title: Selection of inhibitor-resistant viral potassium channels identifies a selectivity filter site that affects barium and amantadine block.
abstract: 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.
authors.affiliation: Cardiovascular Research Institute, University of California San Francisco, San Francisco, California, United States of America.
authors.forename: Franck C
authors.initials: FC
authors.lastname: Chatelain
authors.forename: Sabrina
authors.initials: S
authors.lastname: Gazzarrini
authors.forename: Yuichiro
authors.initials: Y
authors.lastname: Fujiwara
authors.forename: Cristina
authors.initials: C
authors.lastname: Arrigoni
authors.forename: Courtney
authors.initials: C
authors.lastname: Domigan
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Ferrara
authors.forename: Carlos
authors.initials: C
authors.lastname: Pantoja
authors.forename: Gerhard
authors.initials: G
authors.lastname: Thiel
authors.forename: Anna
authors.initials: A
authors.lastname: Moroni
authors.forename: Daniel L
authors.initials: DL
authors.lastname: Minor
mesh_terms: 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
chemical_list: C405463:Kcv potassium channel, Chlorella virus; D026902:Potassium Channel Blockers; D015221:Potassium Channels; D014764:Viral Proteins; D001464:Barium; D000547:Amantadine; D011188:Potassium