Rearrangements in the KcsA cytoplasmic domain underlie its gating

Minako Hirano, Yuko Takeuchi, Takaaki Aoki, Toshio Yanagida, Toru Ide

Research output: Contribution to journalArticle

19 Citations (Scopus)

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 (Po) was ∼1 at pH4 and ∼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.

Original languageEnglish
Pages (from-to)3777-3783
Number of pages7
JournalJournal of Biological Chemistry
Volume285
Issue number6
DOIs
Publication statusPublished - Feb 5 2010
Externally publishedYes

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Fluorophores
Fluorescence
Potassium Channels
Cysteine
Lipids
Amino Acids
tetramethylrhodamine

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology
  • Medicine(all)

Cite this

Rearrangements in the KcsA cytoplasmic domain underlie its gating. / Hirano, Minako; Takeuchi, Yuko; Aoki, Takaaki; Yanagida, Toshio; Ide, Toru.

In: Journal of Biological Chemistry, Vol. 285, No. 6, 05.02.2010, p. 3777-3783.

Research output: Contribution to journalArticle

Hirano, Minako ; Takeuchi, Yuko ; Aoki, Takaaki ; Yanagida, Toshio ; Ide, Toru. / Rearrangements in the KcsA cytoplasmic domain underlie its gating. In: Journal of Biological Chemistry. 2010 ; Vol. 285, No. 6. pp. 3777-3783.
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