title: Identification and characterization of the slowly exchanging pH-dependent conformational rearrangement in KcsA.
abstract: Gating of ion channels is strictly regulated by physiological conditions as well as intra/extracellular ligands. To understand the underlying structures mediating ion channel gating, we investigated the pH-dependent gating of the K(+) channel KcsA under near-physiological conditions, using solution-state NMR. In a series of (1)H(15)N-TROSY HSQC (transverse relaxation optimized spectroscopy-heteronuclear single quantum coherence) spectra measured at various pH values, significant chemical shift changes were detected between pH 3.9 and 5.2, reflecting a conformational rearrangement associated with the gating. The pH-dependent chemical shift changes were mainly observed for the resonances from the residues near the intracellular helix bundle, which has been considered to form the primary gate in the K(+) channel, as well as the intracellular extension of the inner helix. The substitution of His-25 by Ala abolished this pH-dependent conformational rearrangement, indicating that the residue serves as a "pH-sensor" for the channel. Although the electrophysiological open probability of KcsA is less than 10%, the conformations of the intracellular helix bundle between the acidic and neutral conditions seem to be remarkably different. This supports the recently proposed "dual gating" properties of the K(+) channel, in which the activation-coupled inactivation at the selectivity filter determines the channel open probability of the channel. Indeed, a pH-dependent chemical shift change was also observed for the signal from the Trp-67 indole, which is involved in a hydrogen bond network related to the activation-coupled inactivation. The slow kinetic parameter obtained for the intracellular bundle seems to fit better into the time scale for burst duration than very fast fluctuations within a burst period, indicating the existence of another gating element with faster kinetic properties.
authors.affiliation: Graduate School of Pharmaceutical Sciences, University of Tokyo, Hongo, Tokyo 113-0033, Japan.
authors.forename: Koh
authors.initials: K
authors.lastname: Takeuchi
authors.forename: Hideo
authors.initials: H
authors.lastname: Takahashi
authors.forename: Seiko
authors.initials: S
authors.lastname: Kawano
authors.forename: Ichio
authors.initials: I
authors.lastname: Shimada
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D006801:Humans; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating; D007700:Kinetics; D008958:Models, Molecular; D020125:Mutation, Missense; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017433:Protein Structure, Secondary
chemical_list: D024642:Potassium Channels, Voltage-Gated