Fluorescence detection of the movement of single KcsA subunits reveals cooperativity.
The prokaryotic KcsA channel is gated at the helical bundle crossing by intracellular protons and inactivates at the extracellular selectivity filter. The C-terminal transmembrane helix has to undergo a conformational change for potassium ions to access the central cavity. Whereas a partial opening of the tetrameric channel is suggested to be responsible for subconductance levels of ion channels, including KcsA, a cooperative opening of the 4 subunits is postulated as the final opening step. In this study, we used single-channel fluorescence spectroscopy of KcsA to directly observe the movement of each subunit and the temporal correlation between subunits. Purified KcsA channels labeled at the C terminus near the bundle crossing have been inserted into supported lipid bilayer, and the fluorescence traces analyzed by means of a cooperative or independent Markov model. The analysis revealed that the 4 subunits do not move fully independently but instead showed a certain degree of cooperativity. However, the 4 subunits do not simply open in 1 concerted step.
Blunck
Rikard
R
Département de Physique and GEPROM, Université de Montréal, Montréal, QC, Canada H3C 3J7. rikard.blunck@umontreal.ca
McGuire
Hugo
H
Hyde
H Clark
HC
Bezanilla
Francisco
F
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D008051:Lipid Bilayers; D008390:Markov Chains; D009038:Motion; D015221:Potassium Channels / Q000737:chemistry / Q000378:metabolism*; D021122:Protein Subunits; D013050:Spectrometry, Fluorescence / Q000379:methods*
D001426:Bacterial Proteins; D008051:Lipid Bilayers; D015221:Potassium Channels; D021122:Protein Subunits; C107790:prokaryotic potassium channel