Glycerol permeability of mutant aquaporin 1 and other AQP-MIP proteins: Inhibition studies

Abstract

In a recent work, we showed that the aquaporins 1 (AQP1) are permeable to certain small solutes such as glycerol. Here, we have further investigated the permeation pathway of glycerol through human AQP1 (hAQP1) by the use of mutants (C189S, H180A, H209A) and inhibitors such asP-chloromercuribenzene sulphonate (pCMBS), CuSO₄ or phloretin, in comparison with other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel γTIP and bacterial glycerol permease facilitator, G1pF. Glycerol movements were measured inXenopus laevis oocytes. Apparent glycerol permeability coefficients (P ^′_gly ) were calculated from the rates of oocyte swelling upon exposure to an isoosmotic medium containing an inwardly directed gradient of glycerol and from [³H]glycerol uptake measurements. SimilarP ^′_gly values were obtained for hAQP1 and hAQP2, 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. Pof hAQP2P injected oocytes waspCMBS and CuSO₄ sensitive. In contrast, theP ^′_gly value of γTIP was close to that of control, indicating that γTIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gaveP ^′_gly values similar to those obtained for wild hAQP1, indicating that these mutations did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (P _f) value decreased by 50%. The inhibitory effect ofpCMBS onP ^′_gly was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO₄ significantly inhibitedP ^′_gly of oocytes expressing hAQP1, hAQP1-C189S,-H180A, and -H209A mutants and had no effect onP ^′_gly of G1pF-injected oocytes. Phloretin was shown to inhibit by around 80% the glycerol fluxes of wild and mutant hAQP1, hAQP2 and to fully inhibit glycerol uptake in G1pF-injected oocytes.