Rearrangements in the KcsA cytoplasmic domain underlie its gating.
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.
Hirano
Minako
M
From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
Takeuchi
Yuko
Y
From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
Aoki
Takaaki
T
From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
Yanagida
Toshio
T
From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan.
Ide
Toru
T
From the Graduate School of Frontier Biosciences, Osaka University, Osaka 565-0871, Japan. Electronic address: ide@phys1.med.osaka-u.ac.jp.
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
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D008081:Liposomes; D015221:Potassium Channels; D012235:Rhodamines; C107790:prokaryotic potassium channel; C005358:tetramethylrhodamine