Selection of inhibitor-resistant viral potassium channels identifies a selectivity filter site that affects barium and amantadine block.
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.


RESULTS
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
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.
Chatelain
Franck C
FC
Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.
Gazzarrini
Sabrina
S
Fujiwara
Yuichiro
Y
Arrigoni
Cristina
C
Domigan
Courtney
C
Ferrara
Giuseppina
G
Pantoja
Carlos
C
Thiel
Gerhard
G
Moroni
Anna
A
Minor
Daniel L
DL
D000547:Amantadine / Q000494:pharmacology*; D000818:Animals; D001464:Barium / Q000494:pharmacology*; D001665:Binding Sites; D004594:Electrophysiology / Q000379:methods; D008957:Models, Genetic; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010843:Pichia / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D026902:Potassium Channel Blockers / Q000494:pharmacology; D015221:Potassium Channels / Q000737:chemistry* / Q000502:physiology; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D014982:Xenopus laevis / Q000378:metabolism
C405463:Kcv potassium channel, Chlorella virus; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D014764:Viral Proteins; D001464:Barium; D000547:Amantadine; D011188:Potassium