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

Human connexin46 (hCx46) forms gap junctional channels interconnecting lens fiber cells and appears to be critical for normal lens function, because h Cx46 mutations have been linked to congenital cataracts. We studied two hCx 46 mutants, N63S, a missense mutation in the first extracellular domain, an d 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 S DS-PAGE after metabolic labeling with [S-35]methionine or by immunoblotting . Dual two-microelectrode voltage-clamp studies showed that hCx46 formed bo th gap junctional channels in paired Xenopus oocytes and hemi-gap junctiona l channels in single oocytes. In contrast, neither of the two cataract-asso ciated hCx46 mutants could form intercellular channels in paired Xenopus oo cytes. The hCx46 mutants were also impaired in their ability to form hemi-g ap-junctional channels. When N63S or fs380 was coexpressed with wild-type c onnexins, both mutations acted like "loss of function" rather than "dominan t negative" mutations, because they did not affect the gap junctional condu ctance induced by either wild-type hCx46 or wild-type hCx50.