Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes
Mercury chloride decreases the water permeability of aquaporin-4-reconstituted proteoliposomes
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DOI：
10.1042/bc20070132
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发表时间：
2008-06-01
影响因子：
2.7
通讯作者：
Suematsu, Makoto
中科院分区：
文献类型：
--
作者：
Yukutake, Yoshinori;Tsuji, Shoji;Suematsu, Makoto
背景资料。汞剂可抑制水通道蛋白（AQP），定点突变已将Cys（189）确定为汞剂抑制AQP 1的位点。另一方面，AQP 4被认为是汞不敏感的水通道，因为它不具有对应于AQP 1的Cys（189）的反应性半胱氨酸残基。事实上，HgCl 2不会抑制在包括非洲爪蟾卵母细胞在内的各种类型细胞中表达的AQP 4的渗透水渗透性（P-f）。为了研究汞对蛋白脂质体重组系统中AQP 4的直接影响，在酿酒酵母中表达了His标记的rAPR 4（rat AQP 4）M23，用Ni 2 + -nitrilotriacetate亲和柱纯化，并用稀释法重组成脂质体。用停流装置测定了有或无HgCl 2的AQP 4蛋白脂质体的水渗透性。令人惊讶的是，AQP 4蛋白脂质体的P-f显着降低5 μ M氯化汞在30秒内，这种效果是完全逆转的2-巯基乙醇。Hg 2+对AQP 4和AQP 1的抑制作用具有时间和浓度依赖性，提示AQP 4和AQP 1对汞的敏感性不同。对AQP 4的6个半胱氨酸残基的定点突变表明，位于D环面向细胞内侧的Cys（178）是Hg 2+的反应靶点。我们证实了AQP 4以双向取向重组到脂质体中。我们的研究结果表明，汞抑制AQP 4的P-f的机制与AQP 1不同，AQP 4可能是通过修饰细胞质环D中的半胱氨酸残基来门控的。
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 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 P-f of AQP4 proteoliposomes was significantly decreased by 5 mu M HgCl2 within 30 s, and this effect was completely reversed by 2-mercaptoethanol. The close- 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.