Mercury chloride decreases the water permeability of aquaporin‐4‐reconstituted proteoliposomes

Abstract

Background information . Mercurials inhibit AQPs (aquaporins), and site‐directed mutagenesis has identified Cys 189 as a site of the mercurial inhibition of AQP1. On the other hand, AQP4 has been considered to be a mercury‐insensitive water channel because it does not have the reactive cysteine residue corresponding to Cys 189 of AQP1. Indeed, the osmotic water permeability ( P f ) of AQP4 expressed in various types of cells, including Xenopus oocytes, is not inhibited by HgCl 2 . To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His‐tagged rAPR4 (rat AQP4) M23 was expressed in Saccharomyces cerevisiae , purified with an Ni 2+ ‐nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method. Results . The water permeability of AQP4 proteoliposomes with or without HgCl 2 was measured with a stopped‐flow apparatus. Surprisingly, the P f of AQP4 proteoliposomes was significantly decreased by 5 μM HgCl 2 within 30 s, and this effect was completely reversed by 2‐mercaptoethanol. The dose‐ and time‐dependent inhibitory effects of Hg 2+ suggest that the sensitivity to mercury of AQP4 is different from that of AQP1. Site‐directed mutagenesis of six cysteine residues of AQP4 demonstrated that Cys 178 , which is located at loop D facing the intracellular side, is a target responding to Hg 2+ . We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions . Our results suggest that mercury inhibits the P f of AQP4 by mechanisms different from those for AQP1 and that AQP4 may be gated by modification of a cysteine residue in cytoplasmic loop D.