Tetraoctylammonium: Comparison
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Please note this is a comparison between Version 2 by Vivi Li and Version 1 by Jose Antonio Poveda Larrosa.

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the long-chain, hydrophobic tetraoctylammonium (TOA+) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel. By the combined use of a thermal denaturation assay and the analysis of homo-Förster resonance energy transfer in a mutant channel containing a single tryptophan (W67) per subunit, we found that TOA+ binds the channel cavity with high affinity, either with the inner gate open or closed. Moreover, bound TOA+ induces a decrease in the affinity for K+ in the two characteristic K+ binding events to the channel selectivity filter at pH 7.0, when the channel inner gate is in the closed conformation. This is similar to that observed in the absence of TOA+ upon acidic-pH-induced channel inactivation. Therefore, this suggests that TOA+ binding by itself causes inactivation at pH 7.0 when the inner gate is closed. Furthermore, in apparent agreement with such conclusion, the presence of bound TOA+ in the pH 4.0 samples has only modest effects on the affinity of the two binding events for K+, likely because the channel is already inactivated. Finally, we also observed that TOA+ bound at the cavity, allosterically modifies the conformation of the pore helices, leading to longer W67-W67 intersubunit distances at any K+ concentration and both at pH 7.0 and pH 4.0. The changes in the pore helix conformation, along with the decreased affinity for K+ at pH 7.0 caused by TOA+, seen in both homo-FRET and thermal denaturation experiments, are very similar to those effects caused by inactivation at pH 4.0.

  • potassium channels
  • tetraalkylammonium salts
  • protein thermal stability
  • homo-FRET
  • C-type inactivation
  • binding affinity
  • selectivity filter conformation
  • steady-state and time-resolved fluorescence anisotropy
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