Driving Forces underlying Selectivity Filter Gating in the MthK Potassium Channel

K+ channel activity can be limited by C-type inactivation, which is likely initiated in part by dissociation of K+ ions from the selectivity filter, and modulated by side chains surrounding the selectivity filter. Whereas crystallographic and computational studies have linked inactivation to a ‘collapsed’ selectivity filter conformation in the KcsA channel, the structural basis for selectivity filter gating in other K+ channels has been less clear. Here, we combined electrophysiological recordings with molecular dynamics based, in silico electrophysiology simulations, to study selectivity filter gating in the model potassium channel MthK and its V55E mutant (analogous to KcsA E71) in the pore-helix. Experimentally, we find that MthK V55E has a lower open probability than the WT channel, due to decreased stability of the open state, as well as a lower unitary conductance. Simulations account for both aspects of these observations on the atomistic scale, showing that ion permeation in V55E is altered by two distinct orientations of the E55 side chain. In the ‘vertical’ orientation of E55, in which E55 forms a hydrogen bond with D64 (as observed with KcsA WT channels), the filter displays reduced conductance compared to MthK WT. In contrast, with ‘horizontal’ orientation, K+ conductance is closer to MthK WT; however the selectivity filter stability in the conducting conformation is lowered, and the filter more readily transitions to the inactivated conformation. Surprisingly, these transitions of MthK WT and V55E channels to the non-conducting (inactivated) state observed in simulations are associated with a widening selectivity filter, unlike its narrowing seen in KcsA, and reminisce the recent structures of stably-inactivated, voltage-gated potassium channels: Shaker W434F and Kv1.2 W362F mutants, as well as WT Kv1.3 channels. ### Competing Interest Statement The authors have declared no competing interest.