Figures & data
Figure 1. Effect of LAs on temperature dependence of KcsA tetramer stability in the presence and absence of K+. KcsA tetramer was measured by SDS-PAGE analysis of a series of identical samples incubated at various temperatures for 10 min before addition of SDS-PAGE sample buffer. The sample assay mixture contained 10 mM Hepes-Tris, pH 7.4, 100 mM cholineCl, either 5 mM KCl (A) or no added KCl (B) and either no LA (○), 20 mM lidocaine (●) or 5 mM tetracaine (△). Solid lines indicate nonlinear regression fits to a logistic function of temperature described in Materials and Methods. Fit parameters: (A) 20 mM lidocaine (●) : T0.5 = 98.0 ± 2.1 °C, n = 14.1 ± 4.8; (A) 5 mM tetracaine (△): T0.5 = 75.9 ± 1.7 °C, n = 10.7 ± 2.3; (B) no LA (○): T0.5 = 45.7 ± 0.9 °C, n = 6.4 ± 0.7; (B) 20 mM lidocaine (●) : T0.5 = 41.9 ± 0.7 °C, n = 6.4 ± 0.6.
Figure 2. Destabilization of KcsA tetramer as function of LA concentration. KcsA tetramer was measured by SDS-PAGE analysis of samples titrated with increasing concentrations of lidocaine (●) or tetracaine (△, □ ). The sample assay mixture contained 10 mM Hepes-Tris, pH 7.5, 100 mM cholineCl and either 5 mM KCl (● , △) or no added KCl (□ ). Samples were incubated either at 90°C (● , △) or 22 °C (□ ) for 10 min before addition of SDS-PAGE sample buffer. Solid lines indicate nonlinear regression fits to a logistic function of LA concentration as described in Materials and Methods. Fit parameters: lidocaine, 5 mM KCl, 90°C (● ): IC50 = 25.1 ± 2.6 mM, n = 1.14 ± 0.13; tetracaine, 5 mM KCl, 90 °C (△): IC50 = 4.2 ± 0.6 mM, n = 1.43 ± 0.33; tetracaine, 0 KCl, 22 °C (□ ): 1.2 ± 0.2 mM, n = 0.98 ± 0.22.
Figure 3. Stabilization of KcsA tetramer as a function of K+ concentration in the absence and presence of LA. KcsA tetramer was measured by SDS-PAGE analysis of samples titrated with increasing K+ concentration in the absence of LA (○) or in the presence of 20 mM lidocaine (● ) or 5 mM tetracaine (△). The sample assay mixture contained ~500 ng KcsA, 10 mM Hepes-Tris, pH 7.4, 100 mM choline Cl, indicated concentrations of KCl and either no LA, 20 mM lidocaine or 5 mM tetracaine. All samples were incubated at 90 °C for 10 min before addition of SDS-PAGE sample buffer. Solid lines indicate nonlinear regression fits to a logistic function of K+ concentration as described in Materials and Methods. Fit parameters: no LA (○): K0.5 = 1.48 ± 0.04 mM, n = 3.19 ± 0.26; 20 mM lidocaine (● ): K0.5 = 6.65 ± 0.8 mM, n = 1.05 ± 0.13; 5 mM tetracaine (△): K0.5 = 5.03 ± 0.90, n = 1.04 ± 0.19.
Figure 4. Example of energy-minimized structure simulation of lidocaine (A) and tetracaine (B) docked in the open KcsA structure (PDB entry 3PJS) with S1/S3 K+ occupancy of the selectivity filter. Front subunit is removed for clarity. Right panel is an enlargement of the docked LA molecule zoomed from the left panel. In this and subsequent structure figures, the side chains of Thr75, Ile100 and Phe103 residues as well as the backbone carbonyls of Thr75 are shown as thin sticks. LA molecules and water molecules in the selectivity filter are shown as thick sticks. Potassium ions are shown as yellow spheres.
Figure 5. Examples of energy minimized complexes of lidocaine (A) and tetracaine (B and C) with the open KcsA (PDB entry 3PJS) and S2/S4 K+ occupancy of the selectivity filter. Two different docked locations of tetracaine are shown in (B) and (C).
Figure 6. Examples of energy-minimized complexes of lidocaine (A) and tetracaine (B) with the open-inactivated KcsA structure (PDB entry 3F5W) and S1/S4 K+ occupancy of the selectivity filter. Note that the apparently different crossing angles of inner helices in the right panels of (A) vs. (B) are due to different views of the ligand-channel complex chosen for an optimal display of the LA molecule.
Figure 7. Examples of energy-minimized complexes of lidocaine (A) and tetracaine (B) with closed KcsA (PDB entry 1J95) and K+ ions at sites S2 and S4. Right panel of (B) shows detailed enlargement of tetracaine, which forms H-bonds with Thr75 and Thr107.
Table 1. Ligand-channel energy (E, kcal/mol) and top contributions to ita
Figure 8. Sequence alignment of residues corresponding to the inner core pore domain of KcsA and related K+ channels. Range of residue numbers listed for aligned sequences corresponds to NCBI protein database entries for KcsA (Acc. P0A334), human Kv1.2 (Acc. NP_004965), human Kv1.5 (Acc. NP_002225) and hERG/Kv11.1 (Acc. Q12809). Residues identical to KcsA are colored red. Asterisks (*) correspond to residues of KcsA located at monomer-monomer interfaces of closed (PDB entry 1K4C) or open (PDB entry 3FB7) conformations of KcsA crystal structures as determined by the EMBL-EBI PISA (Protein Interfaces, Surfaces and Assemblies) analysis program (www.ebi.ac.uk/msd-srv/prot_int/pistart.html).Citation71,Citation72 Drug-binding residues of Kv1.5Citation10,Citation25,Citation33 and hERGCitation17,Citation19,Citation20 identified by mutational analysis are highlighted in yellow. Residues of KcsA found to exhibit the highest interaction energy with lidocaine and tetracaine in docking simulations performed in this study are also highlighted in yellow.
