The voltage-dependent gate in MthK potassium channels is located at the selectivity filter.

Understanding how ion channels open and close their pores is crucial for comprehending their physiological roles. We used intracellular quaternary ammonium blockers, electrophysiology and X-ray crystallography to locate the voltage-dependent gate in MthK potassium channels from Methanobacterium thermoautotrophicum. 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. Kinetic analysis of tetrabutylammonium block of single MthK channels combined with X-ray crystallographic analysis of the pore with tetrabutyl antimony 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 suggest that MthK inactivation leads to conformational changes within the cavity and intracellular pore entrance.