Mapping the Conformational States of the Outer Vestibule of Kcsa, a Prokaryote Potassium Channel, using the Long Chain Quaternary Ammonium Blocker, Tetraoctylammonium

Abstract

KcsA is a homotetrameric prokaryotic potassium channel, homologous to its eukaryotic counterparts. In this study, we have used a KcsA mutant channel bearing a single tryptophan residue per monomer (W67 KcsA) to investigate the selectivity filter (SF) and pore-helix dynamics using fluorescence spectroscopy. Our steady-state and time-resolved fluorescence measurements indicate that the W67 residues undergo an efficient homo-FRET process among the four channel subunits. An analytical solution describing homo-FRET among a homo-tetramer in a square geometry allowed retrieving W67-W67 lateral distances (R) from the W67 KcsA fluorescence anisotropy decays. Comparison between the distances determined for the closed/conductive (pH 7.0, 200 mM K+, R=15.4 Å), closed/non-conductive (pH 7.0, 0.1 mM K+, R=16.5 Å) and open/inactivated (pH4.0, 200 mM K+, R=15.3 Å) of the detergent-solubilized W67 KcsA, ruled out the hypothesis that the inactivated state is structurally similar to its collapsed state. To further study the inactivated conformation, we added tetraoctylammonium (TOA+), which is a potassium channel blocker that binds to the internal cavity, beneath the SF. TOA+ allosterically triggers a structural rearrangement characterized by a decrease in the affinity for K+ at pH 7 and longer W67-W67 lateral distances, while the shorter alkyl chain salt tetrabutylammonium (TBA+) does not affect the SF conformation of KcsA. Such loss of affinity is similar to that seen for the open/inactivated state at pH 4. This allosteric modification is also linked to a decrease of the thermal stability of KcsA monitored by a thermal denaturation assay. These results reinforce the argument that long chain tetraalkyl ammonium salts, such as TOA+, stabilize an inactivated-like conformation of the SF, which has a lower affinity for K+ and a different conformation than the low-K+ (collapsed) structure.