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

Alkylammonium salts have been used extensively to study the structure and function of potassium channels. Here, we use the hydrophobic tetraoctylammonium (TOA(+)) to shed light on the structure of the inactivated state of KcsA, a tetrameric prokaryotic potassium channel that serves as a model to its homologous eukaryotic counterparts. By the combined use of a thermal denaturation assay and the analysis of homo-Forster 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, TOA(+) bound at the cavity allosterically shifts the equilibrium of the channel's selectivity filter conformation from conductive to an inactivated-like form. The inactivated TOA(+)-KcsA complex exhibits a loss in the affinity towards permeant K+ at pH 7.0, when the channel is in its closed state, but maintains the two sets of K+ binding sites and the W67-W67 intersubunit distances characteristic of the selectivity filter in the channel resting state. Thus, the TOA(+)-bound state differs clearly from the collapsed channel state described by X-ray crystallography and claimed to represent the inactivated form of KcsA.

Automated summary

Alkylammonium salts, particularly tetraoctylammonium, have been used to investigate the structure and function of potassium channels. In this study, it was found that TOA(+) binds to the channel cavity with high affinity and causes a shift in the equilibrium of the channel's selectivity filter conformation towards an inactivated-like form. Additionally, the TOA(+)-bound state differs significantly from the collapsed channel state described by X-ray crystallography as the inactivated form of KcsA.