Cryo-EM structures of heteromeric Kir4.1/5.1 channel suggest mechanisms of inward rectification and channel blockage.

Inwardly rectifying potassium (Kir) channels play key roles in regulating membrane potential and potassium transport through voltage-dependent inhibition by cytoplasmic Mg²⁺ and polyamines. Despite decades of extensive studies, the structural basis for polyamine-mediated inward rectification remains unclear. Here, we present cryo-EM structures of the heteromeric Kir4.1/5.1 channel-which is critical for brain and kidney function and whose dysfunction causes EAST/SeSAME syndrome-in its apo state and in complex with spermine, a channel blocker named VU0134992, and EHop-016, an inhibitor identified in this study. The structures of Kir4.1/5.1, in an opposite 2:2 heterotetrameric assembly, suggest an inner-ring blockage mechanism, where polyamines and channel blockers with different stoichiometries bind in a membrane-parallel orientation at the upper site of the transmembrane central cavity. Together with electrophysiology and molecular dynamics simulations, our findings provide mechanistic insights into inward rectification, channel inhibition, and the pharmacology of Kir channels.