Tetraoctylammonium, a Long Chain Quaternary Ammonium Blocker, Promotes a Noncollapsed, Resting-Like Inactivated State in KcsA

Figures

• 

  Scheme 1. Transmembrane portion of KcsA. Crystallographic structure of the C-terminal truncated KcsA in the closed state with TOA+ (green balls and sticks, with a larger ball representing the quaternary nitrogen) bound at the cavity (PDB: 2W0F). In the top view (panel (A)), two of the four subunits appear faintly drawn to facilitate the observation. The four W67 residues are depicted as blue sticks. In the side view (panel (B)), only two of the four monomers have been drawn as a solid red ribbon for the sake of clarity. Each monomer consists of two transmembrane helices (TM1 and TM2) connected by the P-loop region, a short, tilted pore helix, and the selectivity filter (SF). The thick grey lines indicate the membrane limits. Both views illustrate how the TOA+ alkyl chains traverse the TM2 helix. Panel (C) represents the tetraoctylammonium ion in perspective.

• 

  Figure 1. Effects of tetraoctylammonium (TOA+) on the thermal denaturation of wild-type (WT) KcsA. The intrinsic fluorescence at pH 7.0 (panel (A)) and pH 4.0 (panel (B)) and in the absence of TOA+ (full and empty circles, respectively) or upon the addition of 1 µM (full and empty squares, respectively) or 100 µM (full and empty triangles, respectively) of TOA+. The insets show SDS-PAGE analysis of the different samples to illustrate the integrity of tetrameric WT KcsA at room temperature. Panel (C) shows the dependence of the midpoint temperature of the protein denaturation process (tm) of WT KcsA with increasing concentrations of TOA+, either at pH 7.0 (●) or pH 4.0 (○). The results are the average tm (in Celsius) ± S.D. from three independent titrations. Panel (D) illustrates the fitting of Equation (1) to the experimental data from Panel (C) (see Methods). The apparent dissociation constants for the TOA+-channel complexes and their 95% confidence intervals estimated at pH 7.0 and pH 4.0 were 1.8 (0.54–6.02) × 10−7 M and 3.6 (1.89–6.98) × 10−8 M, respectively. The observed differences between such dissociation constant (KD) values were not statistically significant.

• 

  Figure 2. Effect of TOA+ on Na+ binding to WT KcsA. Panels (A,C) illustrate Na+ binding to the WT KcsA channel at pH 7.0 (panel (A)) or pH 4.0 (panel (C)), in the absence (circles) and presence (squares) of 100 µM TOA+, monitored through the Na+-concentration-dependence of the midpoint temperature of the protein denaturation process (tm). Each experimental point is the average tm (in Celsius) ± S.D. (n = 3). Panels (B,D) show the fitting of Equation (1) to the experimental data from panels (A,C), respectively (see Methods). The apparent KD values estimated for the above binding events are given in Table 1.

• 

  Figure 3. Effect of TOA+ on K+ binding to WT KcsA. Panels (A,D) illustrate K+ binding to the WT KcsA channel in the absence (circles) and presence of 100 µM TOA+ (squares) at pH 7.0 (panel (A)) or pH 4.0 (panel (D)), monitored through the K+-concentration-dependence of the midpoint temperature of the protein denaturation process (tm). Each experimental point is the average tm (in Celsius) ± S.D. (n = 3). When the low (panels (B,E)) and high (panels (C,F)) K+ concentration ranges are analyzed separately, Equation (1) adequately fits the data from panels (A,D), indicating that two different sets of K+ binding sites are present in the WT KcsA. The apparent KD values estimated for the above binding events are given in Table 1.

• 

  Figure 4. Influence of pH and K+ concentration on the homo-Förster resonance energy transfer (homo-FRET) process among the tryptophan residues in the W67 KcsA channel. Panel (A) shows a schematic top view of the tetrameric structure of KcsA (PDB: 1K4C; each monomer numbered 1 to 4) and the location of the W67 residues. Panel (B) illustrates representative fluorescence anisotropy decays obtained for W67 KcsA in the presence of 0.1, 1, and 200 mM K+ at both pH 7.0 (inner gate closed) and pH 4.0 (inner gate open). Equation (3) was then fitted to each data set, and the W67–W67 intersubunit distances were calculated from Equation (4). Panels (C,D) show the changes in the steady-state anisotropy <r>SS (panel (C)) and in the W67–W67 intersubunit lateral distances calculated from the time-resolved anisotropy decays (panel (D)) at different K+ concentrations at both pH 7.0 and pH 4.0. Each titration curve is built from duplicate measurements from independent experiments (average ± S.D.) at each of the 20 to 25 different K+ concentrations covering the indicated concentration range. Panel (E) shows the linear correlation between the <r>SS and W67–W67 lateral distances (distance = 92.165 × <r>SS +…

• 

  Figure 5. Effect of TOA+ on K+ binding to W67 KcsA. Panels (A,D) illustrate K+ binding to the W67 KcsA channel in the absence (circles) and presence of 100 µM TOA+ (squares) at pH 7.0 (panel (A)) or pH 4.0 (panel (D)), monitored through the K+-concentration-dependence of the midpoint temperature of the protein denaturation process (tm). Each experimental point is the average tm (in Celsius) ± S.D. (n = 3). As to the WT KcsA from Figure 3, when the low (panels (B,E)) and high (panels (C,F)) K+ concentration ranges are analyzed separately, Equation (1) adequately fits the data from panels (A,D), which is evidence that the two different sets of K+ binding sites are present in W67 KcsA. The apparent KD values estimated for the above binding events are given in Table 1.

• 

  Figure 6. Effect of TOA+ on the conformation of the W67 KcsA channel. Panels (A) and (C) show a representative experiment of the dependence of the W67 <r>SS on K+ concentration at both pH 7.0 and pH 4.0, in the absence or presence of 100 µM TOA+. The anisotropy values correspond to the average of ten measurements ± S.D. The solid lines represent the best fit of Equation (10) to the data. The apparent KDs for the above binding events were calculated from two independent experiments, and their average values, along with the confidence intervals, are given in Table 1. Panels (B,D) illustrate the dependence of the W67–W67 intersubunit distances on K+ concentration. The results from the samples without TOA+ were obtained experimentally from time-resolved anisotropy decays, as explained in Figure 4. In the samples containing 100 µM TOA+, such distances were obtained by simply interpolating the <r>SS values using the correlation plot from Figure 4E. Nonetheless, the intersubunit distances in several samples containing 100 µM TOA+ were also determined from time-resolved anisotropy decays (black symbols) to test the goodness of the interpolation procedure.