Conformational plasticity in the KcsA potassium channel pore helix revealed by homo-FRET studies

Figures

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  Figure 1. Structure of the wild-type KcsA channel. (A) A side-view of the tetramer is shown (PDB entry: 1K4C). Each monomer consists of two transmembrane helices (TM1 and TM2) connected by the P-loop region and the selectivity filter (SF). The five Trp residues per monomer are depicted as blue sticks. (B) Close-up of the selectivity filter of the KcsA channel. The W67 residue is located just behind the SF structure, participating in the inactivation triad, which involves a complex H-bonding network between D80, E71, W67 and extra interactions with Y78 and G79 from the SF. (C) Schematic representation of the SF binding sites that can be occupied by each ion at pH 7, according to X-ray crystallography available data3,50.

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  Figure 2. The structural and functional properties of the quadruple W67 KcsA mutant are similar to the ones presented by the wild-type channel. (A) SDS-PAGE (13.5%) analysis of the tetramer integrity of the detergent-solubilized protein in the presence of 20 mM Hepes, pH 7 buffer with 5 mM DDM and 200 mM Cs+, Rb+, K+ or 10 mM TBA+ or 10 mM succinic acid, pH 4 buffer with equivalent amounts of DDM and salts. In the particular case of Na+, 200 mM NaCl was added to the W67 KcsA samples and 1 M to the E71A W67 batches, respectively. pH 7 and pH 4 samples represent the closed and open states of the channel, respectively. (B) Protein thermal stability assay (monitoring changes in fluorescence emission at 340 nm) of 1 µM protein solubilized in 20 mM Hepes, pH 7 buffer with 5 mM DDM and 100 mM NaCl indicates that the introduced mutations induced only a 5 °C decrease in stability (tm: 66 °C) when compared to WT channel (tm: 70 °C). The inset reveals that in the presence of 100 mM K+, KcsA W67 is as highly resistant to thermal denaturation as the WT channel. (C) Patch-clamp inside-out patches recordings in continuous mode at + 150 mV of WT, W67, E71A and W67 E71A KcsA channels reconstituted in…

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  Figure 3. Conformational changes of W67 KcsA channel probed by steady-state and time-resolved anisotropy measurements. Steady-state fluorescence anisotropy values of 6 µM W67 (A) and W67 E71A (B) KcsA in 5 mM DDM solutions under saturating concentrations of monovalent cations, either 200 mM of Cs+, Rb+, K+ or 5 mM TBA+. The Na+ concentration used was 200 mM for KcsA W67 and 1 M for W67 E71A KcsA at pH 7. Measurements of both channels were carried out at pH 7 (closed state) or 4 (open state) (C) Representative fluorescence anisotropy decays, r(t), of W67 KcsA at pH 7 in the presence of 5 mM TBA+ (SF average ion occupancy: (0), 200 mM Na+ (SF average ion occupancy: (1) and 200 mM K+ (SF average ion occupancy: (2) (λex = 300 nm; λem = 345 nm). The solid lines are the best fit of Eq. 1 to r(t) with g = 43 ns kept as a fixed parameter in the analyses. The 95% confidence intervals calculated for k1 were [0.85,0.93]×107 s-1, [1.87,1.94]×107 s-1 and [3.60,3.83]×107 s-1 in the presence of 5 mM TBA+, 200 mM Na+ or K+, respectively, at pH 7 (D) Dependence of the reduced χ2 on the energy transfer rate constant k1. The relative χ2 surfaces were obtained for the different ionic conditions by keeping…

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  Figure 4. Topological model used in the determination of lateral inter-W67 distances by homo-FRET measurements. (A) Top view of the KcsA channel (PDB entry: 1K4C) with each W67 residue depicted as blue sticks. The subunits of the tetrameric channel were arbitrarily numbered 1, 2, 3 and 4. (B) Diagram for homo-FRET within a square geometry. The W67 residues in the native symmetric homotetrameric channel define a square with lateral distance, R, and a diagonal distance between opposite monomers across the central pore, \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$${R}_{2}=\sqrt{2\,}R$$\end{document}R2=2R. Considering that the initially excited W67 residue is at subunit 1, ki and k−i are the Förster′s transfer rates for this residue.

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  Figure 5. Conformational dynamics of the detergent-solubilized W67 KcsA channel as a function of the ion occupancy of its selectivity filter. Dependence of the (A) steady-state fluorescence anisotropy, <r>, and (B) lateral inter-W67 distances of the mutant channel on K+ and Na+ concentration. The distances were retrieved from fitting the anisotropy decays obtained for 6 μM W67 KcsA mutant channel with the derived homo-FRET model (Eq. 16) (λex = 300 nm; λem = 345 nm;). The conditions used were pH 7 buffer (closed state) containing 5 mM DDM and varying concentrations of K+ (blue) or Na+ (magenta). As already described for the WT channel26,33, two consecutive binding events with different affinities could also be detected upon K+ binding to W67 KcsA mutant channel. For Na+, only a subtle stabilization of the tetrameric mutant channel could be observed upon increasing its concentration in solution. The solid lines are just a guide to the eye. The concentration range accessible experimentally for this cation was much narrower due to the dissociation of the DDM-solubilized W67 KcsA channel below 1-2 mM of Na+.

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  Figure 6. Comparison between the lateral inter-W67 distances calculated from fitting the anisotropy decays obtained for the detergent-solubilized W67 KcsA channels with the derived homo-FRET model (Eq. 1) and the same distances obtained from the X-ray data available for the closed and open states of KcsA channel. High resolution crystallographic structures include those obtained in the presence of the Fab segment against the extracellular loop (Fab extra) or the C-terminal domain (Fab intra), as presented in detail in Table 1. Homo-FRET measurements were carried out at pH 7 (closed state) or 4 (open state) in 5 mM DDM solutions under saturating concentrations of monovalent cations, either 200 mM of Cs+, Rb+, K+ or Na+. The data represent the mean ± SD of at least three independent experiments.