Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1
Changes in voltage activation, Cs+ sensitivity, and ion permeability in H5 mutants of the plant K+ channel KAT1
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DOI：
10.1073/pnas.93.15.8123
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发表时间：
1996-07-23
影响因子：
11.1
通讯作者：
Hedrich, R
中科院分区：
文献类型：
--
作者：
Becker, D;Dreyer, I;Hedrich, R
KAT 1是从高等植物拟南芥（Arabidopsis thaliana）克隆的电压依赖性内向整流K+通道[安德森，J.A.，Huprikar，S.美国，科钦湖五、卢卡斯，W。J. & Gaber，R. F.等人（1992）Proc. Acad. Sci. USA 89，3736-3740]。它与K+通道的Shaker超家族相关，其特征在于六个跨膜结构域（S1-S6）和S5和S6之间的推定成孔区（H5）。与Shaker相比，KAT 1中Pro-247和Pro-271之间的H5区含有14个额外的氨基酸[Aldrich，R. W.（1993）Nature（伦敦）362，107-108]。我们研究了引入H5的各种点突变，以确定电压依赖性植物和动物K+通道是否具有相似的孔结构。通过在非洲爪蟾卵母细胞中异源表达和电压钳分析结合表型分析，涉及钾转运缺陷的酿酒酵母菌株，我们研究了突变体的选择性过滤器和它们对铯和钙离子抑制的敏感性。关于电生理特性，KAT 1突变体分为三组：（i）野生型样通道，（ii）在选择性和Cs+或Ca 2+敏感性方面修饰的通道，以及（iii）在其电压依赖性方面受到额外影响的组。尽管H5中有另外的14个氨基酸，但K4 T1中的该基序也参与离子传导孔的形成，因为Leu-251、Thr-256、Thr-259和Thr-260处的氨基酸取代导致具有改变的离子选择性和抑制的功能通道。通过窄孔内的突变产生的Ca 2+敏感性和对Cs+阻断的敏感性增加可能表明两种阻断剂都深入到通道中。此外，靠近孔边缘的突变影响半活化电位（U-1/2），表明孔内的氨基酸与电压传感器相互作用或离子渗透反馈门控。
KAT1 is a voltage-dependent inward rectifying K+ channel cloned from the higher plant Arabidopsis thaliana [Anderson, J. A., Huprikar, S. S., Kochian, L. V., Lucas, W. J. & Gaber, R. F. (1992) Proc. Natl. Acad. Sci. USA 89, 3736-3740]. It is related to the Shaker superfamily of K+ channels characterized by six transmembrane spanning domains (S1-S6) and a putative pore-forming region between S5 and S6 (H5). The H5 region between Pro-247 and Pro-271 in KAT1 contains 14 additional amino acids when compared with Shaker [Aldrich, R. W. (1993) Nature (London) 362, 107-108]. We studied various point mutations introduced into H5 to determine whether voltage-dependent plant and animal K+ channels share similar pore structures. Through heterologous expression in Xenopus oocytes and voltage-clamp analysis combined with phenotypic analysis involving a potassium transport-defective Saccharomyces cerevisiae strain, we investigated the selectivity filter of the mutants and their susceptibility toward inhibition by cesium and calcium ions. With respect to electrophysiological properties, KAT1 mutants segregated into three groups: (i) wild-type-like channels, (ii) channels modified in selectivity and Cs+ or Ca2+ sensitivity, and (iii) a group that was additionally affected in its voltage dependence. Despite the additional 14 amino acids in H5, this motif in K4T1 is also involved in the formation of the ion-conducting pore because amino acid substitutions at Leu-251, Thr-256, Thr-259, and Thr-260 resulted in functional channels with modified ionic selectivity and inhibition. Creation of Ca2+ sensitivity and an increased susceptibility to Cs+ block through mutations within the narrow pore might indicate that both blockers move deeply into the channel. Furthermore, mutations close to the rim of the pore affecting the half-activation potential (U-1/2) indicate that amino acids within the pore either interact with the voltage sensor or ion permeation feeds back on gating.