Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons
Point mutations in the aromatic/arginine region in aquaporin 1 allow passage of urea, glycerol, ammonia, and protons
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DOI：
10.1073/pnas.0507225103
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发表时间：
2006-01-10
影响因子：
11.1
通讯作者：
Zeuthen, T
中科院分区：
文献类型：
--
作者：
Beitz, E;Wu, BH;Zeuthen, T
水特异的水通道蛋白(AQP)，如典型的哺乳动物AQP1，严格排除溶质、离子甚至质子的通过。据推测，这是由毛孔内的两个保守区域完成的，一对典型的天冬酰胺-脯氨酸-丙氨酸(NPA)基序(中央收缩)和一个芳香/精氨酸(Ar/R)收缩(外部收缩)。在这里，我们分析了三个残基(Phe-56、His-180和Arg-195)在大鼠AQP1 Ar/R收缩中的作用。分别用丙氨酸和缬氨酸残基(AQP1-H180A、AQP1-R195V和AQP1-H180A/R195V)单独或联合取代His-180和Arg-195不影响透水性。双突变体AQP1-H180A/R195V允许尿素通过。与预测的溶质大小区分一致，Phe-56和His-180(AQP1-F56A/H180A)的替换使Ar/R收缩的最大直径扩大了3倍，并使甘油和尿素得以通过。我们进一步证明，氨通过所有四个AQP1突变体，这是(I)通过酵母缺失菌株与氨的生长互补，(Ii)通过从外部溶液中吸收氨进入卵母细胞，以及(Iii)通过直接记录氨诱导的卵母细胞质子电流。出乎意料的是，AQP1-R195V和AQP1-H180A/R195V的Ar/R收缩中正电荷的去除似乎允许质子通过AQP1。结果表明，Ar/R收缩是决定溶质渗透性的主要关卡，而Ar/R收缩与Ar/R收缩并存，Ar/R收缩与Ar/R收缩并存。
Water-specific aquaporins (AQP), such as the prototypical mammalian AQP1, stringently exclude the passage of solutes, ions, and even protons. Supposedly, this is accomplished by two conserved regions within the pore, a pair of canonical asparagine-proline-alanine (NPA) motifs, the central constriction, and an aromatic/ arginine (ar/R) constriction, the outer constriction. Here, we analyzed the function of three residues in the ar/R constriction (Phe-56, His-180, and Arg-195) in rat AQP1. Individual or joint replacement of His-180 and Arg-195 by alanine and valine residues, respectively (AQP1-H180A, AQP1-R195V, and AQP1-H180A/ R195V), did not affect water permeability. The double mutant AQP1-H180A/R195V allowed urea to pass. In line with the predicted solute discrimination by size, replacement of both Phe-56 and His-180 (AQP1-F56A/H180A) enlarged the maximal diameter of the ar/R constriction 3-fold and enabled glycerol and urea to pass. We further show that ammonia passes through all four AQP1 mutants, as determined (i) by growth complementation of yeast deletion strains with ammonia, (ii) by ammonia uptake from the external solution into oocytes, and (iii) by direct recordings of ammonia induced proton currents in oocytes. Unexpectedly, removal of the positive charge in the ar/R constriction in AQP1-R195V and AQP1-H180A/R195V appeared to allow the passage of protons through AQP1. The data indicate that the ar/R constriction is a major checkpoint for solute permeability, and that the exquisite electrostatic proton barrier in AQPs comprises both the NPA constriction as well as the ar/R constriction.