Single MthK channels were recorded using Axopatch 200A (Molecular Devices) in planar lipid-decane bilayers consisting of 3:1 POPE:POPG lipids (Avanti Polar Lipids), as previously described37 (link). For the experiments in Supplementary Fig. 4 , channels were recorded in symmetric 200 mM K+ (190 mM KCl and 10 mM KOH), 10 mM HEPES (pH 8.5/HCl) and activated by trans 5 mM Ca2+ with and without 0.5 μM Br-bTBA (also in trans) at –100, –25 and +25 mV. Analysis was performed using QuB (www.qub.buffalo.edu ) to fit closed and open dwell time distributions with an open-channel block scheme (Supplementary Fig. 4 ), as described37 (link). Transitions between open (O) and open-blocked (OB) states were used to estimate the open-state dissociation constant Kdopen and the voltage dependence, (V), was fit with a K+-coupling model, equation (13) (see ref. 37 (link)):
where is the maximal blocker affinity and is an affinity amplitude for the exponential term and z is the valence for blocker K+ coupling.
where is the maximal blocker affinity and is an affinity amplitude for the exponential term and z is the valence for blocker K+ coupling.