Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants
Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loop-per-subunit HKT transporters from plants
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DOI：
10.1073/pnas.082123799
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发表时间：
2002-04-30
影响因子：
11.1
通讯作者：
Uozumi, N
中科院分区：
文献类型：
--
作者：
Mäser, P;Hosoo, Y;Uozumi, N
植物HKT蛋白包括阳离子转运蛋白家族以及原核KtrB、TrkH和KdpA转运蛋白亚基和真菌Trk蛋白。这些转运蛋白在一个多肽中含有四个环结构域，与K+通道选择性过滤器具有远端同源性。在酵母和非洲爪蟾卵母细胞中的功能表达表明，小麦HKT 1介导Na+-偶联K+运输。然而，拟南芥AtHKT 1在真核表达系统中仅转运Na+。为了理解这种差异的分子基础，我们构建了一系列AtHKT 1/HKT 1嵌合体，并在预测对K+选择性至关重要的位置处对AtHKT 1和小麦HKT 1引入点突变。一个单点突变，Ser-68甘氨酸，足以恢复K+渗透AtHKT 1。HKT 1的回复突变，Gly-91为丝氨酸，废除了K+渗透性。AtHKT 1和HKT 1的P环A中的甘氨酸可以被建模为K+通道选择性过滤器GYG基序的第一个甘氨酸。通过水稻旁系同源物OsHKT 1和OsHKT 2中Ser-88和Gly-88的相互转化证实了这种过滤甘氨酸对于K+选择性的重要性。令人惊讶的是，所有已知的双子叶植物HKT同源物在P-环A的过滤位置处都具有丝氨酸，这表明这些蛋白质在植物中主要起Na+转运蛋白的作用，HKT蛋白质中的共转运与滤渣中的甘氨酸相关。这些数据提供了实验证据，HKT蛋白的选择性过滤器中的甘氨酸残基在结构上与K+通道的甘氨酸残基相关。
Plant HKT proteins comprise a family of cation transporters together with prokaryotic KtrB, TrkH and KdpA transporter subunits and fungal Trk proteins. These transporters contain four loop domains in one polypeptide with a proposed distant homology to K+ channel selectivity filters. Functional expression in yeast and Xenopus oocytes revealed that wheat HKT1 mediates Na+-coupled K+ transport. Arabidopsis AtHKT1, however, transports only Na+ in eukaryotic expression systems. To understand the molecular basis of this difference we constructed a series of AtHKT1/HKT1 chimeras and introduced point mutations to AtHKT1 and wheat HKT1 at positions predicted to be critical for K+ selectivity. A single-point mutation, Ser-68 to glycine, was sufficient to restore K+ permeability to AtHKT1. The reverse mutation in HKT1, Gly-91 to serine, abrogated K+ permeability. This glycine in P-loop A of AtHKT1 and HKT1 can be modeled as the first glycine of the K+ channel selectivity filter GYG motif. The importance of such filter glycines for K+ selectivity was confirmed by interconversion of Ser-88 and Gly-88 in the rice paralogues OsHKT1 and OsHKT2 Surprisingly, all HKT homologues known from dicots have a serine at the filter position in P-loop A, suggesting that these proteins function mainly as Na+ transporters in plants and that Na+/K+ symport in HKT proteins is associated with a glycine in the filter residue. These data provide experimental evidence that the glycine residues in selectivity filters of HKT proteins are structurally related to those of K+ channels.