Németh-Cahalan KL et al. (2013), Regulation of AQP0 water permeability is enhanc...

XB-ART-46724

J Gen Physiol 2013 Mar 01;1413:287-95. doi: 10.1085/jgp.201210884.

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Regulation of AQP0 water permeability is enhanced by cooperativity.

Németh-Cahalan KL , Clemens DM , Hall JE .

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Aquaporin 0 (AQP0), essential for lens clarity, is a tetrameric protein composed of four identical monomers, each of which has its own water pore. The water permeability of AQP0 expressed in Xenopus laevis oocytes can be approximately doubled by changes in calcium concentration or pH. Although each monomer pore functions as a water channel, under certain conditions the pores act cooperatively. In other words, the tetramer is the functional unit. In this paper, we show that changes in external pH and calcium can induce an increase in water permeability that exhibits either a positive cooperativity switch-like increase in water permeability or an increase in water permeability in which each monomer acts independently and additively. Because the concentrations of calcium and hydrogen ions increase toward the center of the lens, a concentration signal could trigger a regulatory change in AQP0 water permeability. It thus seems plausible that the cooperative modes of water permeability regulation by AQP0 tetramers mediated by decreased pH and elevated calcium are the physiologically important ones in the living lens.

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Species referenced: Xenopus laevis
Genes referenced: aqp1 mip rnf2 tbx2 yap1

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	Figure 1. Topology and structure of AQP0. (A) Each AQP0 monomer is composed of six transmembrane domains (1–6) and five loops (A–E) in rainbow color (ROYGIBV). The pore is formed by two half helices and loops B and E (in green) folded back into the membrane. Major players in the cooperativity are H40 in the external loop A and S235 in the C terminus tail. (B) The tetramer is shown from the extracelluar side with the four monomers (blue and mustard) and the two CaMs (red). Both panels were generated with VMD from a molecular dynamics simulation based on crystal structures (deposited in the Protein Data Bank under accession no. 1TM8 for AQP0; Harries et al., 2004; and Protein Data Bank accession no. 1NWD for CaM; Yap et al., 2003) originally modeled together in Reichow and Gonen (2008).
	Figure 2. pH dose–response curve of the water permeability of AQP0. The solid black line is a fit to the Hill equation, with n = 4 (χ2/DOF = 0.0068 and R2 = 0.994).
	Figure 3. pH cooperativity. (A) Effect of acid and alkaline pH on the water permeability of mixtures of AQP0 (WT) and the H40C mutant. WT responds to acid pH, whereas the H40C mutant responds to alkaline pH. Mix 5:1 represents 10 ng AQP0 and 2 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.166), and mix 1:1 represents 10 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 10/20 = 0.5). The Pf of the mix 1:1 at pH 8.5 is greater than the Pf at pH 7.5, with a p-value of 5 × 10−5 (Student’s t test). The horizontal dotted line represents the water permeability of uninjected oocytes. (B) Fraction of increase plotted against the fraction of insensitive monomer. (For acid pH, the H40C mutant is the insensitive monomer. For alkaline pH, WT is the insensitive monomer.) Experimental results for acid pH are plotted as red squares and are well fit by a curve calculated from the binomial distribution of monomers randomly partitioning into each tetramer and the assumption that a single insensitive monomer renders the whole tetramer insensitive to acid pH (dashed red line; χ2 = 0.840). The curve assuming that two insensitive monomers are required to render the tetramer insensitive to acid pH (dashed black line; χ2 = 8.88) does not fit the experimental data. Experimental results for alkaline pH are plotted as green circles and are well fit by a straight line (the theoretical prediction assuming each monomer acts independently of the others in the tetramer; χ2/DOF = 0.071 and R2 = 0.910; solid green line). Each data point is the average of experiments using nine oocytes from three different batches.
	Figure 4. Alkaline pH increase of the Pf of AQP1 D48H is not cooperative. (A) Effect of acid and alkaline pH on the water permeability of AQP1 (WT) and a mutant D48H. WT does not respond to either acid or alkaline pH, whereas the D48H mutant responds to alkaline pH. Mix 1:1 represents 5 ng AQP1 and 5 ng of mutant D48H (fraction of insensitive monomer = 5/10 = 0.5). The horizontal dotted line represents the water permeability of uninjected oocytes. (B) Fraction of increase plotted against the fraction of insensitive monomer. For alkaline pH, WT is the insensitive monomer. The fraction of increase in Pf induced by alkaline pH is proportional to the fraction of insensitive monomer, indicating a lack of cooperativity between insensitive (WT) and sensitive (D48H) monomers. The data are well fit by a straight black line (R2 = 0.99) indicating that each monomer in a tetramer acts independently of the others.
	Figure 5. Calcium cooperativity. (A) Effect of calcium concentration on the water permeability of mixed injection of AQP0 (WT) and a mutant S235D. Mix 1:1 represents 10 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 10/20 = 0.5), mix 1:5 represents 2 ng AQP0 and 10 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.17), mix 5:1 = 10 ng AQP0 and 2 ng of mutant H40C (fraction of insensitive monomer = 2/12 = 0.17), and mix 1:9 represents 1 ng AQP0 and 9 ng S235D. The left-hand panel shows the response of WT AQP0. Note that Pf increases when the calcium concentration is decreased. In contrast, an increased calcium concentration increases Pf in the S235D mutant. When the two are mixed so as to form hetero tetramers, even 20% of WT is sufficient to suppress completely the Pf increase induced by 5 mM calcium, whereas the proportional increase in Pf induced by lowering calcium remains. When the mixture contains 80% WT, the low calcium response is still present in proportion to the amount of WT, but the elevated calcium response is completely suppressed. The horizontal dotted line represents the water permeability of uninjected oocytes. (B) Fraction of increase plotted against the fraction of insensitive monomer. (For no Ca2+ response, S235D is the insensitive monomer. For 5-mM Ca2+ response, WT is the insensitive monomer.) Experimental results for 5-mM Ca2+ increase are plotted as purple triangles and are well fit by a curve calculated from the binomial distribution assuming one insensitive monomer is sufficient to render the whole tetramer insensitive and with a bias of 0.5 kT for WT to associate with mutants in a tetramer (Ding et al., 2005) (dashed purple curve; χ2 = 1.260). The dotted black curve is calculated assuming no bias and that one insensitive monomer is sufficient to render the entire tetramer insensitive to Ca2+ increase (χ2 = 3.133). Experimental data for zero calcium are plotted as blue squares and are well fit by a straight line (the theoretical prediction assuming each monomer acts independently of the others in the tetramer; χ2/DOF = 0.18774 and R2 = 0.557; blue straight line). Positive and negative errors are equal, but error bars are shown in only one direction to avoid clutter.

References [+] :

Ball, Post-translational modifications of aquaporin 0 (AQP0) in the normal human lens: spatial and temporal occurrence. 2004, Pubmed