PROCESSING OF THE GAP JUNCTION PROTEIN CONNEXIN50 IN THE OCULAR LENS IS ACCOMPLISHED BY CALPAIN

Gap junction channel forming connexins share a common membrane topolog y which has four transmembrane spanning segments with the amino- and c arboxy termini both located on the cytoplasmic side. Both, mutation an d truncation of the carboxyl tail of some connexins have been shown pr eviously to have profound effects on channel function. Truncation of t he carboxyl tail of connexin50 (Cx50) and connexin46 (Cx46) occurs nat urally during the maturation of fiber cells in the mammalian lens. Thi s system therefore offers the unique opportunity to study not only the cleavage process but also the functional role played by the cleaved d omain, in a physiologically relevant context. As a first step, rye now report on the cleavage of the 70 kDa ovine isoform of Cx50. The calci um-activated neutral protease calpain (EC 3.4.22.17) was identified as the enzyme which removed a 32 kDa carboxyl portion from the Cx50 mole cule in mature lens fiber cells. The amino-terminal 38 kDa portion rem ained embedded in the plasma membrane and was isolated and visualized as channel structures. The amino-terminal sequence of the cleaved 32 k Da portion matched an interior portion of the published amino acid seq uence of the ovine Cx50 isoform. Thus, two closely spaced calpain clea vage sites were identified in the Cx50 molecule which were located car boxy-terminal from the predicted exit of the fourth transmembrane span ning segment by 62 or 72 amino acid residues, respectively. These data pro,ide the basic information required for the future construction of Cx50 mutants to explore the functional consequences of this cleavage.