The bundle crossing region is responsible for the inwardly rectifying internal spermine block of the Kir2.1 channel.

Inward rectifier potassium channels conduct K(+) across the cell membrane more efficiently in the inward than outward direction in physiological conditions. Voltage-dependent and flow-dependent blocks of outward K(+) currents by intracellular polyamines (e.g., spermine (SPM)) have been proposed as the major mechanisms underlying inward rectification. In this study, we show that the SPM blocking affinity curve is shifted according to the shift in K(+) reversal potential. Moreover, the kinetics of SPM entry to and exit from the binding site are correlatively slowed by specific E224 and E299 mutations, which always also disrupt the flux coupling feature of SPM block. The entry rates carry little voltage dependence, whereas the exit rates are e-fold decelerated per ∼15 mV depolarization. Interestingly, the voltage dependence remains rather constant among WT and quite a few different mutant channels. This voltage dependence offers an unprecedented chance of mapping the location (electrical distance) of the SPM site in the pore because these kinetic data were obtained along the preponderant direction of K(+) current flow (outward currents for the entry rate and inward currents for the exit rate) and thus contamination from flow dependence should be negligible. Moreover, double mutations involving E224 and A178 or M183 seem to alter the height of the same asymmetrical barrier between the SPM binding site and the intracellular milieu. We conclude that the SPM site responsible for the inward rectifying block is located at an electrical distance of ∼0.5 from the inside and is involved in a flux coupling segment in the bundle crossing region of the pore. With preponderant outward K(+) flow, SPM is "pushed" to the outmost site of this segment (∼D172). On the other hand, the blocking SPM would be pushed to the inner end of this segment (∼M183-A184) with preponderant inward K(+) flow. Moreover, E224 and E299 very likely electrostatically interact with the other residues (e.g., R228, R260) in the cytoplasmic domain and then allosterically keep the bundle crossing region in an open conformation appropriate for the flux coupling block of SPM.