Connexin46 mutations linked to congenital cataract show loss of gap junction channel function

1 Department of Physiology and Biophysics, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064; 2 Department of Ophthalmology and Visual Sciences, Washington University, St. Louis, Missouri 63110; and 3 Department of Pediatrics, University of Chicago, Chicago, Illinois 60637 Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because hCx46 mutations have been linked to congenital cataracts. We studied two hCx46 mutants, N63S, a missense mutation in the first extracellular domain, and fs380, a frame-shift mutation that shifts the translational reading frame at amino acid residue 380. We expressed wild-type Cx46 and the two mutants in Xenopus oocytes. Production of the expressed proteins was verified by SDS-PAGE after metabolic labeling with [ 35 S]methionine or by immunoblotting. Dual two-microelectrode voltage-clamp studies showed that hCx46 formed both gap junctional channels in paired Xenopus oocytes and hemi-gap junctional channels in single oocytes. In contrast, neither of the two cataract-associated hCx46 mutants could form intercellular channels in paired Xenopus oocytes. The hCx46 mutants were also impaired in their ability to form hemi-gap-junctional channels. When N63S or fs380 was coexpressed with wild-type connexins, both mutations acted like "loss of function" rather than "dominant negative" mutations, because they did not affect the gap junctional conductance induced by either wild-type hCx46 or wild-type hCx50. human connexin 46; intercellular communication; lens