Bimane fluorescence scanning suggests secondary structure near the S3-S4 linker of BK channels.
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.
Semenova
Nina P
NP
Department of Physiology, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, USA.
Abarca-Heidemann
Karin
K
Loranc
Eva
E
Rothberg
Brad S
BS
D000595:Amino Acid Sequence; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005456:Fluorescent Dyes / Q000737:chemistry; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000737:chemistry* / Q000235:genetics; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D013050:Spectrometry, Fluorescence / Q000379:methods*; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005456:Fluorescent Dyes; D051036:Large-Conductance Calcium-Activated Potassium Channels; C021149:bimanes; D014443:Tyrosine; D014364:Tryptophan