title: Fast and slow voltage sensor rearrangements during activation gating in Kv1.2 channels detected using tetramethylrhodamine fluorescence.
abstract: The Kv1.2 channel, with its high resolution crystal structure, provides an ideal model for investigating conformational changes associated with channel gating, and fluorescent probes attached at the extracellular end of S4 are a powerful way to gain a more complete understanding of the voltage-dependent activity of these dynamic proteins. Tetramethylrhodamine-5-maleimide (TMRM) attached at A291C reports two distinct rearrangements of the voltage sensor domains, and a comparative fluorescence scan of the S4 and S3-S4 linker residues in Shaker and Kv1.2 shows important differences in their emission at other homologous residues. Kv1.2 shows a rapid decrease in A291C emission with a time constant of 1.5 +/- 0.1 ms at 60 mV (n = 11) that correlates with gating currents and reports on translocation of the S4 and S3-S4 linker. However, unlike any Kv channel studied to date, this fast component is dwarfed by a larger, slower quenching of TMRM emission during depolarizations between -120 and -50 mV (tau = 21.4 +/- 2.1 ms at 60 mV, V(1/2) of -73.9 +/- 1.4 mV) that is not seen in either Shaker or Kv1.5 and that comprises >60% of the total signal at all activating potentials. The slow fluorescence relaxes after repolarization in a voltage-dependent manner that matches the time course of Kv1.2 ionic current deactivation. Fluorophores placed directly in S1 and S2 at I187 and T219 recapitulate the time course and voltage dependence of slow quenching. The slow component is lost when the extracellular S1-S2 linker of Kv1.2 is replaced with that of Kv1.5 or Shaker, suggesting that it arises from a continuous internal rearrangement within the voltage sensor, initiated at negative potentials but prevalent throughout the activation process, and which must be reversed for the channel to close.
authors.affiliation: Department of Anesthesiology, Pharmacology, and Therapeutics, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada.
authors.forename: Andrew James
authors.initials: AJ
authors.lastname: Horne
authors.forename: Christian Joseph
authors.initials: CJ
authors.lastname: Peters
authors.forename: Thomas William
authors.initials: TW
authors.lastname: Claydon
authors.forename: David
authors.initials: D
authors.lastname: Fedida
mesh_terms: D019943:Amino Acid Substitution / Q000502:physiology; D000818:Animals; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology*; D007700:Kinetics; D051663:Kv1.2 Potassium Channel / Q000502:physiology*; D051666:Kv1.5 Potassium Channel / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D008958:Models, Molecular; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012235:Rhodamines / Q000737:chemistry* / Q000378:metabolism; D051637:Shaker Superfamily of Potassium Channels / Q000502:physiology; D013050:Spectrometry, Fluorescence; D013194:Staining and Labeling; D014162:Transfection; D014982:Xenopus laevis
chemical_list: D051663:Kv1.2 Potassium Channel; D051666:Kv1.5 Potassium Channel; D011993:Recombinant Fusion Proteins; D012235:Rhodamines; D051637:Shaker Superfamily of Potassium Channels; C005358:tetramethylrhodamine