannels) shortly after their cotranslational insertion into the endoplasmic reticulum membrane (6Laird D.W. The gap junction proteome and its relationship to disease.Trends Cell Biol. 2010; 20: 92-101Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 7Laird D.W. Life cycle of connexins in health and disease.Biochem. 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Transport vesicles actively and constitutively carry connexons to the cell surface, where they dock with compatible connexons from a juxtaposed cell to form intercellular channels that allow for gap junctional intercellular communication (GJIC) (11Koval M. Molina S.A. Burt J.M. Mix and match: investigating heteromeric and heterotypic gap junction channels in model systems and native tissues.FEBS Lett. 2014; 588: 1193-1204Crossref PubMed Scopus (101) Google Scholar). These intercellular channels quickly cluster into tightly packed arrays that have historically been termed GJs, structures that are interchangeably referred to as GJ plaques (12Goodenough D.A. Goliger J.A. Paul D.L. Connexins, connexons, and intercellular communication.Annu. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Google Scholar) (Fig. 2C). A single GJ can easily contain hundreds to thousands of channels allowing for a massive site of regulated intercellular exchange of ions and potentially thousands of members of the metabolome that are less than 1 kD in size (13Veenstra R.D. Wang H.Z. Beblo D.A. Chilton M.G. Harris A.L. Beyer E.C. et al.Selectivity of connexin-specific gap junctions does not correlate with channel conductance.Circ. Res. 1995; 77: 1156-1165Crossref PubMed Google Scholar, 14Ek-Vitorin J.F. Burt J.M. Quantification of gap junction selectivity.Am. J. Physiol. Cell Physiol. 2005; 289: C1535-C1546Crossref PubMed Scopus (0) Google Scholar, 15Ek-Vitorin J.F. Burt J.M. Structural basis for the selective permeability of channels made of communicating junction proteins.Biochim. Biophys. Acta. 2013; 1828: 51-68Crossref PubMed Scopus (55) Google Scholar, 16Alexander D.B. Goldberg G.S. Transfer of biologically important molecules between cells through gap junction channels.Curr. Med. Chem. 2003; 10: 2045-2058Crossref PubMed Scopus (196) Google Scholar)(Fig. 2, A–C). While it is well known that small molecules can pass through GJ channels, direct evidence of which molecules do is limited to less than two dozen, including ATP, cAMP, IP3, GSH, nucleotides, ions, and at least some amino acids (17Harris A.L. Connexin channel permeability to cytoplasmic molecules.Prog. Biophys. Mol. Biol. 2007; 94: 120-143Crossref PubMed Scopus (368) Google Scholar, 18Harris A.L. Electrical coupling and its channels.J. Gen. Physiol. 2018; 150: 1606-1639Crossref PubMed Scopus (18) Google Scholar) (Fig. 3). It is further unclear which of these transjunctional molecules can pass through GJ channel conduits composed of different connexin constituents. Given the brevity of the present article, this aspect of channel complexity is reviewed in more detail elsewhere (17Harris A.L. Connexin channel permeability to cytoplasmic molecules.Prog. Biophys. Mol. 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GJIC, gap junctional intercellular communication.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Within a decade of connexins being identified as the proteins that lined the pore of GJ channels, they were proposed to have a second function in serving as single membrane channels at the plasma membrane (hemichannels) (23Beyer E.C. Steinberg T.H. Evidence that the gap junction protein connexin-43 is the ATP-induced pore of mouse macrophages.J. Biol. Chem. 1991; 266: 7971-7974Abstract Full Text PDF PubMed Google Scholar). Early on this concept met considerable resistance in the GJ community as cell surface connexons were proposed to be transient structures that remained closed as they quickly proceeded to dock with connexons from an adjacent cell to form GJ channels. Opposition to connexins acting in a hemichannel context could also stem from an evolutionary perspective as this might be considered a "step-down" function to connexins acting as unique direct cell–cell channel conduits. It is notable that in addition to connexins, several membrane transport proteins and channels are capable of actively or passively moving small regulator molecules across the plasma membrane of human cells (e.g., ion selective channels, pannexins, calcium homeostasis modulator) (24Koval M. Schug W.J. Isakson B.E. Pharmacology of pannexin channels.Curr. Opin. Pharmacol. 2023; 69102359Crossref PubMed Scopus (7) Google Scholar, 25Syrjanen J. Michalski K. Kawate T. Furukawa H. On the molecular nature of large-pore channels.J. Mol. Biol. 2021; 433166994Crossref PubMed Scopus (21) Google Scholar). Hemichannel redundancy arguments also pointed to the notion that connexins are not unique in forming large-pore channels that allow molecules larger than ions to pass (25Syrjanen J. Michalski K. Kawate T. Furukawa H. On the molecular nature of large-pore channels.J. Mol. Biol. 2021; 433166994Crossref PubMed Scopus (21) Google Scholar). However, in vertebrates, other than connexins, there is no other protein family that has acquired the evolutionary advantage of direct and regulated intercellular exchange of signaling molecules barring the exception that pannexin 1 potentially acquires this capacity in rare situations (26Welzel G. Schuster S. Connexins evolved after early chordates lost innexin diversity.Elife. 2022; 11e74422Crossref PubMed Scopus (4) Google Scholar, 27Palacios-Prado N. Soto P.A. Lopez X. Choi E.J. Marquez-Miranda V. Rojas M. et al.Endogenous pannexin1 channels form functional intercellular cell-cell channels with characteristic voltage-dependent properties.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2202104119Crossref PubMed Scopus (12) Google Scholar). Nevertheless, overwhelming experimental evidence now points to cell surface connexin hemichannels having key roles in normal chordate physiology, pathology, and tissue injury, all of which are reviewed in detail elsewhere (28Kiani L. Targeting connexin hemichannels to treat temporal lobe epilepsy.Nat. Rev. Neurol. 2023; 19: 1Crossref PubMed Scopus (1) Google Scholar, 29Leybaert L. De Smet M.A. Lissoni A. Allewaert R. Roderick H.L. Bultynck G. et al.Connexin hemichannels as candidate targets for cardioprotective and anti-arrhythmic treatments.J. Clin. Invest. 2023; 133e168117Crossref PubMed Scopus (2) Google Scholar, 30Delvaeye T. Vandenabeele P. Bultynck G. Leybaert L. Krysko D.V. Therapeutic targeting of connexin channels: new views and challenges.Trends Mol. Med. 2018; 24: 1036-1053Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 31Aasen T. Connexins, innexins, and pannexins: from biology to clinical targets.Biomolecules. 2021; 11: 155Crossref PubMed Scopus (1) Google Scholar). The regulated opening of cell surface hemichannels provides a conduit for the release of many small molecules (e.g., GSH, glutamate, others) from the cytosol to the extracellular milieu with the best characterized of these being ATP (32Yang D. Chen M. Yang S. Deng F. Guo X. Connexin hemichannels and pannexin channels in toxicity: recent advances and mechanistic insights.Toxicology. 2023; 488153488Crossref Scopus (1) Google Scholar). Hemichannels not only enable bidirectional diffusion of small molecules such as a plethora of informative dyes (e.g., ethidium bromide, YO-PRO) (33Saez J.C. Vargas A.A. Hernandez D.E. Ortiz F.C. Giaume C. Orellana J.A. Permeation of molecules through Astroglial connexin 43 hemichannels is modulated by cytokines with parameters depending on the permeant species.Int. J. Mol. 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While it is not our intent here to cover the breadth of evidence that supports a key role for connexin hemichannels, we would be remiss not to give a couple of examples. The first is in the lens where Cx46 hemichannels play a critical role in maintaining lens homeostasis (36Retamal M.A. Altenberg G.A. Role and Posttranslational regulation of Cx46 hemichannels and gap junction channels in the eye lens.Front. Physiol. 2022; 13864948Crossref Scopus (4) Google Scholar, 37Beyer E.C. Berthoud V.M. Connexin hemichannels in the lens.Front. Physiol. 2014; 5: 20Crossref PubMed Scopus (68) Google Scholar, 38Berthoud V.M. Gao J. Minogue P.J. Jara O. Mathias R.T. Beyer E.C. Connexin mutants compromise the lens circulation and cause cataracts through Biomineralization.Int. J. Mol. Sci. 2020; 21: 5822Crossref PubMed Scopus (27) Google Scholar). The precise roles that Cx43, Cx46, or Cx50 hemichannels play in normal human lens remains somewhat elusive. However, models have been proposed where mechanosensitive hemichannels accommodate a steady-state fluid equilibrium and pathways for the influx of calcium and sodium, and the efflux of potassium (37Beyer E.C. Berthoud V.M. Connexin hemichannels in the lens.Front. Physiol. 2014; 5: 20Crossref PubMed Scopus (68) Google Scholar, 38Berthoud V.M. Gao J. Minogue P.J. Jara O. Mathias R.T. Beyer E.C. Connexin mutants compromise the lens circulation and cause cataracts through Biomineralization.Int. J. Mol. Sci. 2020; 21: 5822Crossref PubMed Scopus (27) Google Scholar) required for normal lens function and maintenance of transparency. Amplified hemichannel activity, as seen for some diseased-linked Cx46 and Cx50 gene mutations (39Beyer E.C. Mathias R.T. Berthoud V.M. Loss of fiber cell communication may contribute to the development of cataracts of many different etiologies.Front. Physiol. 2022; 13989524Crossref Scopus (5) Google Scholar), may lead to ATP and GSH leaking from lens cells harboring these mutants culminating in cytotoxicity and/or cataracts. In another example, mechanosensitive hemichannels are found in bone osteocytes, where they appear to play a role in bone remodeling and plasticity through their regulated release of ATP, NAD+, and prostaglandin E2 (40Zhao D. Riquelme M.A. Guda T. Tu C. Xu H. Gu S. et al.Connexin hemichannels with prostaglandin release in anabolic function of bone to mechanical loading.Elife. 2022; 11e74365Crossref Google Scholar). While it is expected that few hemichannels are open at steady state, they can be regulated to open in response to changes in the microenvironment such as calcium reduction. In this context, connexin hemichannels would be expected to react to oxidative stress as a molecular means to protect osteocytes from cell death (41Hua R. Zhang J. Riquelme M.A. Jiang J.X. Connexin gap junctions and hemichannels link oxidative stress to skeletal physiology and pathology.Curr. Osteoporos. Rep. 2021; 19: 66-74Crossref PubMed Scopus (24) Google Scholar, 42Kar R. Riquelme M.A. Werner S. Jiang J.X. Connexin 43 channels protect osteocytes against oxidative stress-induced cell death.J. Bone Miner. Res. 2013; 28: 1611-1621Crossref PubMed Scopus (72) Google Scholar). Given the recognition that hyperactive or leaky hemichannels are linked to pathologies in the brain, eyes, bone, skin, lens, and other organs (43Cocozzelli A.G. White T.W. Connexin 43 mutations lead to increased hemichannel functionality in skin disease.Int. J. Mol. Sci. 2019; 20: 6186Crossref PubMed Scopus (23) Google Scholar, 44Retamal M.A. Reyes E.P. Garcia I.E. Pinto B. Martinez A.D. Gonzalez C. Diseases associated with leaky hemichannels.Front. Cell. Neurosci. 2015; 9: 267Crossref PubMed Google Scholar), they are now being considered as viable targets for therapeutics (21Laird D.W. Lampe P.D. 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Connexin43 hemichannels: a potential drug target for the treatment of diabetic retinopathy.Drug Discov. Today. 2019; 24: 1627-1636Crossref PubMed Scopus (0) Google Scholar). Of note, a recent study using a newly developed hemichannel blocker D4 was found to greatly attenuate seizures in a mouse model of temporal lobe epilepsy by reducing neuroinflammation (49Guo A. Zhang H. Li H. Chiu A. Garcia-Rodriguez C. Lagos C.F. et al.Inhibition of connexin hemichannels alleviates neuroinflammation and hyperexcitability in temporal lobe epilepsy.Proc. Natl. Acad. Sci. U. S. A. 2022; 119e2213162119Crossref Scopus (11) Google Scholar). Another preclinical study found that an antibody directed to Cx26 potently improved the skin pathology found in a mouse model of keratitis ichthyosis deafness syndrome (46Peres C. Sellitto C. Nardin C. Putti S. Orsini T. Di Pietro C. et al.Antibody gene transfer treatment drastically improves epidermal pathology in a keratitis ichthyosis deafness syndrome model using male mice.EBioMedicine. 2023; 89104453Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar). Preclinical studies using inhibitors of hemichannels continue to arise that show promise in having efficacy in pathologies and injury scenarios. However, caution needs to be exercised in using pharmacological inhibitors that are known to have a broad spectrum of effects on diseased and healthy cells. Complexity in GJ channel formation begins at the expression level as most human cells express two or more connexin isoforms, resulting in the potential for a mixed array of connexons that can be either homomeric or heteromeric (50Defourny J. Thiry M. Recent insights into gap junction biogenesis in the cochlea.Dev. Dyn. 2023; 252: 239-246Crossref PubMed Scopus (1) Google Scholar) (Fig. 2B). 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