Mutations in position I131 reduces K sensitivity of KirBac1.1 SF dynamics. (A) Left: smFRET imaging of KirBac1.1 proteins in liposomes labeled with Alexa Fluor 555/647 c2 maleimide pair. Proteoliposomes were immobilized on a polyethylene glycol (PEG)-coated coverslip surface with biotin-POPE and then permeabilized by 50 µM β-escin to achieve symmetrical ionic conditions. Side view of KirBac1.1 crystal structure indicating diagonally opposed T120C labeling sites (purple and magenta) in the SF loop and I131 position in red. Tetrameric channels are formed from pairs of tandem dimers in which only one protomer contains the cysteine mutation. Due to the asymmetric structures, the two dimers can only assemble anti-parallel, with two cysteine residues at diagonal subunits. Right: Representative smFRET trajectories in different [ion] for KirBac1.1 WT and I131M mutant. Fluorescence intensities of the donor Alexa Fluor 555 and acceptor Alexa Fluor 647 (AF555/647) are colored cyan and red, respectively; calculated FRET is colored blue. FU, fluorescence uptake. (B) FRET histograms for AF555/647 fluorophores labeled at diagonal T120C sites in the SF loop in the WT and I131M protein, at increasing [K⁺] from 0 to 50 mM on a background of 150 mM Na⁺, as indicated (n indicates number of traces in each case). In all histograms, over the whole range of [K⁺] and [Na⁺], the FRET amplitude distributions show clear peaks, requiring a minimum sum of three Gaussians for adequate fitting, with FRET peaks at ∼0.2, ∼0.45, and ∼0.8.