Abstract
Although a functional pore domain is required for connexin 37 (Cx37)–mediated suppression of rat insulinoma (Rin) cell proliferation, it is unknown whether functional hemichannels would be sufficient or if Cx37 gap junction channels are required for growth suppression. To test this possibility, we targeted extracellular loop cysteines for mutation, expecting that the mutated protein would retain hemichannel, but not gap junction channel, functionality. Cysteines at positions 61 and 65 in the first extracellular loop of Cx37 were mutated to alanine and the mutant protein (Cx37-C61,65A) expressed in Rin cells. Although the resulting iRin37-C61,65A cells expressed the mutant protein comparably to Cx37 wild-type (Cx37-WT)–expressing Rin cells (iRin37), Cx37-C61,65A expression did not suppress the proliferation of Rin cells. As expected, iRin37-C61,65A cells did not form functional gap junction channels. However, functional hemichannels also could not be detected in iRin37-C61,65A cells by either dye uptake or electrophysiological approaches. Thus, failure of Cx37-C61,65A to suppress the proliferation of Rin cells is consistent with previous data demonstrating the importance of channel functionality to Cx37’s growth-suppressive function. Moreover, failure of the Cx37-C61,65A hemichannel to function, even in low external calcium, emphasizes the importance of extracellular loop cysteines not only in hemichannel docking but also in determining the ability of the hemichannel to adopt a closed configuration that can open in response to triggers, such as low external calcium, effective at opening Cx37-WT hemichannels.
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References
Bao X, Chen Y, Reuss L, Altenberg GA (2004) Functional expression in Xenopus oocytes of gap-junctional hemichannels formed by a cysteine-less connexin 43. J Biol Chem 279:9689–9692
Beahm DL, Oshima A, Gaietta GM, Hand GM, Smock AE, Zucker SN, Toloue MM, Chandrasekhar A, Nicholson BJ, Sosinsky GE (2006) Mutation of a conserved threonine in the third transmembrane helix of alpha- and beta-connexins creates a dominant-negative closed gap junction channel. J Biol Chem 281:7994–8009
Bednarczyk D, Mash EA, Aavula BR, Wright SH (2000) NBD-TMA: a novel fluorescent substrate of the peritubular organic cation transporter of renal proximal tubules. Pflugers Arch 440:184–192
Burt JM, Fletcher AM, Steele TD, Wu Y, Cottrell GT, Kurjiaka DT (2001) Alteration of Cx43:Cx40 expression ratio in A7r5 cells. Am J Physiol Cell Physiol 280:C500–C508
Burt JM, Nelson TK, Simon AM, Fang JS (2008) Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells. Am J Physiol Cell Physiol 295:C1103–C1112
Dahl G, Levine E, Rabadan-Diehl C, Werner R (1991) Cell/cell channel formation involves disulfide exchange. Eur J Biochem 197:141–144
Dahl G, Werner R, Levine E, Rabadan-Diehl C (1992) Mutational analysis of gap junction formation. Biophys J 62:172–180
Ek-Vitorín JF, Burt JM (2005) Quantification of gap junction selectivity. Am J Physiol Cell Physiol 289:C1535–C1546
Foote CI, Zhou L, Zhu X, Nicholson BJ (1998) The pattern of disulfide linkages in the extracellular loop regions of connexin 32 suggests a model for the docking interface of gap junctions. J Cell Biol 140:1187–1197
Good ME, Nelson TK, Simon AM, Burt JM (2011) A functional channel is necessary for growth suppression by Cx37. J Cell Sci 124:2448–2456
Goodenough DA, Paul DL (2003) Beyond the gap: functions of unpaired connexon channels. Nat Rev Mol Cell Biol 4:285–294
Harris AL (2001) Emerging issues of connexin channels: biophysics fills the gap. Q Rev Biophys 34:325–472
Kardami E, Dang X, Iacobas DA, Nickel BE, Jeyaraman M, Srisakuldee W, Makazan J, Tanguy S, Spray DC (2007) The role of connexins in controlling cell growth and gene expression. Prog Biophys Mol Biol 94:245–264
King TJ, Bertram JS (2005) Connexins as targets for cancer chemoprevention and chemotherapy. Biochim Biophys Acta 1719:146–160
Nakagawa S, Maeda S, Tsukihara T (2010) Structural and functional studies of gap junction channels. Curr Opin Struct Biol 20:423–430
Plum A, Hallas G, Magin T, Dombrowski F, Hagendorff A, Schumacher B, Wolpert C, Kim J-S, Lamers WH, Evert M, Meda P, Traub O, Willecke K (2000) Unique and shared functions of different connexins in mice. Curr Biol 10:1083–1091
Scherer SS, Xu YT, Nelles E, Fischbeck K, Willecke K, Bone LJ (1998) Connexin32-null mice develop demyelinating peripheral neuropathy. Glia 24:8–20
Simon AM, Goodenough DA, Li E, Paul DL (1997) Female infertility in mice lacking connexin 37. Nature 385:525–529
Simon AM, Chen H, Jackson CL (2006) Cx37 and Cx43 localize to zona pellucida in mouse ovarian follicles. Cell Commun Adhes 13:61–77
Sohl G, Willecke K (2004) Gap junctions and the connexin protein family. Cardiovasc Res 62:228–232
Solan JL, Lampe PD (2007) Key connexin 43 phosphorylation events regulate the gap junction life cycle. J Membr Biol 217:35–41
Sonntag S, Sohl G, Dobrowolski R, Zhang J, Theis M, Winterhager E, Bukauskas FF, Willecke K (2009) Mouse lens connexin23 (Gje1) does not form functional gap junction channels but causes enhanced ATP release from HeLa cells. Eur J Cell Biol 88:65–77
Tong D, Li TY, Naus KE, Bai D, Kidder GM (2007) In vivo analysis of undocked connexin43 gap junction hemichannels in ovarian granulosa cells. J Cell Sci 120:4016–4024
Weber PA, Chang HC, Spaeth KE, Nitsche JM, Nicholson BJ (2004) The permeability of gap junction channels to probes of different size is dependent on connexin composition and permeant-pore affinities. Biophys J 87:958–973
Wong CW, Christen T, Roth I, Chadjichristos CE, Derouette JP, Foglia BF, Chanson M, Goodenough DA, Kwak BR (2006) Connexin37 protects against atherosclerosis by regulating monocyte adhesion. Nat Med 12:950–954
Acknowledgments
The authors acknowledge Dr. Ross Johnson for encouraging, in a hands-on manner and from a distance, our pursuit of hemichannel function. The authors also acknowledge the technical support of Tasha K. Nelson. These studies were supported by the National Institutes of Health, grants (to J.M.B.) R01HL057832, R01HL077675 and T32HL007249 (supporting M.E.G.).
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Good, M.E., Ek-Vitorín, J.F. & Burt, J.M. Extracellular Loop Cysteine Mutant of Cx37 Fails to Suppress Proliferation of Rat Insulinoma Cells. J Membrane Biol 245, 369–380 (2012). https://doi.org/10.1007/s00232-012-9459-x
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DOI: https://doi.org/10.1007/s00232-012-9459-x