Lipid trapping slows ball-and-chain inactivation in a calcium-activated potassium channel.

Ion channel inactivation is a key modulatory mechanism that shapes action potentials and cellular excitability. In N-type (ball-and-chain) inactivation, a tethered N-terminal domain occludes the open pore. The prokaryotic MthK channel, a homolog of BK channels, undergoes such inactivation via its N-terminus. Notably, MthK inactivation was observed in liposome assays but not in decane-containing planar bilayer recordings, suggesting membrane dependence. We found that MthK inactivation progressively slowed with increasing bilayer thickness in liposomes composed of varying acyl-chain length lipids. Pore size was not a determining factor, as cryo-EM structures and molecular dynamics (MD) simulations showed similar pore dimensions across conditions, and block of a non-inactivating mutant by a peptide mimicking the N-terminal domain was largely bilayer thickness-independent. Instead, MD simulations, later confirmed with mutagenesis, revealed that N-terminal arginines form stronger interactions with lipid phosphates in thicker bilayers, thus limiting the access of the N-terminus to the pore and slowing inactivation.