Németh-Cahalan KL et al. (2007), Zinc modulation of water permeability reveals t...

XB-ART-36614

J Gen Physiol 2007 Nov 01;1305:457-64. doi: 10.1085/jgp.200709826.

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Zinc modulation of water permeability reveals that aquaporin 0 functions as a cooperative tetramer.

Németh-Cahalan KL , Kalman K , Froger A , Hall JE .

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We previously showed that the water permeability of AQP0, the water channel of the lens, increases with acid pH and that His40 is required (Németh-Cahalan, K.L., and J.E. Hall. 2000. J. Biol. Chem. 275:6777-6782; Németh-Cahalan, K.L., K. Kalman, and J.E. Hall. 2004. J. Gen. Physiol. 123:573-580). We have now investigated the effect of zinc (and other transition metals) on the water permeability of AQP0 expressed in Xenopus oocytes and determined the amino acid residues that facilitate zinc modulation. Zinc (1 mM) increased AQP0 water permeability by a factor of two and prevented any additional increase induced by acid pH. Zinc had no effect on water permeability of AQP1, AQP4 or MIPfun (AQP0 from killifish), or on mutants of AQP1 and MIPfun with added external histidines. Nickel, but not copper, had the same effect on AQP0 water permeability as zinc. A fit of the concentration dependence of the zinc effect to the Hill equation gives a coefficient greater than three, suggesting that binding of more than one zinc ion is necessary to enhance water permeability. His40 and His122 are necessary for zinc modulation of AQP0 water permeability, implying structural constraints for zinc binding and functional modulation. The change in water permeability was highly sensitive to a coinjected zinc-insensitive mutant and a single insensitive monomer completely abolished zinc modulation. Our results suggest a model in which positive cooperativity among subunits of the AQP0 tetramer is required for zinc modulation, implying that the tetramer is the functional unit. The results also offer the possibility of a pharmacological approach to manipulate the water permeability and transparency of the lens.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 aqp4 mip

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	Figure 1. (A) Effect of 1 mM ZnCl2 on the water permeability of AQP0, MIPfun, AQP1, and AQP4. Only AQP0 showed zinc-induced increase in water permeability (from 35.9 ± 2.5 to 62.3 ± 5.5 μm/s). (B) Effect of 1 mM ZnCl2 on the water permeability of mutants of AQP0, AQP1, and MIPfun. They all lacked the zinc sensitivity. *, different from control at a significance of P < 10−5 using the Student's t distribution. Unless otherwise noted, each data point is the average of experiments using nine oocytes from three different batches.
	Figure 2. (A) Effect of pH 6.5, 1 mM ZnCl2, NiCl2, or CuCl2 on the water permeability of AQP0. Zinc did not have any additional effect on water permeability at pH 6.5, suggesting that low pH and zinc act at the same site or sites to increase AQP0 water permeability. Nickel increased Pf (from 47 ± 4.2 to 77.3 ± 6.6 μm/s). By contrast, copper had no effect. , different from control at a significance of P < 10−5; *, not significantly different from control P > 0.5 using the Student's t distribution. (B) Dose–response curve of the water permeability of AQP0. Fitting with no weighting (continuous line) gives a Hill coefficient of three, suggesting a highly cooperative binding for zinc. Statistical parameters for the no-weighting fit are as follows: reduced ξ2 = 17.1, R2 = 0.9, ΔPfMax = 30.3 ± 4.8, n = 3.0 ± 1.2, K = 0.6 ± 0.1, Pf0 = 35.3 ± 2.7. Using instrumental weighting, where the points are weighted according to their standard errors (dotted curve) gives a Hill coefficient of 5. Statistical parameters for the weighted fit are as follows: reduced ξ2 = 1.4, R2 = 0.9, ΔPfMax = 25.4 ± 3.0, n = 5.1 ± 0.9, K = 0.54 ± 0.02, Pf0 = 35.3 ± 2.7. While both curves appear to fit the data well, the instrumental fit has considerably better statistics and also gives a greater Hill coefficient. Each data point is the average of experiments using nine oocytes from three different batches.
	Figure 3. (A) Effect of 1 mM ZnCl2 on the water permeability of coinjected AQP0 and a mutant insensitive to zinc H122Q or H40C. Mix 1:1 represents 10 ng of wild type and 10 ng of mutant H122Q (fraction of insensitive monomer = 10 divided by [10+10] = 0.5) and mix 5:1 represents 10 ng of wild type and 2 ng of mutant H122Q (fraction of insensitive monomer = 2 divided by [10+2] = 0.166). In both mixtures, the effect of zinc was dramatically diminished but the calcium sensitivity remained intact. Mix 1:1 represents 5 ng of wild type and 5 ng of mutant H40C. (B) Western Blot of uninjected oocyte membranes, AQP0, mutant H122Q, and mutant H40C. A band, lower than the 28 kD marker, was seen in AQP0, H122Q, and H40C and not seen in uninjected oocytes. The bar graphs represent the average of scanned bands from three different experiments. There was no noticeable difference between the wild type and the mutant expression level. (C) Theoretical curves predicting the factor of increase, assuming the monomers behave independently (gray dashed line) or one or two insensitive monomers can block zinc sensitivity (black continuous line and gray dotted line respectively). Experimental results are plotted as filled squares and are well fit by the one insensitive monomer curve. Each data point is the average of experiments using nine oocytes from three different batches.
	Figure 4. (A) Plots of biased binomial predictions of the factor of increase assuming that two insensitive monomers are required to render the heterotetramer insensitive to zinc. Curves are shown for incorporation of mutant AQP0 in the heterotetramer biased by 0, 1, 2, 3, or 4 kT per contact face. Only the 2 kT curve fits the experimental data. (B) Overview of AQP0 tetramer structure. His40 is shown in blue and His122 in red. The tetramer is shown from the extracellular side looking down the pores. The coordinates used to generate the figure were from the published structure of AQP0 (PDB: 6B0P). We used proteinexplorer.org (http://molvis.sdsc.edu/protexpl/frntdoor.htm).

References [+] :

Agre, Aquaporin CHIP: the archetypal molecular water channel. 1993, Pubmed, Xenbase