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PMID: 19244238 Published · ppublish English Journal Article Research Support, N.I.H., Extramural

Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels.

The Journal of biological chemistry ·Vol. 284 ·No. 16 ·2009-04-17 ·Pages 10684-93

Semenova NP, Abarca-Heidemann K, Loranc E, Rothberg BS

Abstract

Gating of large conductance Ca(2+)-activated K(+) channels (BK or maxi-K channels) is controlled by a Ca(2+)-sensor, formed by the channel cytoplasmic C-terminal domain, and a voltage sensor, formed by its S0-S4 transmembrane helices. Here we analyze structural properties of a portion of the BK channel voltage sensing domain, the S3-S4 linker, using fluorescence lifetime spectroscopy. Single residues in the S3-S4 linker region were substituted with cysteine, and the cysteine-substituted mutants were expressed in CHO cells and covalently labeled with the sulfhydryl-reactive fluorophore monobromo-trimethylammonio-bimane (qBBr). qBBr fluorescence is quenched by tryptophan and, to a lesser extent, tyrosine side chains. We found that qBBr fluorescence in several of the labeled cysteine-substituted channels shows position-specific quenching, as indicated by increase of the brief lifetime component of the qBBr fluorescence decay. Quenching was reduced with the mutation W203F (in the S4 segment), suggesting that Trp-203 acts as a quenching group. Our results suggest a working hypothesis for the secondary structure of the BK channel S3-S4 region, and places residues Leu-204, Gly-205, and Leu-206 within the extracellular end of the S4 helix.

MeSH Terms
Amino Acid Sequence Animals Bridged Bicyclo Compounds, Heterocyclic/chemistry CHO Cells Cricetinae Cricetulus Fluorescent Dyes/chemistry Large-Conductance Calcium-Activated Potassium Channels/chemistry,genetics Mice Models, Molecular Molecular Sequence Data Molecular Structure Mutagenesis, Site-Directed Protein Structure, Secondary Sequence Alignment Spectrometry, Fluorescence/methods Tryptophan/chemistry Tyrosine/chemistry
Chemicals
Bridged Bicyclo Compounds, Heterocyclic Fluorescent Dyes Large-Conductance Calcium-Activated Potassium Channels bimanes Tyrosine Tryptophan
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Semenova Nina P
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Abarca-Heidemann Karin
Loranc Eva
Rothberg Brad S
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Article Info
Journal
The Journal of biological chemistry
Abbr.
J Biol Chem
ISSN
0021-9258
DOI
10.1074/jbc.M808891200
Published
2009-04-17
Epub
2009-00-25
Pages
10684-93
Language
English
Region
United States
NLM ID
2985121R
PMCID
PMC2667755
Subset
IM
Grants
NIGMS NIH HHS · R01 GM068523 · United States
NIGMS NIH HHS · GM68523 · United States
External Links
PubMed source DOI: 10.1074/jbc.M808891200 PMC Full Text View journal details
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