Németh-Cahalan KL et al. (2004), Molecular basis of pH and Ca2+ regulation of aq...

XB-ART-3702

J Gen Physiol 2004 May 01;1235:573-80. doi: 10.1085/jgp.200308990.

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Molecular basis of pH and Ca2+ regulation of aquaporin water permeability.

Németh-Cahalan KL , Kalman K , Hall JE .

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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.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 aqp4 mip tbx2

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	Figure 1. . The predicted membrane topology of AQPs. (A) In the hourglass model, confirmed by high-resolution structures, loops B and E containing highly conserved NPA motifs fold into the bilayer from opposite sides of the membrane to form the water pore. (B) Sequence alignment of AQPs (using ClustalW at http://clustalw.genome.ad.jp). In loop A, bAQP0 contains His40, MIPfun contains His39 and His43, and hAQP1 and rAQP4 both contain Asp48 and Asp47, respectively. In loop C, both bAQP0 and rAQP4 present a His at the position 122 and 129, respectively. In both bAQP0 and MIPfun, the C-tail contains a consensus CaM binding site with alternating hydrophobic (underlined) and positive residues (in bold).
	Figure 2. . Role of histidines in loop A on bAQP0 and MIPfun water permeability. (A) The permeability of bAQP0-injected oocytes increases by a factor of 1.8 at pH 6.5 and by a factor of 2.1 with no added Ca2+. MIPfun-injected oocytes have a water permeability 11 times higher than oocytes injected with the same quantity of AQP0 cRNA under control conditions. The permeability of MIPfun-injected oocytes increases by a factor of 2.2 at pH 8.5 and by a factor of 1.9 with no added Ca2+. (B) Moving the His39 to the position 40 restores the acid pH sensitivity, but replacing His43 does not affect the pH sensitivity. Acid pH has no effect on the water permeability of bAQP0/H40C-injected oocytes, but no added Ca2+ or alkaline pH increases the permeability by a factor of 1.8 and 1.9, respectively. Note the different scales for bAQP0 and MIPfun.
	Figure 3. . Role of histidines in loop C on bAQP0 and MIPfun water permeability. Adding a histidine at position 122 in MIPfun does not affect alkaline pH sensitivity, but the factor of increase is only 1.5 (32% less than wild-type increase). Removing the His122 in bAQP0 does not affect acid pH sensitivity but the factor of increase is only of 1.5 (17% less than wild-type increase). Note the different scales.
	Figure 4. . Effect of pH and Ca2+ on AQP4 and mutants of AQP1. (A) The water permeability of rAQP4-injected oocytes increases at alkaline pH by a factor of 1.9, but acid pH and low Ca2+ have no effect. (B) In contrast to hAQP1 wild-type, the water permeability of hAQP1/D48H-injected oocytes increases with alkaline pH by a factor of 1.7. (C) The water permeability of hAQP1/D48H/A130H-injected oocytes increases with acid pH and alkaline pH by a factor of 1.9 and 1.6, respectively.
	Figure 5. . Elimination of Ca2+ sensitivity of bAQP0, bAQP0/H40C and MIPfun by crippled CaM. (A) The water permeability of bAQP0 and crippled CaM coinjected oocytes is still increased by low pH but not by low Ca2+. (B) The water permeability of bAQP0/H40C and crippled CaM coinjected oocytes is not increased by low pH or low Ca2+. (C) The water permeability of MIPfun and crippled CaM coinjected oocytes is not increased by low pH or low Ca2+. Note the different scale for MIPfun.
	Figure 6. . The aqueous pore. (A) A cartoon shows the three different regions seen by a transiting water molecule: the outer vestibule, the maximum constriction near the NPA boxes, and the inner vestibule. Water molecules must change their orientations to pass from region to region. We propose that histidines (His) in loops A and C act on water in the outer vestibule under the influence of pH and that the COOH-terminal tail containing the CaM-binding site acts on water in the inner vestibule under the influence of Ca2+. (B) A section through the pore based on the crystal structure of AQP1 using the coordinates of Sui et al. (2001). The section shows the permeation pathway (with seven water molecules), the positions of Asp48 and Ala130 (homologues of His40 and His122 in bAQP0), and the NPA boxes. Ser249 is the last resolved residue visible in the crystal and is situated in the middle of the COOH terminus tail. This figure was prepared using Protein Explorer (http://proteinexplorer.org) (Martz, 2002).

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