Hydrophobic and lipid-mediated gating mechanism revealed by low-conductance MthK mutants

Large-conductance calcium (Ca²⁺)-activated potassium (K⁺) channels are involved in several essential cellular processes. This requires the ability to alternate between conductive and non-conductive states, yet the molecular mechanism of their gating remains incompletely understood. Bundle-crossing, selectivity filter, and hydrophobic gating are some of the mechanisms previously proposed for these channels, including the archaeal Ca²⁺-activated K⁺ channel MthK. Here, we took advantage of two MthK mutants that show dramatically-reduced conductance to gain insights into how the channels gate at the molecular level by combining cryo-electron microscopy, functional assays, and extensive molecular dynamics simulations. Functional and computational analyses reveal distinct mechanisms of reduced conductance between the two mutants. One mutant, where a glutamate residing at the intracellular mouth of the pore was replaced by an alanine (E92A) reduced ion density in the cavity and dramatically accelerated spontaneous channel closure. The second mutant, where an alanine residing in the channel's aqueous cavity below the selectivity filter was replaced by a phenylalanine (A88F), introduced a hydrophobic constriction that partially dehydrates the cavity, imposing a barrier to ion permeation. The cryo-EM structures of these mutants, virtually identical to those of WT MthK, were used as models to investigate gating using molecular dynamics (MD) simulations. Long-timescale simulations captured complete open-to-closed transitions in both mutants, uncovering a dehydrated intermediate state and a tightly coupled lipid-entry mechanism that drove final pore closure. Our results show that hydrophobicity and lipid interactions are central determinants of MthK gating.