Functional Characterization Of A Connexin50Mutation(V44A)that Leads To A Congenital Cataract

Purpose:To elucidate the basis of suture-sparing autosomal dominant congenital nuclear cataracts in a Chinese family caused by a novel connexin50(Cx50) mutant, Cx50V44A, by determining its cellular distribution and functional behavior.Methods:Mutation Screening:Family history and clinical data were recorded. Lens material was aspirated during cataract surgery from the proband and was examined using transmission electron microscopy. Direct gene sequencing was applied to identify the disease-causing mutation. The structure of wild type protein and mutant type one were predicted by Antheprot4.3and Swiss-model software.Gene function study:Cx50gene was acquired from human lens cDNA library, Cx50mutant was generated by PCR. Cx50-EGFP, Cx50V44A-EGFP and PEGFP-N1plasmids were transfected into Hek293cell, and selected by G418. Stable expressed Cx50was localized by laser scanning confocal microscope. Hemichannel function was analyzed by dye uptake in external free Ca2+HBSS, HBSS containing1.8mM Ca2+or HBSS containing10uM FFA, and whole cell patch clamp technique in Ca2+free bath solution. Gap junction function was analyzed by dye transfer experiments in normal [Ca2+] bath solution, using4%neurobiotin and5%lucifer yellow. Results:Lens examination in the affected members revealed suture-sparing congenital nuclear cataracts. Sequencing of the candidate genes detected a heterozygous c.131T>C change in GJA8gene, resulting in the substitution of a highly conserved Valine by Alanine (V44A). This mutation was co-segregated with the affected individuals, but was not found in the unaffected family members or in the100unrelated controls. In stable transfectants, both wild-type Cx50and Cx50V44A protein fluorescence were detected at appositional membranes and perinuclear cytoplasmic locations. And both two groups could be observed some gap junction plaques between adjacent cells. After hemichannel was opened in the absence of external calcium for30min with PI and10min with DAPI, Hek293cells expressing wild type Cx50took up both dyes, but cells expressing Cx50V44A or GFP took up no dyes. Dye loading could be blocked by1.8mM Ca2+and10uM FFA. Using whole cell patch clamp technique, we found currents in Cx50wild cells was bigger than which in Cx50V44A cells. Both neurobiotin and lucifer yellow could transfered in wild type cells, but only neurobiotin could transdered in mutant type cells.Conclusions:To our knowledge, Cx50V44A is reported as a novel mutation related to suture-sparing congenital nuclear cataracts. These findings suggest that amino acid44in Cx50is important for hemichannel and gap junction function.