Specific interaction between lens MIP/Aquaporin-0 and two members of the γ-crystallin family

Lens major intrinsic protein (MIP), also known as Aquaporin 0, is the most abundant protein of the ocular lens fiber membrane. It belongs to an ancient family of membrane channel proteins [1-3]. Mutations in the MIP gene have been linked to genetic cataracts in mice and humans, suggesting an important role for MIP in maintaining lens transparency [49]. MIP may play a role in reducing the interfiber space, as tightly packed fibers are required for lens transparency and vacuolated fibers are observed in the mouse mutant lenses. Four mouse MIP mutations, including a point mutation at amino acid 51 (A51P, lop), replacement of the last 61 amino acids (amino acids 203 to 263) at the MIP C-terminus by a transposon sequence (Cat Fr), deletion of MIP amino acids 121 to 175 (Hfi) and deletion of amino acids 46 to 49 (Cat) result in autosomal dominant cataracts and MIP trapping in the endoplasmic reticulum without being inserted into the plasma membrane [4-7]. Similarly, two different point mutations in MIP identified in two human cataract families (E134G, T138R) do not integrate into the plasma membrane when expressed in Xenopus oocytes [8,9]. Water channel activity measurements of reconstituted lens membrane vesicles derived from Cat Fr or MIP null mice, showed a marked decrease in water channel activity compared with those from the wild type lens [10,11]. Severe changes in lens fiber structure with dominant phenotype are observed in MIP mutant mice, suggesting there maybe additional functions for MIP in the lens, such as maintenance of fiber structure and arrangement required for optimal focusing of the lens [11,12]. MIP forms pH and Ca dependent water channels when expressed in Xenopus oocytes [13,14] and pH dependent voltage dependent channels in mammalian and insect cells [15]. MIP voltage dependent channels in lipid bilayer vesicles are regulated by protein kinase A dependent phosphorylation [16]. MIP may also function as an adhesion molecule and may play a role in gap junction formation [17-21]. MIP localizes to thin junctions of lens fibers, where it appears to be in a closed water pore configuration [22,23]. Both the N-terminus and the C-terminus of MIP are located in the cytoplasmic side of the plasma membrane. The MIP C-terminal polypeptide may play an important role in its physiological function as it is cleaved in cataractogenesis and aging [24-28], is serine phosphorylated [29], and may interact with calmodulin [30,31]. In our previous report, we demonstrated that γE-crystallin, a soluble protein that is also specifically expressed in lens fibers, is an MIP binding protein that interacts with the MIP C-terminal domain. We also demonstrated that this specific interaction results in the recruitment of γE-crystallin to the ©2005 Molecular Vision