| Summary: | Understanding how ion channels open and close their pores is crucial for understanding their physiological roles. We used intracellular quaternary ammonium blockers to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology and X-ray crystallography. Blockers bind in an aqueous cavity between two putative gates, an intracellular gate and the selectivity filter. Thus, these blockers directly probe gate location: an intracellular gate will prevent binding when closed, whereas a selectivity filter gate will always allow binding. A kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutylantimony unequivocally determined that the voltage-dependent gate, like the C-type inactivation gate in eukaryotic channels, is located at the selectivity filter. State-dependent binding kinetics suggests that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.
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