Developmental Regulation of the Direct Interaction between the Intracellular Loop of Connexin 45.6 and the C Terminus of Major Intrinsic Protein (Aquaporin-0)

Abstract

The eye lens is dependent upon a network of gap junction-mediated intercellular communication to facilitate its homeostasis and development. Three gap junction-forming proteins are expressed in the lens of which two are in lens fibers, namely connexin (Cx) 45.6 and 56. Major intrinsic protein (MIP), also known as aquaporin-0 (AQP0), is the most abundant membrane protein in lens fibers. However, its role in the lens is not clear. Our previous studies show that MIP(AQP0) associates with gap junction plaques formed by Cx45.6 and Cx56 during the early stages of embryonic chick lens development but not in late embryonic and adult lenses. We report here that MIP(AQP0) directly interacts with Cx45.6 but not with Cx56. We further identified the intracellular loop of Cx45.6 as the interacting domain for the MIP(AQP0) C terminus. Surface plasmon resonance experiments indicated that the C-terminal domain of MIP(AQP0) interacts with two binding sites within the intracellular loop region of Cx45.6 with a K(D(app)) of 7.5 and 10.3 microm, respectively. The K(D(app)) for the full-length loop region is 7.7 microm. The cleavage at the intracellular loop of Cx45.6 was observed during lens development, and the C terminus of MIP(AQP0) did not interact with the loop-cleaved form of Cx45.6. Thus, the dissociation between these two proteins that occurs in the mature fibers of late lens development is likely caused by this cleavage. Finally this interaction had no impact on Cx45.6-mediated intercellular communication, suggesting that the Cx45.6-MIP(AQP0) interaction plays a novel unidentified role in lens fibers.