Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block

被引:39
作者
Chatelain, Franck C.
Gazzarrini, Sabrina
Fujiwara, Yuichiro
Arrigoni, Cristina
Domigan, Courtney
Ferrara, Giuseppina
Pantoja, Carlos
Thiel, Gerhard
Moroni, Anna
Minor, Daniel L., Jr.
机构
[1] Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA
[2] Dipartimento di Biologia, Istituto di Biofisica del Consiglio Nazionale delle Ricerche, Università degli Studi di Milano, Milan
[3] Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, CA
[4] Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA
[5] Department of California, Institute for Quantitative Biomedical Research, University of California San Francisco, San Francisco, CA
[6] Technische Universität Darmstadt, Institute für Botanik, Darmstadt
[7] Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA
来源
PLOS ONE | 2009年 / 4卷 / 10期
关键词
KCSA K+ CHANNEL; PROTON CHANNEL; IN-VITRO; MOLECULAR EVOLUTION; STRUCTURAL BASIS; VOLTAGE SENSOR; ION CHANNELS; PORE HELIX; VIRUS; PROTEIN;
D O I
10.1371/journal.pone.0007496
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
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.
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页数:12
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