Ion permeation of AQP6 water channel protein -: Single-channel recordings after Hg2+ activation
Ion permeation of AQP6 water channel protein -: Single-channel recordings after Hg2+ activation
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DOI：
10.1074/jbc.m204258200
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发表时间：
2002-08-09
影响因子：
4.8
通讯作者：
Yasui, M
中科院分区：
文献类型：
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作者：
Hazama, A;Kozono, D;Yasui, M
水通道蛋白6（Aquaporin-6，AQP 6）是一种具有阴离子通透性的细胞内水通道，可被低pH或HgCl 2激活。在这里，我们提出了直接的证据，AQP 6通道门控使用膜片钳技术。非洲爪蟾卵母细胞中表达的AQP 6的细胞贴附贴片记录表明，AQP 6是由10 pm HgCl 2诱导的具有中等电导（在100 mm NaCl中为49皮西门子）的门控通道。电流-电压关系是线性的，并且在任何给定的电压下开放概率是相当恒定的，表明Hg 2+诱导的AQP 6电导是电压无关的。切除的外向补丁记录显示快速激活AQP 6通道后立即应用10 μ M氯化汞。减少Na+和Cl-浓度从100至30毫米并没有改变汞2+诱导的AQP 6电流的逆转电位，这表明Na+是作为渗透性的Cl-。Na-22（+）内流测量进一步证实了Hg 2+诱导的AQP 6电流的Na+渗透性。定点突变确定Cys-155和Cys-190残基作为Hg 2+激活位点，用于水渗透性和离子电导。Hg 2+诱导电导的浓度-响应曲线的希尔系数为1.1 +/- 0.3。这些数据提供了在单通道水平上AQP 6通道门控的第一个证据，并表明每个单体包含基于Hg 2+结合位点数量和Hg 2+激活通道动力学的离子孔区域。
Aquaporin-6 (AQP6) has recently been identified as an intracellular vesicle water channel with anion permeability that is activated by low pH or HgCl2. Here we present direct evidence of AQP6 channel gating using patch clamp techniques. Cell-attached patch recordings of AQP6 expressed in Xenopus laevis oocytes indicated that AQP6 is a gated channel with intermediate conductance (49 picosiemens in 100 mm NaCl) induced by 10 pm HgCl2. Current-voltage relationships were linear, and open probability was fairly constant at any given voltage, indicating that Hg2+-induced AQP6 conductance is voltage-independent. The excised outside-out patch recording revealed rapid activation of AQP6 channels immediately after application of 10 muM HgCl2. Reduction of both Na+ and Cl- concentrations from 100 to 30 mm did not shift the reversal potential of the Hg2+-induced AQP6 current, suggesting that Na+ is as permeable as Cl-. The Na+ permeability of Hg2+-induced AQP6 current was further demonstrated by Na-22(+) influx measurements. Site-directed mutagenesis identified Cys-155 and Cys-190 residues as the sites of Hg2+ activation both for water permeability and ion conductance. The Hill coefficient from the concentration-response curve for Hg2+-induced conductance was 1.1 +/- 0.3. These data provide the first evidence of AQP6 channel gating at a single-channel level and suggest that each monomer contains the pore region for ions based on the number of Hg2+-binding sites and the kinetics of Hg2+-activation of the channel.