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

Yoshinori Yukutake, Shoji Tsuji, Yoshinori Hirano, Takeshi Adachi, Toshiyuki Takahashi, Kazuo Fujihara, Peter Agre, Masato Yasui, Makoto Suematsu

Research output: Contribution to journalArticle

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Abstract

Background information. Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys189 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 Cys189 of AQP1. Indeed, the osmotic water permeability (Pf) 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 rAPR4 (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 Pf of AQP4 proteoliposomes was significantly decreased by 5 μM 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 Cys178 which is located at loop D facing the intracellular side, is a target responding to Hg26+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the Pf 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.

Original languageEnglish
Pages (from-to)355-363
Number of pages9
JournalBiology of the Cell
Volume100
Issue number6
DOIs
Publication statusPublished - 2008 Jun

Fingerprint

Aquaporin 4
Mercuric Chloride
Mercury
Cysteine
Chlorides
Permeability
Aquaporins
Site-Directed Mutagenesis
Liposomes
Water
Mercaptoethanol
Xenopus
Oocytes
Saccharomyces cerevisiae
proteoliposomes

Keywords

  • Aquaporin-4 (AQP4)
  • Mercury
  • Oocyte
  • Proteoliposome
  • Water permeability

ASJC Scopus subject areas

  • Cell Biology

Cite this

Yukutake, Y., Tsuji, S., Hirano, Y., Adachi, T., Takahashi, T., Fujihara, K., ... Suematsu, M. (2008). Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. Biology of the Cell, 100(6), 355-363. https://doi.org/10.1042/BC20070132

Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. / Yukutake, Yoshinori; Tsuji, Shoji; Hirano, Yoshinori; Adachi, Takeshi; Takahashi, Toshiyuki; Fujihara, Kazuo; Agre, Peter; Yasui, Masato; Suematsu, Makoto.

In: Biology of the Cell, Vol. 100, No. 6, 06.2008, p. 355-363.

Research output: Contribution to journalArticle

Yukutake, Y, Tsuji, S, Hirano, Y, Adachi, T, Takahashi, T, Fujihara, K, Agre, P, Yasui, M & Suematsu, M 2008, 'Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes', Biology of the Cell, vol. 100, no. 6, pp. 355-363. https://doi.org/10.1042/BC20070132
Yukutake Y, Tsuji S, Hirano Y, Adachi T, Takahashi T, Fujihara K et al. Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. Biology of the Cell. 2008 Jun;100(6):355-363. https://doi.org/10.1042/BC20070132
Yukutake, Yoshinori ; Tsuji, Shoji ; Hirano, Yoshinori ; Adachi, Takeshi ; Takahashi, Toshiyuki ; Fujihara, Kazuo ; Agre, Peter ; Yasui, Masato ; Suematsu, Makoto. / Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes. In: Biology of the Cell. 2008 ; Vol. 100, No. 6. pp. 355-363.
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AB - Background information. Mercurials inhibit AQPs (aquaporins), and site-directed mutagenesis has identified Cys189 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 Cys189 of AQP1. Indeed, the osmotic water permeability (Pf) 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 rAPR4 (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 Pf of AQP4 proteoliposomes was significantly decreased by 5 μM 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 Cys178 which is located at loop D facing the intracellular side, is a target responding to Hg26+. We confirmed that AQP4 is reconstituted into liposome in a bidirectional orientation. Conclusions. Our results suggest that mercury inhibits the Pf 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.

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