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

Franck C Chatelain,Sabrina Gazzarrini,Gerhard Thiel,Courtney Domigan,Daniel L Minor,Giuseppina Ferrara,Carlos Pantoja,Cristina Arrigoni,Anna Moroni,Yuichiro Fujiwara

doi:10.1371/journal.pone.0007496

Abstract

BackgroundUnderstanding 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 FindingsWe 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/SignificanceThe 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.

Highlights

Ion channels produce the bioelectrical signals that drive sensory signals, locomotion, cognition, and in some cases viral infection [1]
Further studies using a plate assay in which blocking compounds are applied via Whatman filter discs [9] indicated that both barium and amantadine, two reagents that block Kcv function [16], prevented Kcv-mediated rescue but did not affect the ability of heterologous expression of the yeast potassium transporter TRK1 to rescue growth (Figure 1B). These observations provide further evidence that Kcv forms functional potassium channels in yeast that can be affected by known channel blockers and gave the opportunity to explore the nature of the channelblocker interactions using selection methods
Previous application of the yeast genetic selection approach to the study of block of the mammalian inward rectifier Kir2.1 yielded a mutation in a residue adjacent to site 4 that does not coordinate the permeant ion but that caused an electrostatic disturbance at site 4

Summary

Introduction

Ion channels produce the bioelectrical signals that drive sensory signals, locomotion, cognition, and in some cases viral infection [1]. The development of new approaches to identify and map the sites of action of agents that can be used to control channel function remains an important goal. Genetic selections provide a potentially powerful means to identify and uncover the mechanisms of action of ion channel modifiers. Such approaches have the additional advantage of finding compounds that directly affect function [4]. 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

Methods

Results

Conclusion