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 KD(app) of 7.5 and 10.3 μm, respectively. The KD(app) for the full-length loop region is 7.7 μm. 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. 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 KD(app) of 7.5 and 10.3 μm, respectively. The KD(app) for the full-length loop region is 7.7 μm. 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. The vertebrate eye lens is a valuable model system in the study of the function and regulation of gap junctions. The lens is an avascular organ formed by an anterior epithelial cell layer with highly differentiated fiber cells constituting the remaining lenticular mass. Mitotically active epithelial cells at the lens equator differentiate to give rise to the lens fibers, which lose intracellular organelles and accumulate high concentrations of soluble proteins known as crystallins (1Beebe D.C. Piatigorsky J. Dev. Biol. 1981; 84: 96-101Crossref PubMed Scopus (31) Google Scholar). With the loss of cellular organelles, lens fibers lose the ability to support an active metabolism. In addition, there is no blood supply inside the lens. To maintain their metabolic activities and homeostasis, cells inside the lens depend fully on extensive networks of gap junction-mediated intercellular communications with the cells on the lens surface (2Goodenough D.A. Goliger J.A. Paul D.L. Annu. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1088) Google Scholar). Gap junctions are channels connecting neighboring cells and allowing passage of small molecules (molecular mass, ≤1,000 Da) such as small metabolites, ions, and second messengers between the cytoplasm of two adjacent cells (3Kumar N. Gilula N.B. Cell. 1996; 84: 381-388Abstract Full Text Full Text PDF PubMed Scopus (1652) Google Scholar). The structural components of gap junctions are membrane proteins of a multigene family called connexins. All connexins have four conserved transmembrane domains and two extracellular loops, whereas their intracellular loops and C termini are the most variable regions (2Goodenough D.A. Goliger J.A. Paul D.L. Annu. Rev. Biochem. 1996; 65: 475-502Crossref PubMed Scopus (1088) Google Scholar). Three types of connexins are expressed in lens. Junctions between lens epithelial cells contain predominately Cx43 1The abbreviations used are: Cx, connexin; AQP0, aquaporin-0; CEF, chick embryonic fibroblast; GST, glutathione S-transferase; LY, Lucifer yellow; MIP, major intrinsic protein; RD, rhodamine dextran; SPR, surface plasmon resonance; Ni-NTA, nickel-nitrilotriacetic acid; PBS, phosphate-buffered saline. in most of the animal species (4Beyer E.C. Kistler J. Paul D.L. Goodenough D.A. J. Cell Biol. 1989; 108: 595-605Crossref PubMed Scopus (415) Google Scholar), whereas in human and rodent Cx50 and Cx46 are co-expressed predominately in differentiated fiber cells (5White T.W. Bruzzone R. Goodenough D.A. Paul D.L. Mol. Biol. Cell. 1992; 3: 711-720Crossref PubMed Scopus (273) Google Scholar, 6Paul D.L. Ebihara L. Takemoto L.J. Swenson K.I. Goodenough D.A. J. Cell Biol. 1991; 115: 1077-1089Crossref PubMed Scopus (501) Google Scholar). Distinctly different physiological properties of gap junction channels formed by individual types of connexins confer unique functions in maintaining normal lens physiology (7Donaldson P.J. Dong Y.M. Roos M. Green C. Goodenough D.A. Kistler J. Am. J. Physiol. 1995; 38: C590-C600Crossref Google Scholar). Targeted deletion of Cx46 results in nuclear cataracts (8Gong X. Li E. Klier G. Huang Q. Wu Y. Lei H. Kumar N.M. Horwitz J. Gilua N.B. Cell. 1997; 91: 833-843Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar) while deletion of Cx50 causes microphthalmia and pulverulent cataracts (9White T.W. Goodenough D.A. Paul D.L. J. Cell Biol. 1998; 143: 815-825Crossref PubMed Scopus (301) Google Scholar). This implies that Cx46 and Cx50 are likely to be differentially regulated by post-translational modifications and interaction with protein partners for the fulfillment of their unique roles in lens (10Thomas M.A. Huang S. Cokoja A. Riccio O. Staub O. Suter S. Chanson M. Biol. Cell. 2002; 94: 445-456Crossref PubMed Scopus (41) Google Scholar). Another major fiber membrane protein, MIP, also known as aquaporin-0 (AQP0), belongs to a family of water transporter proteins including aquaporin-1 in kidney (11Fischbard J. Investig. Opthalmol. Vis. Sci. 1995; 36: 758-763PubMed Google Scholar). MIP(AQP0) is the most abundant membrane protein in lens fibers, yet its function is still not clear. MIP(AQP0) does not form gap junction-like channels in the Xenopus oocyte pairing assay and baby hamster kidney cell transfection assay (12Kushmerick C. Rice S.J. Baldo G.J. Haspel H.C. Mathias R.T. Exp. Eye Res. 1995; 61: 351-362Crossref PubMed Scopus (76) Google Scholar, 13Miller A.G. Zampighi G.A. Hall J.E. J. Membr. Biol. 1992; 128: 91-102PubMed Google Scholar). In addition, MIP(AQP0) has a very limited ability to transport water compared with other members of the family (14Nemeth-Cahalan K.L. Hall J.E. J. Biol. Chem. 2000; 275: 6777-6782Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 15Virkki L.V. Cooper G.J. Boron W.F. Am. J. Physiol. 2001; 281: R1994-R2003Crossref PubMed Google Scholar), implying that the protein may have novel functions in lens. Previous reports have shown that MIP(AQP0) appears to transiently associate with differentiating fiber gap junction plaques in the adult lens (16Yancey S.B. Koh K. Chung J. Revel J.P. J. Cell Biol. 1988; 106: 705-714Crossref PubMed Scopus (57) Google Scholar), localizing predominantly at the periphery of junctional domains when large junctional plaques are assembled. Association of non-connexin proteins with lens gap junctions has been reported in limited cases. The interaction has been documented between lens connexins and A. Kumar N.M. Mol. Biol. Cell. PubMed Scopus Google Scholar). Our previous has that MIP(AQP0) associates with fiber gap junctions formed by Cx45.6 and chick of Cx50 and in early embryonic and that these the C terminus of MIP(AQP0) J. Cell Sci. PubMed Scopus Google Scholar). The chick lens was as system as is very for allowing and study at stages of lens development H. D.C. Piatigorsky J. Dev. Biol. 1981; PubMed Scopus Google Scholar, Goodenough D.A. J. Cell Biol. PubMed Scopus Google Scholar, Goodenough D.A. J. Cell Biol. PubMed Scopus Google Scholar). In the for the that the C terminus of MIP(AQP0) directly interacts with the intracellular loop domain of Cx45.6. We that the dissociation of these two proteins in lens development was by the cleavage at the intracellular loop domain of Cx45.6. In addition, the interaction between MIP(AQP0) and Cx45.6 was not observed to be in the function of gap junction-mediated intercellular communication, suggesting that the of these two proteins plays a yet novel role in lens fibers. chick and for the in a was The and The was membrane was The was The and for experiments The was molecules Lucifer rhodamine and was All other of and proteins a intracellular loop domain of intracellular loop domain of C terminus of MIP(AQP0) as the intracellular loop of Cx45.6 intracellular loop of Cx56 C terminus of MIP(AQP0) was by chick MIP(AQP0) as a and the of and and and was the The protein was expressed in as J. Cell Sci. PubMed Scopus Google Scholar), with and and with which the proteins the C terminus of Cx45.6 C terminus of MIP(AQP0) as T.W. Goodenough D.A. Paul D.L. Mol. Biol. Cell. PubMed Scopus Google Scholar, X. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). a protein the intracellular loop of Cx45.6 was by the at the C-terminal of Cx45.6 loop domain to to facilitate the of the protein a To the at the of at of and of Cell cells at cells in with chick and cells on the with high Goodenough D.A. Investig. Vis. Sci. 1998; Google Scholar, Bruzzone R. C. and Scholar). protein cells with Cx45.6 Cx56 cells with two Cx45.6 and MIP(AQP0) cells with and cells and in and and at for and in and lens membrane was and chick as as and adult and of and of and of the intracellular loop C terminus domains and the C terminus as T.W. Goodenough D.A. Paul D.L. Mol. Biol. Cell. PubMed Scopus Google Scholar, X. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). cell used for of the MIP(AQP0) the of and at the of cells to the of and E. Scholar). was at for and the the was The of the was by to MIP(AQP0) protein in chick lens membrane as Eye Res. PubMed Scopus Google Scholar). and embryonic chick cells with at to binding to the The of the was and with the protein at to the protein and its interacting a the was to a and the was The with and and the binding proteins and components of the and by by was and to a as E. Scholar). was to to Cx45.6 protein the cell was to to MIP(AQP0) protein, which a at its C-terminal was to which The membrane with these in the of at The with and The the by in for and to and on of by with with for and with for The of proteins on was to the and in to the intracellular loop domain of Cx45.6 with at the of by the of at was to the terminus of to facilitate the of the on a Chem. Biol. 2001; PubMed Scopus Google Scholar). The is in resonance resonance the in of on the the full-length intracellular loop domain was at a of resonance the of between the and a in a system a proteins the C terminus of MIP(AQP0) at the indicated concentrations the and the in and with at was used at a of with the of of in the the protein was with the indicated of at for to The Cx45.6 the intracellular loop domain was used as a to that the had been to the to as the was at the to a surface to that are caused by in the of the and G. Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google Scholar). cells on with Cx45.6 and MIP(AQP0) as of MIP(AQP0) and cells in for at for in PBS, cells in normal and in for of of and its intracellular loop domain in and at four for in and with in for at four for in PBS, cells with in for at four in for a was a with a of The a was at by an and rhodamine was at with a The used for and for rhodamine for Gap assay was on a J.E. Exp. Cell Res. PubMed Scopus Google Scholar). cells in the of two types of (molecular mass, and (molecular mass, Da) (molecular mass, is large to gap junction channels and as a for the cells cells with Cx45.6 Cx45.6 MIP(AQP0) with for and in to the cells that with a for cells with and in for The results with an in which and be with the for and be with the for MIP(AQP0) with Cx45.6 but with have shown that and Cx56 form a on the membrane of chick lens fiber cells in and that the C terminus of MIP(AQP0) Cx45.6 and Cx56 embryonic lens membrane J. Cell Sci. PubMed Scopus Google Scholar). Cx45.6 and Cx56 in the lens J. Cell Sci. PubMed Scopus Google Scholar, Goodenough D.A. Sci. S. A. 1996; PubMed Scopus Google Scholar), is to this system to which of these two fiber connexins interacts with To this Cx45.6 Cx56 was expressed in a cell was by Cx45.6 Cx56 Goodenough D.A. Investig. Vis. Sci. 1998; Google Scholar, Bruzzone R. C. and Scholar). protein the C terminus of MIP(AQP0) was used in experiments with membrane cells Cx45.6 Cx56 protein observed in the of the MIP(AQP0) C terminus cells was to be Cx45.6 by protein was by MIP(AQP0) C terminus cells and in a of Cx56 in the of of Cx45.6 and Cx56 in cell observed by and In to the binding of the C other domains of such as the terminus two intracellular loops, may interact with Cx56. However, domains have very limited in which such J. Cell Sci. PubMed Scopus Google Scholar, S. Sci. S. A. 1996; PubMed Scopus Google Scholar). results that MIP(AQP0) does not directly interact with Cx56 and that the of Cx56 in of lens is of its with Cx45.6 J. Cell Sci. PubMed Scopus Google Scholar). was to the interaction between MIP(AQP0) and Cx45.6. MIP(AQP0) a at its C Cx45.6 was by membrane to with and protein with a of was also and to be MIP(AQP0) by Cx45.6 was in of that was used to MIP(AQP0) and of MIP(AQP0) and Cx45.6 indicated by of cell membrane and and In to MIP(AQP0) and two proteins also by the by but not by the This may be by the that MIP(AQP0) is to to proteins in cells that these proteins contain to that of that may be by the results a interaction between MIP(AQP0) and Cx45.6 in and in of Cx45.6 MIP(AQP0) which of Cx45.6 protein interacts with proteins domains of Cx45.6 We observed that when a protein the intracellular loop of Cx45.6 was with lens protein shown in a protein with of was and as MIP(AQP0) by limited of MIP(AQP0) in the whereas the protein was in the lens proteins in the of lens membrane by the Cx45.6 intracellular loop protein a a protein the C-terminal domain of Cx56 was used in and no of MIP(AQP0) observed in the by with the The of MIP(AQP0) in lens is shown in Finally the C terminus of Cx45.6 to MIP(AQP0) as indicated by the of MIP(AQP0) in the by a protein the C terminus of Cx45.6 not The results that the intracellular loop domain of Cx45.6 directly to The and of the of Cx45.6 with the C of was to the interaction between MIP(AQP0) and Cx45.6 Chem. Biol. 2001; PubMed Scopus Google Scholar). The full-length intracellular loop region of Cx45.6 was on surface of a adjacent surface was and with protein the MIP(AQP0) C terminus was was observed on the but not on the a between the full-length intracellular loop of Cx45.6 and the C terminus of MIP(AQP0) To further the binding of Cx45.6 on its loop the intracellular loop of Cx45.6 and a The four to adjacent of a and C terminus protein was the surface MIP(AQP0) with the and but did not interact with the The observed on the and are to as which are caused by small in between the and that a rise in at the of the that is for the of the and to at the of the J. Mol. PubMed Scopus Google Scholar). are not of a was the as a no binding was observed This that the interaction between the MIP(AQP0) C terminus and the Cx45.6 loop domain is the the of the lens fiber cells is at lens surface to at the of the lens of differentiated fiber cells R.T. G. J. Physiol. 1991; PubMed Scopus Google Scholar). To the intracellular the between MIP(AQP0) and a binding was at We observed that the to did not have on the by the between the MIP(AQP0) C terminus and the suggesting that the are of the during fiber cell also had no on the interaction between Cx45.6 and MIP(AQP0) when the of MIP(AQP0) protein was used not To the that the of for the was caused by of the and to the of the an for the intracellular loop domain of Cx45.6 at two different and was a with and The loop to the and but not to the and This that the was the and that the was in the that was by the Our also that the by the loop are within the and of the intracellular loop of Cx45.6. these results that the C terminus of MIP(AQP0) directly interacts with the and of the intracellular loop domain of and this interaction at the physiological in the differentiated lens fibers. at the and of of with on the C of MIP(AQP0) C terminus interacts with and of the intracellular loop of experiments to these two regions interact with the on MIP(AQP0) C terminus two different sites MIP(AQP0) C terminus protein was with by the with the The MIP(AQP0) C terminus binding to the in a binding between MIP(AQP0) C terminus and was also by the in a In the interaction between the protein and the was by a of the a with the did not the interaction between MIP(AQP0) and not Thus, that the interaction two binding sites on the loop domain of Cx45.6 and on the C terminus of concentrations of MIP(AQP0) C terminus protein a surface with full-length Cx45.6 loop The the surface was the on and the for MIP(AQP0) binding to full-length Cx45.6 loop and are Three of these experiments and the shown in Cx45.6 and by the of the at MIP(AQP0) The to a function by and the KD(app) was as the of MIP(AQP0) to give binding G. Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, J. 1995; PubMed Scopus Google Scholar). The KD(app) of the interaction between MIP(AQP0) C terminus and Cx45.6 intracellular loop was to be 7.7 μm, between MIP(AQP0) C terminus and the was to be 7.5 μm, and between MIP(AQP0) C terminus and was to be 10.3 μm. studies that that the interaction between the intracellular loop of Cx45.6 and C terminus of MIP(AQP0) is and of of of Cx45.6 in the and between the C of MIP(AQP0) and have observed that between MIP(AQP0) and Cx45.6 directly with late stages of lens development and the of lens cell mature fibers J. Cell Sci. PubMed Scopus Google Scholar). shown in the of differentiated fibers of the lens to the dissociation of the interaction between MIP(AQP0) and Cx45.6. To the that the dissociation of these two proteins in differentiated lens fibers, the of Cx45.6 during lens development by of have that are for the intracellular loop and the C-terminal regions of Cx45.6 T.W. Goodenough D.A. Paul D.L. Mol. Biol. Cell. PubMed Scopus Google Scholar, X. S. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). Cx45.6 was and a during chick lens development. embryonic a form of Cx45.6 was by its C terminus indicated by The the form of Cx45.6 in stages suggesting that the cleavage is directly with lens fiber and The cleavage was most likely to at the intracellular loop domain of Cx45.6 as indicated by the with a of this was not by the its intracellular loop region To such cleavage the interaction of Cx45.6 and the C terminus of MIP(AQP0) was used to experiments embryonic lens the full-length Cx45.6 and the the C the of the full-length and the of Cx45.6 in lens membrane and the the full-length Cx45.6 was in the no of Cx45.6 was with the the of the Cx45.6 in the on the but no of this was observed in the of the proteins This a for the dissociation of the two proteins that have observed in late embryonic and adult in J. Cell Sci. PubMed Scopus Google Scholar). between MIP(AQP0) and Cx45.6 on Gap the interaction of MIP(AQP0) and Cx45.6 is in the function of gap junction channels in differentiating fibers, the was in which have a very limited cell shown in Cx45.6 and MIP(AQP0) with other in of Cx45.6 and MIP(AQP0) also the and most of these two proteins the two types of and Cx45.6 was a with a MIP(AQP0) The was as a The within the the that the cells was between and Cx45.6 MIP(AQP0) Our results that the interaction between Cx45.6 and MIP(AQP0) has no impact on Cx45.6-mediated intercellular This for the a interaction between the most abundant lens fiber membrane protein, and gap junction-forming Cx45.6 and the of the in differentiating and differentiated lens fibers. We the properties and physiological regulation of the the interaction as between MIP(AQP0) and Cx45.6. We further two binding sites on the and of the intracellular loop of Cx45.6 and a on the C terminus of the of the and the of the dissociation of the two proteins to cleavage of the intracellular loop of Cx45.6. the most abundant membrane protein in vertebrate lens fiber the function of MIP(AQP0) has been a of The MIP(AQP0) as the protein for lens fibers (14Nemeth-Cahalan K.L. Hall J.E. J. Biol. Chem. 2000; 275: 6777-6782Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar) protein MIP(AQP0) to the water transporter However, the MIP(AQP0) a water with water that for aquaporin-1 (14Nemeth-Cahalan K.L. Hall J.E. J. Biol. Chem. 2000; 275: 6777-6782Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar, 15Virkki L.V. Cooper G.J. Boron W.F. Am. J. Physiol. 2001; 281: R1994-R2003Crossref PubMed Google Scholar). be for by the of the protein expressed in lens fiber cells as of membrane has been that MIP(AQP0) plays other unidentified functions in lens. Our study reported here implies that this interaction appears not to be to the water of MIP(AQP0) L. C. S. A. G. J. Biol. Chem. 2002; Full Text Full Text PDF Scopus Google Scholar) report that the C terminus of the Cx45.6 binding does not with the transport of the by M. Kumar N. H. J. Cell Sci. 1998; Google Scholar) and by J. Cell Sci. PubMed Scopus Google Scholar) that the of MIP(AQP0) is with formed gap junctional plaques at the of the lens fiber cells are implying that the interaction between MIP(AQP0) and Cx45.6 may facilitate the of the Cx45.6 gap junction The between MIP(AQP0) and lens gap junctions has been by by Y. A. Scopus Google Scholar) in that the of fiber gap junctions is in and and that the of membrane in the of lens fibers as gap junctional plaques is in lens as compared with in lens. studies by M. S. S. Investig. Vis. Sci. Google Scholar) also show a of the and of MIP(AQP0) with a in gap junction during development. However, of MIP(AQP0) and Cx45.6 in cells did not in Cx45.6-mediated intercellular that interaction between the two proteins is to be in the of gap junction J. Investig. Opthalmol. Vis. Sci. PubMed Scopus (31) Google Scholar) show that is to the membrane by interacting with MIP(AQP0) in MIP(AQP0) may have a role in the of Cx45.6 on the lens fiber MIP(AQP0) does not fiber this interaction may to gap junctional plaques which to the unique functions of this connexin in cell and lens development (9White T.W. Goodenough D.A. Paul D.L. J. Cell Biol. 1998; 143: 815-825Crossref PubMed Scopus (301) Google Scholar). be to further the of MIP(AQP0) on gap junctions and on lens In this observed that Cx45.6 was during lens development. The cleavage was most likely in the intracellular loop domain on the of the form of Cx45.6 as by as as on the of of the by the intracellular loop the of the the loop domain by experiments are in the and but not in the implying that the cleavage is likely to within the of the which to the loss of for the the loop region of Cx45.6. have observed is the dissociation of the two proteins in the differentiated lens fibers of stages J. Cell Sci. PubMed Scopus Google Scholar). that the interaction be to the cleavage of the loop region of Cx45.6 that the MIP(AQP0) C terminus no associates to the form of Cx45.6. This is further by other results in this the cleavage at the intracellular loop domain of Cx45.6 of embryonic development the when the dissociation of MIP(AQP0) and Cx45.6 was In addition, the cleavage at stages when dissociation is also observed J. Cell Sci. PubMed Scopus Google Scholar). the lens is the surface and such a in is of the most during development R.T. G. J. Physiol. 1991; PubMed Scopus Google Scholar). in this study that MIP(AQP0) has a binding with Cx45.6 at as has at suggesting that the of lens fiber cells during is not the major that to such a In as shown in the model that in differentiating fiber cells in the lens MIP(AQP0) interacts with Cx45.6 at two sites the and of Cx45.6 intracellular loop shown to interact with the on the MIP(AQP0) C terminus. Cx45.6 loop most likely at the of the intracellular during lens cell and causes the dissociation and of the two proteins in differentiated mature fibers of lens most of the interaction is is of that the of the Cx45.6 loop domain be to further the properties and function of the between MIP(AQP0) and Cx45.6. We at the of at for for valuable and in We also C. for and the members of the for of the