All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus
All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus
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DOI：
10.1074/jbc.m507647200
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发表时间：
2005-12-16
影响因子：
4.8
通讯作者：
Bakker, EP
中科院分区：
文献类型：
--
作者：
Tholema, N;Vor der Brüggen, M;Bakker, EP
细菌Na+依赖性K+转运KtrAB系统的KtrB亚基属于K+转运蛋白超家族。这些蛋白质含有四个重复的结构域，每个结构域由两个跨膜螺旋组成，由一个假定的孔环（p环）连接。四个p-环在相当于K+通道中甘氨酸选择性过滤残基的位置处具有保守的甘氨酸残基。我们研究了这些甘氨酸是否也在KtrB中形成选择性过滤器。将溶藻弧菌KtrB的P环P-A至P-D的单个残基Gly（70）、Gly（185）、Gly（290）和Gly（402）替换为丙氨酸、丝氨酸或天冬氨酸。在大肠杆菌中，三种丙氨酸变体KtrB（A70）、KtrB（A185）和KtrB（A290）在KtrAB介导的K+摄取中保持实质性活性。该活性与对K+的亲和力降低2个数量级相关，对V-max几乎没有影响。还观察到其他三种变体的轻微活性：KtrB（A402）、KtrB（S70）和KtrB（D185）。在所有这些变体的情况下，K+运输的Na+依赖性的性质被保留。只有四个丝氨酸的变体介导的Na+的摄取，这些变体在其K+/Na+的选择性有很大的不同。对pBAD 18载体中克隆的ktrB的实验表明，单独的溶藻弧菌KtrB在E.杆菌它介导Na+非依赖性、缓慢、高亲和力和突变特异性K+摄取以及K+非依赖性Na+摄取。这些数据表明，KtrB包含一个选择性过滤器的K+离子和所有四个保守的p-环甘氨酸残基是这个过滤器的一部分。他们还表明，KtrA的作用在于赋予速度和离子耦合的KTR复合物。
The subunit KtrB of bacterial Na+- dependent K+- translocating KtrAB systems belongs to a superfamily of K+ transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly(70), Gly(185), Gly(290), and Gly(402) from p-loops P-A to P-D of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB(A70), KtrB(A185), and KtrB(A290) maintained a substantial activity in KtrAB-mediated K+ uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on V-max. Minor activities were also observed for three other variants: KtrB(A402), KtrB(S70), and KtrB(D185). With all of these variants, the property of Na+ dependence of K+ transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K+/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.