Oral Presentation 2014 International Biophysics Congress

Molecular locations of gates in MthK potassium channels (#203)

Crina Nimigean 1
  1. Weill Cornell Medical College, New York, NY, USA

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 and calcium-dependent gates in MthK potassium channels from Methanobacterium thermoautotrophicum with electrophysiology, stopped-flow spectrofluorometry, 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 single-channel analysis of tetrabutylammonium block of 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 gating with voltage also leads to conformational changes within the cavity and intracellular pore entrance.
For locating the calcium gate, we employed a Tl+ flux assay using a stopped-flow spectrofluorometer. MthK channel activity was estimated from the rate of a MthK-containing liposome-trapped fluorophore quenching due to Tl+ influx through the channels. The high-affinity blocker tetrapentylammonium, applied prior to channel activation using a sequential mixing protocol, was able to fully block closed channels, indicating that the blocker can reach its binding site in closed channels. Given that the blocker binding site is in the cytoplasmic access below the selectivity filter, these results suggest that there is also a calcium-dependent gate at the selectivity filter in MthK.