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

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.

Role of methyl-induced polarization in ion binding

“…A drop in K + binding affinity at the S4 site could therefore destabilize the filter's conductive state and/or impede the channel's ability to form tetramers. Indeed, electrophoresis studies show that T→S substitutions in Kcv reduce the channel's ability to assemble into tetramers (16). In addition, electrophysiological studies show that T→S substitutions in Kir 2.1 alter the channel's mean open probabilities (16).…”

“…Genetic selection and site-directed mutagenesis experiments on a viral K channel, Kcv, and the inward rectifier K channel, Kir 2.1, show that a threonine-to-serine (T→S) substitution in the S4 sites reduces channel susceptibility to Ba 2+ block by over two orders in magnitude (16). In addition, this substitution reduces channel stability, including the channel's mean open probabilities.…”

Potassium Ion Channels: Could They Have Evolved from Viruses?

“…1C; Pagliuca et al, 2007;Chatelain et al, 2009;Tayefeh et al, 2009). As expected from their predicted structures, many studies in the past 15 years have demonstrated that the chlorovirus channels have the basic properties of a K + channel; they are ion selective, exhibit gating, and are sensitive to typical K + channel blockers .…”

“…They can also complement yeast (Saccharomyces cerevisiae) cells that are defective in K + uptake (Balss et al, 2008;Chatelain et al, 2009).…”

How to resolve microsecond current fluctuations in single ion channels: The power of beta distributions

“…Xray crystallography, 98 energy minimization studies, 99 MD simulations 100 and tetramerization experiments. 101,102 An early hint to the interaction between selectivity filter and K C ion was the very slow exit rate of a lonely ion, when the repulsive forces of other ions do not help to overcome the binding to the pore wall. 103 Theoretical studies 99,100 suggest that the selectivity filter, especially the carbonyl group in the second binding site (S2 site) changes its orientation during ion depletion.…”