May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
Analysis of the Cataract-Associated Connexin Mutant, CX50R23T, Reveals an N-Terminal Positive Charge Critical for Gap Junction Function
Author Affiliations & Notes
  • B. C. Haemmerling
    Pediatrics, University of Chicago, Chicago, Illinois
  • P. J. Minogue
    Pediatrics, University of Chicago, Chicago, Illinois
  • V. M. Berthoud
    Pediatrics, University of Chicago, Chicago, Illinois
  • E. C. Beyer
    Pediatrics, University of Chicago, Chicago, Illinois
  • Footnotes
    Commercial Relationships B.C. Haemmerling, None; P.J. Minogue, None; V.M. Berthoud, None; E.C. Beyer, None.
  • Footnotes
    Support Postdoctoral Fellowship from the German Academic Exchange Service (to BCH) and NIH Grant EY08368 (to ECB)
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 4220. doi:
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      B. C. Haemmerling, P. J. Minogue, V. M. Berthoud, E. C. Beyer; Analysis of the Cataract-Associated Connexin Mutant, CX50R23T, Reveals an N-Terminal Positive Charge Critical for Gap Junction Function. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4220.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: To study the cellular distribution and functional behavior of a mutant human connexin50, CX50R23T, associated with autosomal dominant congenital nuclear cataracts and to determine the consequences of substituting other amino acid residues for R23.

Methods:: CX50R23T and other CX50 mutants differing in size, charge, and polarity of amino acid residue 23 were generated by PCR. HeLa cells were transiently or stably transfected with wild-type or mutant CX50. Expressed CX50 protein was detected by immunoblotting and localized by immunofluorescence. Intercellular communication was quantified by counting the number of gap junction tracer-containing neighbors after microinjection of Lucifer yellow and neurobiotin into one cell.

Results:: HeLa cells stably transfected with CX50R23T produced immunoreactive CX50 protein of identical electrophoretic mobility to wild-type CX50. CX50R23T localized predominantly in the cytoplasm with very rare formation of small gap junction plaques. In contrast, HeLa cells stably expressing CX50 contained abundant gap junction plaques. Neither HeLaCX50R23T nor HeLaCX50 cells showed significant intercellular transfer of Lucifer yellow. Cells expressing wild-type CX50 showed extensive transfer of neurobiotin (19.54 ± 3.40 coupled cells), whereas those expressing CX50R23T did not allow significant neurobiotin transfer (0.37 ± 0.10 coupled cells). When transiently transfected into HeLa cells, three CX50R23 substitution mutants formed gap junction plaques (R23K, R23L, R23W), while two mutants with substitutions to negatively charged residues (R23D, R23E) failed to form detectable plaques. The mutant containing the positive charge substitution (R23K) allowed neurobiotin transfer at levels similar to wild-type CX50, while none of the other mutants induced intercellular transfer above the levels observed in untransfected HeLa cells.

Conclusions:: These results demonstrate that (1) the cataract-associated mutant CX50R23T is non-functional and severely impaired in trafficking and clustering into plaques, (2) plaque formation by a connexin variant does not insure gap junction function, and (3) the presence of a positive charge at position 23 is critical for formation of functional CX50 gap junction channels. This suggests that substitution of amino acid residue 23 in CX50 by an amino acid that is not positively charged would lead to cataract formation.

Keywords: gap junctions/coupling • cataract • cell-cell communication 

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