Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters
Role of positively charged amino acids in the M2D transmembrane helix of Ktr/Trk/HKT type cation transporters
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DOI：
10.4161/chan.4374
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发表时间：
2007-05-01
期刊：
影响因子：
3.3
通讯作者：
Uozumi, Nobuyuki
中科院分区：
文献类型：
--
作者：
Kato, Naoki;Akai, Masaro;Uozumi, Nobuyuki
研究表明，Ktr/Trk/HKT型转运蛋白已经从KcsA型简单K+通道的多个基因融合进化成至少跨越膜8次的蛋白质。在转运蛋白的第八个跨膜片段M2（D）中存在几个带正电荷的残基，但不存在K+通道。一些离子转运蛋白模型需要屏障来防止离子沿其电化学梯度自由扩散，并且转运蛋白孔内的带正电荷的残基可能会阻止转运蛋白成为通道。本研究通过检测大肠杆菌K+吸收速率，研究了这些阳性残基在三种Ktr/Trk/HKT型转运蛋白（集胞藻KtrB介导的K+单向转运蛋白、拟南芥AtHKT 1介导的Na+单向转运蛋白和小麦TaHKT 1介导的K+/Na+同向转运蛋白）中的功能作用。coli的卵母细胞电生理测定和E.大肠杆菌和酵母菌。M2（D）片段中部附近的保守Arg是KtrB和植物HKT的K+转运活性所必需的。TaHKT 1中几个阳性残基的组合替换表明，在许多K+通道中保守的M2（D）开始处的阳性残基也有助于阳离子转运活性。该正残基和保守的Arg都面向离子传导孔侧。我们介绍了一个预测氨基酸相互作用的原子级同源模型。基于实验结果和模型，我们提出了一个盐桥（S）之间存在的M2 D中的正残基和保守的负残基的孔区域，以减少由正残基（S）引起的对阳离子渗透的静电排斥。这种盐桥可以帮助稳定转运蛋白构型，也可以防止通道中发生的构象变化。
Studies suggest that Ktr/Trk/HKT-type transporters have evolved from multiple gene fusions of simple K+ channels of the KcsA type into proteins that span the membrane at least eight times. Several positively charged residues are present in the eighth transmembrane segment, M2(D), in the transporters but not K+ channels. Some models of ion transporters require a barrier to prevent free diffusion of ions down their electrochemical gradient, and it is possible that the positively charged residues within the transporter pore may prevent transporters from being channels. Here we studied the functional role of these positive residues in three Ktr/Trk/HKT-type transporters (Synechocystis KtrB-mediated K+ uniporter, Arabidopsis AtHKT1-mediated Na+ uniporter and wheat TaHKT1-mediated K+/Na+ symporter) by examining K+ uptake rates in E. coli, electrophysiological measurements in oocytes and growth rates of E. coli and yeast. The conserved Arg near the middle of the M2(D) segment was essential for the K+ transport activity of KtrB and plant HKTs. Combined replacement of several positive residues in TaHKT1 showed that the positive residue at the beginning of the M2(D), which is conserved in many K+ channels, also contributed to cation transport activity. This positive residue and the conserved Arg both face towards the ion conducting pore side. We introduced an atomic-scale homology model for predicting amino acid interactions. Based on the experimental results and the model, we propose that a salt bridge(s) exists between positive residues in the M2D and conserved negative residues in the pore region to reduce electrostatic repulsion against cation permeation caused by the positive residue(s). This salt bridge may help stabilize the transporter configuration, and may also prevent the conformational change that occurs in channels.