Aquaporins increase the water permeability in many cell types across many species. We investigated the effects of external pH and Ca 2؉ on water permeability of Xenopus oocytes injected with aquaporin cRNA by measuring the rate of swelling in hypotonic solutions. Lowering pH to 6.5 increased the water permeability of aquaporin (AQP0) 3.4 ؎ 0.4-fold. Diethylpyrocarbonate pretreatment increased water permeability 4.2 ؎ 0.5-fold and abolished pH sensitivity, suggesting that the pH regulation is mediated by an external histidine. Lowering Ca 2؉ increased water permeability 4.1 ؎ 0. acts at an internal site. Three different calmodulin inhibitors each increased AQP0 water permeability, suggesting that Ca 2؉ may act through calmodulin. None of the above altered the water permeability induced by AQP1 or AQP4. Because the greatest change in AQP0 water permeability is in the normal pH range found in the lens (7.2-6.5), this paper provides evidence for regulation of an aquaporin by pH under physiological conditions.The major intrinsic protein (MIP, now designated aquaporin 0 and abbreviated AQP0) 1 of the optical lens was the first sequenced member of the aquaporins, an ancient family of proteins found in bacteria, plants, and animals (1-4). Preston et al. (5) discovered that CHIP 28 (now called aquaporin 1 (AQP1)), a protein abundant in red blood cells, facilitates the diffusion of water across the plasma membrane when expressed in Xenopus oocytes. AQP4 (previously called MIWC for mercurial-insensitive water channel) is expressed strongly in the brain and kidney collecting duct (6, 7). Work in several laboratories subsequently demonstrated that many members of the aquaporin family facilitate the diffusion of water and other nonelectrolytes (3, 8 -12). Among the aquaporins, AQP0 forms a water channel with a relatively low water permeability (13,14); the water permeability per molecule is 40 times higher for AQP1 (13,15). The structural basis of this large difference in water permeability is unknown. AQP0 and AQP1 form tetrameric arrays in their native membranes and when reconstituted in lipid vesicles (6 -18). AQP0, AQP1, and AQP4 share ϳ40% sequence identity with each other. Attempts to increase AQP0 water permeability by exchanging parts of AQP0 for corresponding parts of AQP1 have been ineffective (19).Although low in water permeability per molecule, AQP0 comprises more than 60% of the membrane protein in the normal vertebrate lens and therefore provides the major permeability pathway for water movement across the membranes of lens fiber cells. If it is defective or missing from an otherwise normal lens, a cataract results (20, 21). In a chimeric mouse model, cataract can be prevented by the presence of 20% normal cells, which presumably supply the requisite AQP0 (22). The role of AQP0 in maintaining normal lens conditions is uncertain, but it likely facilitates the intrinsic circulation of fluid in the lens that maintains lens transparency and homeostasis in the absence of blood vessels (23). pH and Ca 2ϩ are likely ca...

Aquaporins facilitate the diffusion of water across cell membranes. We previously showed that acid pH or low Ca2+ 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, Ca2+ sensitivity was lost but pH sensitivity was maintained. These results demonstrate that Ca2+ and pH modulation are separable and arise from processes on opposite sides of the membrane.

Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, our understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudo-atomic structure of full-length mammalian aquaporin-0 (AQP0, Bos Taurus) in complex with CaM using electron microscopy to understand how this signaling protein modulates water channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.

These results suggest that Aqp0a is the primary water channel of the lens and that Aqp0b, though possibly a secondary water channel, has an unidentified function in the lens.

In the zebrafish lens, the two closely related AQP0s have acquired distinct functions that are both necessary for lens development and clarity. Regulation of AQP0 water permeability, a well-studied phenomenon in vitro, may be physiologically relevant in the living lens.