Mercury-Sensitive Residues and Pore Site in AQP3 Water Channel

Abstract

Water channel function of all aquaporins (AQPs) but AQP4 can be inhibited by mercurial reagents. Mercurial reagents are believed to bind specifically to cysteine residues and block the aqueous pore of AQPs. Because of the low homology of AQP3 to other AQPs, it is not certain whether the pore structure of AQP3 is similar to that of the others. Determination of mercury-sensitive cysteine residues in AQP3 and comparison with those in other AQPs will help to resolve this question. When AQP3 was expressed in Xenopus oocytes, incubation with 0.3 mM HgCl₂ decreased its osmotic water permability (P_f) by ∼30%. To identify the mercury-sensitive site, six individual cysteine residues in human AQP3 (at positions 11, 29, 40, 91, 174, and 267) were altered by site-directed mutagenesis. Mutants of C11S and C11A had a similar basal P_f to wild-type but acquired mercury resistance. Replacement of Cys-11 with Trp, which possesses a large side chain, did not change P_f. Mercurial inhibition of P_f was still observed in five other Cys-to-Ser mutants. These results suggest that Cys-11 is the mercury-sensitive residue in AQP3 and that this residue might be independent of water channel function. Mutation of Tyr-212, a position corresponding to the mercury-sensitive residues in AQP1 and AQP2, to cysteine enhanced the mercurial inhibition of P_f. Y212W had no water channel activity. Expression of AQP3 increased glycerol permeability (P_gly) 3.1-fold, whereas P_gly of Y212W-expressing oocytes was similar to P_gly of control oocytes. Cysteine mutation at Tyr-212 increased the inhibitory effect of mercury on P_gly. These results suggest that the structure of the aqueous pore of AQP3 resembles those of AQP1 and AQP2 and support the hypothesis that water and small molecules share a common pore in AQP3.