PURPOSE: To elucidate the basis of the autosomal dominant congenital nuclear cataracts caused by the connexin50 mutant, CX50R23T, by determining its cellular distribution and functional behavior and the consequences of substituting other amino acids for arginine-23. METHODS: Connexin50 (CX50) mutants were generated by PCR and transfected into HeLa or N2a cells. Expressed CX50 protein was detected by immunoblot analysis and localized by immunofluorescence. Intercellular communication was assessed by microinjection of neurobiotin or by double whole-cell patch-clamp recording. RESULTS: HeLa cells stably transfected with CX50R23T or wild-type CX50 produced immunoreactive CX50 bands of identical electrophoretic mobility. Whereas HeLa cells stably expressing CX50 contained abundant gap junction plaques, CX50R23T localized predominantly in the cytoplasm. HeLa cells expressing wild-type CX50 showed large gap junctional conductances and extensive transfer of neurobiotin, but those expressing CX50R23T did not show significant intercellular communication by either assay. Moreover, CX50R23T inhibited the function of coexpressed wild-type CX50. Three CX50R23 substitution mutants (CX50R23K, CX50R23L, and CX50R23W) formed gap junction plaques, whereas two mutant substitutions with negatively charged residues (CX50R23D, CX50R23E) did not form detectable plaques. Only the mutant with a positive charge substitution (CX50R23K) allowed neurobiotin transfer at levels similar to those of wild-type CX50; none of the other mutants induced transfer. CONCLUSIONS: These results suggest that replacement of amino acid 23 in CX50 by any residue that is not positively charged would lead to cataract formation.
PURPOSE: To elucidate the basis of the autosomal dominant congenital nuclear cataracts caused by the connexin50 mutant, CX50R23T, by determining its cellular distribution and functional behavior and the consequences of substituting other amino acids for arginine-23. METHODS:Connexin50 (CX50) mutants were generated by PCR and transfected into HeLa or N2a cells. Expressed CX50 protein was detected by immunoblot analysis and localized by immunofluorescence. Intercellular communication was assessed by microinjection of neurobiotin or by double whole-cell patch-clamp recording. RESULTS:HeLa cells stably transfected with CX50R23T or wild-type CX50 produced immunoreactive CX50 bands of identical electrophoretic mobility. Whereas HeLa cells stably expressing CX50 contained abundant gap junction plaques, CX50R23T localized predominantly in the cytoplasm. HeLa cells expressing wild-type CX50 showed large gap junctional conductances and extensive transfer of neurobiotin, but those expressing CX50R23T did not show significant intercellular communication by either assay. Moreover, CX50R23T inhibited the function of coexpressed wild-type CX50. Three CX50R23 substitution mutants (CX50R23K, CX50R23L, and CX50R23W) formed gap junction plaques, whereas two mutant substitutions with negatively charged residues (CX50R23D, CX50R23E) did not form detectable plaques. Only the mutant with a positive charge substitution (CX50R23K) allowed neurobiotin transfer at levels similar to those of wild-type CX50; none of the other mutants induced transfer. CONCLUSIONS: These results suggest that replacement of amino acid 23 in CX50 by any residue that is not positively charged would lead to cataract formation.
Authors: C E Willoughby; Sara Arab; R Gandhi; S Zeinali; Seddigheh Arab; D Luk; G Billingsley; F L Munier; E Héon Journal: J Med Genet Date: 2003-11 Impact factor: 6.318
Authors: William A Paznekas; Simeon A Boyadjiev; Robert E Shapiro; Otto Daniels; Bernd Wollnik; Catherine E Keegan; Jeffrey W Innis; Mary Beth Dinulos; Cathy Christian; Mark C Hannibal; Ethylin Wang Jabs Journal: Am J Hum Genet Date: 2002-11-27 Impact factor: 11.025
Authors: Gabriele Richard; Nkecha Brown; Fatima Rouan; Jan-Gerrit Van der Schroeff; Emilia Bijlsma; Lawrence F Eichenfield; Virginia P Sybert; Kenneth E Greer; Peter Hogan; Carmen Campanelli; John G Compton; Sherri J Bale; John J DiGiovanna; Jouni Uitto Journal: J Invest Dermatol Date: 2003-04 Impact factor: 8.551
Authors: Viviana M Berthoud; Peter J Minogue; Jun Guo; Edward K Williamson; Xiaorong Xu; Lisa Ebihara; Eric C Beyer Journal: Eur J Cell Biol Date: 2003-05 Impact factor: 4.492
Authors: Jun-Jie Tong; Peter J Minogue; Wenji Guo; Tung-Ling Chen; Eric C Beyer; Viviana M Berthoud; Lisa Ebihara Journal: Am J Physiol Cell Physiol Date: 2011-01-12 Impact factor: 4.249
Authors: John W Kyle; Peter J Minogue; Bettina C Thomas; Denise A Lopez Domowicz; Viviana M Berthoud; Dorothy A Hanck; Eric C Beyer Journal: J Cell Sci Date: 2008-07-29 Impact factor: 5.285
Authors: John W Kyle; Viviana M Berthoud; Josh Kurutz; Peter J Minogue; Michael Greenspan; Dorothy A Hanck; Eric C Beyer Journal: J Biol Chem Date: 2009-05-28 Impact factor: 5.157
Authors: Peter J Minogue; Jun-Jie Tong; Anita Arora; Isabelle Russell-Eggitt; David M Hunt; Anthony T Moore; Lisa Ebihara; Eric C Beyer; Viviana M Berthoud Journal: Invest Ophthalmol Vis Sci Date: 2009-08-13 Impact factor: 4.799
Authors: Jun-Jie Tong; Bonnie C H Sohn; Anh Lam; D Eric Walters; Barbara M Vertel; Lisa Ebihara Journal: Am J Physiol Cell Physiol Date: 2013-01-09 Impact factor: 4.249
Authors: Maria D Mayan; Raquel Gago-Fuentes; Paula Carpintero-Fernandez; Patricia Fernandez-Puente; Purificacion Filgueira-Fernandez; Noa Goyanes; Virginijus Valiunas; Peter R Brink; Gary S Goldberg; Francisco J Blanco Journal: Ann Rheum Dis Date: 2013-11-13 Impact factor: 19.103
Authors: Jochen Graw; Werner Schmidt; Peter J Minogue; Jessica Rodriguez; Jun-Jie Tong; Norman Klopp; Thomas Illig; Lisa Ebihara; Viviana M Berthoud; Eric C Beyer Journal: Mol Vis Date: 2009-09-14 Impact factor: 2.367