Rearrangements in the KcsA Cytoplasmic Domain Underlie Its Gating

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

doi:10.1074/jbc.m109.084368

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.

Highlights

That changes in the cytoplasmic domain are transmitted through a linker to the channel core region, which includes the channel pore and leads to gate opening
The part of the channel ranging from the C terminus of TM2 to the adjacent part of the cytoplasmic domain was found to be in a hydrophobic environment when the channel was activated at pH 4, whereas at pH 7, when the channel was closed, it was in a hydrophilic environment
The Cytoplasmic Domain Is in a Hydrophobic Environment When the Gate Opens—We found that the C terminus of TM2 and the adjacent part of the cytoplasmic domain, in which the aforementioned five residues locate, are in a hydrophobic environment while in the open state

Summary

The abbreviations used are

KcsA, potassium channels from S. lividans; DPA, dipicrylamine; TMR, tetramethylrhodamine; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio] propane sulfonate; POPE, 1-palmitoyl-2-oleoyl-snglycero-3-phosphoethanolamine; POPG, 1-palmitoyl-2-oleoyl-sn-glycero3-[phospho-rac-(1-glycerol)]; MES, 4-morpholineethanesulfonic acid; TM, transmembrane; pS, picosiemens. Conformational Changes in KcsA with gating in the cytoplasmic domain. The part of the channel ranging from the C terminus of TM2 to the adjacent part of the cytoplasmic domain was found to be in a hydrophobic environment when the channel was activated at pH 4, whereas at pH 7, when the channel was closed, it was in a hydrophilic environment. We measured the fluorescence resonance energy transfer between fluorophores at the cytoplasmic domain in the presence of dipicrylamide (DPA), a membranelocalized quencher (14) that lowers fluorescence intensities at acidic pH. These results suggest that the region from the C terminus of TM2 to the adjacent part of the cytoplasmic domain moves largely toward the membrane when the gate opens

EXPERIMENTAL PROCEDURES

RESULTS

92 Ϯ 8 50 Ϯ 11 43–90 144 Ϯ 18

DISCUSSION