Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block
Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block
复制标题
DOI：
10.1371/journal.pone.0007496
复制
发表时间：
2009-10-16
期刊：
影响因子：
3.7
通讯作者：
Minor, Daniel L., Jr.
中科院分区：
文献类型：
--
作者：
Chatelain, Franck C.;Gazzarrini, Sabrina;Minor, Daniel L., Jr.
背景：了解离子通道和阻滞剂之间的相互作用仍然是一个重要的目标，这对于阐明离子通道功能的基本机制和离子通道导向药物的发现和开发具有重要意义。方法/主要发现：我们使用遗传选择方法探索了两种离子通道阻滞剂Ba2+和金刚烷胺与微型病毒钾通道Kcv的相互作用。对抗任一种阻断剂的Kcv突变体的选择鉴定出了一个对这两种阻断剂都具有抗性的突变株。实施PCR改组和回交程序发现，阻滞剂抗性可能归因于T63S的单一改变，该改变位于可能形成选择性过滤器中内离子的结合位置(位置4)。电生理和生化分析的组合显示，突变通道与阻滞剂相互作用的能力有明显的差异。对哺乳动物内向整流基因Kir2.1中类似突变的研究表明，T->；S突变不仅影响Ba2+阻断，而且影响传导状态的稳定性。比较Kcv和Kir2.1中相似的抗钡突变的效果表明，Kcv选择性过滤器中邻近的氨基酸影响阻滞剂的结合。结论/意义：数据支持这样的观点，即离子在稳定钾通道结构中起着不可或缺的作用，提示钡和金刚烷胺在相似的位置起作用，并展示了如何利用遗传选择来定位阻滞剂结合部位和揭示机制特征。
Background: Understanding the interactions between ion channels and blockers remains an important goal that has implications for delineating the basic mechanisms of ion channel function and for the discovery and development of ion channel directed drugs.Methodology/Principal Findings: We used genetic selection methods to probe the interaction of two ion channel blockers, barium and amantadine, with the miniature viral potassium channel Kcv. Selection for Kcv mutants that were resistant to either blocker identified a mutant bearing multiple changes that was resistant to both. Implementation of a PCR shuffling and backcrossing procedure uncovered that the blocker resistance could be attributed to a single change, T63S, at a position that is likely to form the binding site for the inner ion in the selectivity filter (site 4). A combination of electrophysiological and biochemical assays revealed a distinct difference in the ability of the mutant channel to interact with the blockers. Studies of the analogous mutation in the mammalian inward rectifier Kir2.1 show that the T -> S mutation affects barium block as well as the stability of the conductive state. Comparison of the effects of similar barium resistant mutations in Kcv and Kir2.1 shows that neighboring amino acids in the Kcv selectivity filter affect blocker binding.Conclusions/Significance: The data support the idea that permeant ions have an integral role in stabilizing potassium channel structure, suggest that both barium and amantadine act at a similar site, and demonstrate how genetic selections can be used to map blocker binding sites and reveal mechanistic features.