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

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, H280A, H209A) and inhibitors such as P-chloromercuribenzene sulphonate (pCMBS), CuSO4 or phloretin, in comparison with Other AQP-MIP (where MIP denotes major intrinsic protein) proteins: hAQP2, plant water channel gamma TIP and bacterial glycerol permease facilitator, GlpF. Glycerol movements were measured in Xenopus 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 [H-3]glycerol uptake measurements. Similar P'(gly) values were obtained for hAQP1 and hAQP2, 6 to 8 times greater than control indicating that hAQP2 also transports glycerol. P'(gly) of hAQP2-injected oocytes was pCMBS and CuSO4 sensitive. In contrast, the P'(gly). value of gamma TIP was close to that of control, indicating that gamma TIP does not transport glycerol. The hAQP1-C189S, -H180A and -H209A mutants gave P'(gly) values similar to those obtained for wild hAQP1, indicating that did not affect glycerol movements. However, the H209A mutant has an osmotic water permeability coefficient (P-f) value decreased by 50%. The inhibitory effect of pCMBS on P'(gly) was maintained for the 2 His mutants and, more interestingly, was also conserved for the C189S mutant. CuSO4 significantly inhibited P'(gly) of oocytes expressing hAQP1, hAQP1-C189S, -H180A, and -H209A mutants and had no effect on P'(gly) of GlpF-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 GlpF-injected oocytes.