title: Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes.
abstract: BACKGROUND
  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 HgCl2. To examine the direct effects of mercurials on AQP4 in a proteoliposome reconstitution system, His-tagged rAQP4 [corrected] (rat AQP4) M23 was expressed in Saccharomyces cerevisiae, purified with an Ni2+-nitrilotriacetate affinity column, and reconstituted into liposomes with the dilution method.
  
  
  RESULTS
  The water permeability of AQP4 proteoliposomes with or without HgCl2 was measured with a stopped-flow apparatus. Surprisingly, the P(f) of AQP4 proteoliposomes was significantly decreased by 5 microM HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The dose- and time-dependent inhibitory effects of Hg2+ 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 Hg2+. 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.
authors.affiliation: Department of Biochemistry and Integrative Medical Biology, School of Medicine, Keio University, Shinanomachi, Shinjyuku-ku, Tokyo 160-8582, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Yukutake
authors.forename: Shoji
authors.initials: S
authors.lastname: Tsuji
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Hirano
authors.forename: Takeshi
authors.initials: T
authors.lastname: Adachi
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Takahashi
authors.forename: Kazuo
authors.initials: K
authors.lastname: Fujihara
authors.forename: Peter
authors.initials: P
authors.lastname: Agre
authors.forename: Masato
authors.initials: M
authors.lastname: Yasui
authors.forename: Makoto
authors.initials: M
authors.lastname: Suematsu
mesh_terms: D000818:Animals; D051398:Aquaporin 1 / Q000378:metabolism; D051401:Aquaporin 4 / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D017612:Gold Compounds / Q000494:pharmacology; D006801:Humans; D008627:Mercuric Chloride / Q000378:metabolism / Q000494:pharmacology*; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000187:drug effects / Q000378:metabolism; D009995:Osmosis; D011510:Proteolipids / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D018030:Silver Compounds / Q000494:pharmacology; D014867:Water / Q000378:metabolism*; D014982:Xenopus laevis / Q000378:metabolism
chemical_list: D051401:Aquaporin 4; D017612:Gold Compounds; D011510:Proteolipids; D018030:Silver Compounds; C020485:proteoliposomes; D014867:Water; D051398:Aquaporin 1; D008627:Mercuric Chloride; D003545:Cysteine