Cataract-associated D3Y mutation of human connexin46 (hCx46) increases the dye coupling of gap junction channels and suppresses the voltage sensitivity of hemichannels
- 282 Downloads
Connexin46 (Cx46), together with Cx50, forms gap junction channels between lens fibers and participates in the lens pump-leak system, which is essential for the homeostasis of this avascular organ. Mutations in Cx50 and Cx46 correlate with cataracts, but the functional relationship between the mutations and cataract formation is not always clear. Recently, it was found that a mutation at the third position of hCx46 that substituted an aspartic acid residue with a tyrosine residue (hCx46D3Y) caused an autosomal dominant zonular pulverulent cataract. We expressed EGFP-labeled hCx46wt and hCx46D3Y in HeLa cells and found that the mutation did not affect the formation of gap junction plaques. Dye transfer experiments using Lucifer Yellow (LY) and ethidium bromide (EthBr) showed an increased degree of dye coupling between the cell pairs expressing hCx46D3Y in comparison to the cell pairs expressing hCx46wt. In Xenopus oocytes, two-electrode voltage-clamp experiments revealed that hCx46wt formed voltage-sensitive hemichannels. This was not observed in the oocytes expressing hCx46D3Y. The replacement of the aspartic acid residue at the third position by another negatively charged residue, glutamic acid, to generate the mutant hCx46D3E, restored the voltage sensitivity of the resultant hemichannels. Moreover, HeLa cell pairs expressing hCx46D3E and hCx46wt showed a similar degree of dye coupling. These results indicate that the negatively charged aspartic acid residue at the third position of the N-terminus of hCx46 could be involved in the determination of the degree of metabolite cell-to-cell coupling and is essential for the voltage sensitivity of the hCx46 hemichannels.
KeywordshCx46 N-terminus Voltage sensitivity Dye transfer Hemichannels Cataract
Unable to display preview. Download preview PDF.
- Addison PK, Berry V, Holden KR, Espinal D, Rivera B, Su H, Srivastava AK, Bhattacharya SS (2006) A novel mutation in the connexin 46 gene (GJA3) causes autosomal dominant zonular pulverulent cataract in a Hispanic family. Mol Vis 12:791–795Google Scholar
- Arora, A., Minogue, P.J., Liu, X., Reddy, M.A., Ainsworth, J.R., Bhattacharya, S.S., Webster, A.R., Hunt, D.M., Ebihara, L., Moore, A.T., Beyer, E.C. and Berthoud, V.M. (2006) A novel GJA8 mutation is associated with autosomal dominant lamellar pulverulent cataract: further evidence for gap junction dysfunction in human cataract. J Med Genet. 43:e2 (http://www.jmedgenet.com/cgi/content/full/43/1/e2)
- Donaldson P, Kistler J, Mathias RT (2001) Molecular solutions to mammalian lens transparency. News Physiol Sci 16:118–123Google Scholar
- Ebihara, L., Berthoud, V.M. and Beyer, E.C. (1995) Distinct behavior of connexin56 and connexin46 gap junctional channels can be predicted from the behavior of their hemi-gap-junctional channels. Biophys J.68:1796-1803Google Scholar
- Hansen L, Yao W, Eiberg H, Funding M, Riise R, Kjaer KW, Hejtmancik JF, Rosenberg T (2006) The congenital “ant-egg” cataract phenotype is caused by a missense mutation in connexin46. Mol Vis 12:1033–1039Google Scholar
- Mathias RT, Rae JL, Baldo GJ (1997) Physiological properties of the normal lens. Physiol Rev 77:21–50Google Scholar
- Pal JD, Liu X, Mackay D, Shiels A, Berthoud VM, Beyer EC, Ebihara L (2000) Connexin46 mutations linked to congenital cataract show loss of gap junction channel function. Am J Physiol Cell Physiol 279:C596–C602Google Scholar