Regulation of Aquaporin Water Permeability

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca 2 increase the water permeability of bovine AQP0 expressed in Xenopus oocytes. We now show that external histidines in loops A and C mediate the pH dependence. Furthermore, the position of histidines in different members of the aquaporin family can “tune” the pH sensitivity toward alkaline or acid pH ranges. In bovine AQP0, replacement of His40 in loop A by Cys, while keeping His122 in loop C, shifted the pH sensitivity from acid to alkaline. In the killifish AQP0 homologue, MIPfun, with His at position 39 in loop A, alkaline rather than acid pH increased water permeability. Moving His39 to His40 in MIPfun, to mimic bovine AQP0 loop A, shifted the pH sensitivity back to the acid range. pH regulation was also found in two other members of the aquaporin family. Alkaline pH increased the water permeability of AQP4 that contains His at position 129 in loop C. Acid and alkaline pH sensitivity was induced in AQP1 by adding histidines 48 (in loop A) and 130 (in loop C). We conclude that external histidines in loops A and C that span the outer vestibule contribute to pH sensitivity. In addition, we show that when AQP0 (bovine or killifish) and a crippled calmodulin mutant were coexpressed, Ca 2 sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca 2 and pH modulation are separable and arise from processes on opposite sides of the membrane. key words: calmodulin • MIP • histidine • lens • killifish I N T R O D U C T I O N Aquaporins (AQPs) facilitate the rapid and selective movement of small neutral molecules, such as water and glycerol, across cell membranes. AQP1 from red blood cells was the first member of the family shown to be a functional water channel (Preston et al., 1992). AQP0, the major intrinsic protein of the lens, is expressed only in terminally differentiated fiber cells. The killifish ( Fundulus heteroclitus ) lens homologue of AQP0, designated MIPfun, was recently cloned (Virkki et al., 2001). MIPfun has 70% sequence identity with bAQP0 and 46% with human AQP1 (hAQP1) and exhibits a water permeability much higher than that of bAQP0 but close to that of hAQP1 in the oocyte expression system. Another member, AQP4, functions as a water-selective channel in epithelial cells in the kidneycollecting duct as well as in brain astrocytes (Shi and Verkman, 1996). Rat AQP4 (rAQP4) has 37% identity with bAQP0 and 41% identity with hAQP1 and also exhibits water permeability close to that of hAQP1 in the oocyte expression system. Some AQPs are sensitive to change in pH, including AQP0 (Németh-Cahalan and Hall, 2000), AQP3 (Zeuthen and Klaerke, 1999), and AQP6 (Yasui et al., 1999), but bovine AQP0 (bAQP0) is the only water channel shown to be modulated also by low Ca 2 (Németh-Cahalan and Hall, 2000). Fig. 1 A shows the predicted topology of aquaporins, confirmed by X-ray (Sui et al., 2001) and electron crystallography (Murata et al., 2000) with six transmembrane helices (1–6) and five connecting loops (A–E). Two highly conserved loops (B and E) each contain the signature motif present in every aquaporin, asparagineproline-alanine (NPA), which forms one wall of the water pore. Fig. 1 B shows the sequence alignments of the critical regions that contribute to pH and Ca 2 sensitivity, loop A, loop C, and the COOH terminus tail. Loop A of bAQP0 contains the critical His40 residue, while two histidines are present in MIPfun at positions 39 and 43. In loop C, only bAQP0 and rAQP4 present an aligned His at positions 122 and 129, respectively. In both bAQP0 and MIPfun, the indicated sequence within the COOH terminus tail is atypical for a CaMbinding site, but fits the general requirement of an amphiphilic chain with alternation of positive charges (Peracchia and Girsch, 1989). Moreover, several reports demonstrate that calmodulin binds AQP0 (van den Eijnden-van Raaij et al., 1985, 1986; Peracchia and Girsch, 1989; Louis et al., 1990; Girsch and Peracchia, 1991; Swamy-Mruthinti, 2001). Address correspondence to James E. Hall, Department of Physiology and Biophysics, University of California, Irvine, CA 92697. Fax: (949) 824-3143; email: [email protected] Abbreviations used in this paper: AQP, aquaporin; bAQP0, bovine AQP-0; CaM, calmodulin; hAQP1, human AQP1; NPA, asparagine-prolinealanine; rAQP4, rat AQP4. on Jne 9, 2017 D ow nladed fom Published April 12, 2004