title: Rearrangements in the KcsA cytoplasmic domain underlie its gating.
abstract: A change of cytosolic pH 7 to 4 opens the bacterial potassium channel KcsA. However, the overall gating mechanism leading to channel opening, especially the contribution of the cytoplasmic domain, remains unsolved. Here we report that deletion of the cytoplasmic domain resulted in changes in channel conductance and gating behavior at pH 4 without channel opening at pH 7. To probe for rearrangements in the cytoplasmic domain during channel opening, amino acid residues were substituted with cysteines and labeled with a fluorophore (tetramethylrhodamine maleimide) that exhibits increased fluorescence intensity upon transfer from a hydrophilic to hydrophobic environment. In all cases channel open probability (P(o)) was approximately 1 at pH 4 and approximately 0 at pH 7. Major increases in fluorescence intensity were observed for tetramethylrhodamine maleimide-labeled residues in the cytoplasmic domain as pH changed from 7 to 4, which suggests the fluorophores shifted from a hydrophilic to hydrophobic environment. Dipicrylamide, a lipid soluble quencher, reduced the fluorescence intensities of labeled residues in the cytosolic domain at pH 4. These results reveal that a decrease in pH introduces major conformational rearrangements associated with channel opening in the KcsA cytoplasmic domain.
authors: From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
authors: Minako
authors: M
authors: Hirano
authors: From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
authors: Yuko
authors: Y
authors: Takeuchi
authors: From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
authors: Takaaki
authors: T
authors: Aoki
authors: From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
authors: Toshio
authors: T
authors: Yanagida
authors: From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan. Electronic address: ide@phys1.med.osaka-u.ac.jp.
authors: Toru
authors: T
authors: Ide
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001665:Binding Sites / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D006863:Hydrogen-Ion Concentration; D015640:Ion Channel Gating / Q000235:genetics / Q000502:physiology*; D008051:Lipid Bilayers; D008081:Liposomes; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012235:Rhodamines / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D015221:Potassium Channels; D012235:Rhodamines; C107790:prokaryotic potassium channel; C005358:tetramethylrhodamine