Voltage Dependence of Kir2.1 Block by Intracellular Spermine

Strong voltage sensitivity of inward-rectifier K+ (Kir) channels, such as Kir2.1, has been hypothesized to arise primarily from an intracellular spermine molecule displacing K+ ions from the wide, intracellular part of the ion conduction pore outwardly across the narrow ion selectivity filter. This hypothesis anticipates: i) that mutations intracellular to the ion selectivity filter can abolish any high-affinity spermine block, and ii) that the blocker can force essentially unidirectional K+ movement in a pore region generally wider than the combined dimensions of the blocker plus a K+ ion. We find that simultaneous mutation of five residues, all located internal to the selectivity filter, abolishes specific spermine block. Thus, the selectivity filter itself evidently must have little inherent affinity for spermine. We also find that a constriction near the intracellular end of the pore, acting as a gasket, prevents K+ ions from bypassing the blocker. This heretofore unrecognized gasket ensures that the blocker can effectively displace K+ ions across the selectivity filter to generate exceedingly strong voltage sensitivity.