Mechanism for attenuated outward conductance induced by mutations in the cytoplasmic pore of Kir2.1 channels

Outward currents through Kir2.1 channels regulate the electrical properties of excitable cells. These currents are subject to voltage-dependent attenuation by the binding of polyamines to high-and low-affinity sites, which leads to inward rectification, thereby controlling cell excitability. To examine the effects of positive charges at the low-affinity site in the cytoplasmic pore on inward rectification, we studied a mutant Kir channel (E224K/H226E) and measured single-channel currents and streaming potentials (V-stream), the latter provide the ratio of water to ions queued in a single-file permeation process in the selectivity filter. The water-ion coupling ratio was near one at a high K+ concentration ([K+]) for the wild-type channel and increased substantially as [K+] decreased. On the other hand, fewer ions occupied the selectivity filter in the mutant at all [K+]. A model for the Kir channel involving a K+ binding site in the wide pore was introduced. Model analyses revealed that the rate constants associated with the binding and release to and from the wide-pore K+ binding site was modified in the mutant. These effects lead to the reduced contribution of a conventional two-ion permeation mode to total conductance, especially at positive potentials, thereby inward rectification.