title: Cataracts are caused by alterations of a critical N-terminal positive charge in connexin50.
abstract: OBJECTIVE
  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.affiliation: Department of Pediatrics, University of Chicago, Chicago, Illinois 60637-1470, USA.
authors.forename: Bettina C
authors.initials: BC
authors.lastname: Thomas
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Minogue
authors.forename: Virginijus
authors.initials: V
authors.lastname: Valiunas
authors.forename: Giedrius
authors.initials: G
authors.lastname: Kanaporis
authors.forename: Peter R
authors.initials: PR
authors.lastname: Brink
authors.forename: Viviana M
authors.initials: VM
authors.lastname: Berthoud
authors.forename: Eric C
authors.initials: EC
authors.lastname: Beyer
mesh_terms: D019943:Amino Acid Substitution; D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002386:Cataract / Q000235:genetics*; D017630:Connexins / Q000235:genetics*; D004594:Electrophysiology; D005136:Eye Proteins / Q000235:genetics*; D019084:Fluorescent Antibody Technique, Indirect; D017629:Gap Junctions / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007546:Isoquinolines / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D014162:Transfection; D000099264:Connexin 50
chemical_list: D001710:Biotin; D017630:Connexins; D005136:Eye Proteins; D007546:Isoquinolines; D000099264:Connexin 50; C070614:neurobiotin; C017475:lucifer yellow