Selection of Inhibitor-Resistant Viral Potassium Channels Identifies a Selectivity Filter Site that Affects Barium and Amantadine Block
Open Access
- 16 October 2009
- journal article
- research article
- Published by Public Library of Science (PLoS) in PLOS ONE
- Vol. 4 (10), e7496
- https://doi.org/10.1371/journal.pone.0007496
Abstract
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. 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. 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.This publication has 64 references indexed in Scilit:
- Fast and slow gating are inherent properties of the pore module of the K+ channel KcvThe Journal of general physiology, 2009
- Searching for interesting channels: pairing selection and molecular evolution methods to study ion channel structure and functionMolecular BioSystems, 2009
- Specific and Slow Inhibition of the Kir2.1 K+ Channel by Gambogic AcidOnline Journal of Public Health Informatics, 2009
- Chlorella virus ATCV-1 encodes a functional potassium channel of 82 amino acidsBiochemical Journal, 2009
- Detergent binding explains anomalous SDS-PAGE migration of membrane proteinsProceedings of the National Academy of Sciences of the United States of America, 2009
- Differential Roles of Blocking Ions in KirBac1.1 Tetramer StabilityOnline Journal of Public Health Informatics, 2009
- Integrating the discovery pipeline for novel compounds targeting ion channelsCurrent Opinion in Chemical Biology, 2008
- Transmembrane domain length of viral K + channels is a signal for mitochondria targetingProceedings of the National Academy of Sciences of the United States of America, 2008
- Ion Channels as Drug Targets: The Next GPCRsThe Journal of general physiology, 2008
- Structure and mechanism of the M2 proton channel of influenza A virusNature, 2008