Potassium channels can become nonconducting via inactivation at a gate inside the highly conserved selectivity filter (SF) region near the extracellular side of the membrane. In certain ligand-gated channels, such as BK channels and MthK, a Ca²⁺-activated K⁺ channel from Methanobacterium thermoautotrophicum, the SF has been proposed to play a role in opening and closing rather than inactivation, although the underlying conformational changes are unknown. Using X-ray crystallography, identical conductive MthK structures were obtained in wide-ranging K⁺ concentrations (6 to 150 mM), unlike KcsA, whose SF collapses at low permeant ion concentrations. Surprisingly, three of the SF’s four binding sites remained almost fully occupied throughout this range, indicating high affinities (likely submillimolar), while only the central S2 site titrated, losing its ion at 6 mM, indicating low K⁺ affinity (∼50 mM). Molecular simulations showed that the MthK SF can also collapse in the absence of K⁺, similar to KcsA, but that even a single K⁺ binding at any of the SF sites, except S4, can rescue the conductive state. The uneven titration across binding sites differs from KcsA, where SF sites display a uniform decrease in occupancy with K⁺ concentration, in the low millimolar range, leading to SF collapse. We found that ions were disfavored in MthK’s S2 site due to weaker coordination by carbonyl groups, arising from different interactions with the pore helix and water behind the SF. We conclude that these differences in interactions endow the seemingly identical SFs of KcsA and MthK with strikingly different inactivating phenotypes.

钾通道可通过在膜的细胞外侧附近的高度保守的选择性过滤器（SF）区域内的栅极失活而变得不导电。在某些配体门控的通道中，例如BK通道和MthK（来自嗜热甲烷杆菌的Ca ²⁺激活的K ⁺通道），尽管潜在的构象变化是，SF也被认为在打开和关闭而不是失活中起作用。未知。使用X射线晶体学，在宽范围的K ⁺中获得了相同的导电MthK结构浓度（6至150 mM），与KcsA不同，后者的SF在低渗透离子浓度下会崩溃。出人意料的是，SF的四个结合位点中的三个仍在此范围内几乎完全被占据，表明高亲和力（可能为亚毫摩尔级），而只有中央S2位点被滴定，在6 mM处失去离子，表明K ⁺亲和力低（约50 mM）。 。分子模拟显示，与KcsA相似，在没有K ⁺的情况下MthK SF也会坍塌，但即使在除S4之外的任何SF位点上，即使单个K ⁺结合也可以挽救导电状态。跨结合位点的滴定不均匀不同于KcsA，后者的SF位点对K ^+的占用率均匀降低浓度在低毫摩尔范围内，导致SF塌陷。我们发现，由于与SF后面的孔隙螺旋和水的不同相互作用，羰基的配位作用较弱，因此MthK的S2位点处的离子不利。我们得出的结论是，相互作用的这些差异赋予了KcsA和MthK似乎相同的SF，并具有明显不同的失活表型。