Inward rectifier K+ channels commonly exhibit long openings (slow gating) punctuated by rapid open-close transitions (fast gating), suggesting that two separate gates may control channel open-closed transitions. Previous studies have suggested possible gate locations at the selectivity filter and at the 'bundle crossing', where the two transmembrane segments (M1 and M2) cross near the cytoplasmic end of the pore. Wild-type Kir2.1 channels exhibit only slow gating, but mutations in the cytoplasmic pore domain at E224 and E299 have been shown to induce fast flickery gating. Since these mutations also affect polyamine affinity, we conjectured that the fast gating mechanism might affect the kinetics of polyamine block/unblock if located more intracellularly than the polyamine blocking site in the pore. Neutralization of either E224 or E299 induced fast gating and slowed both block and unblock rates by the polyamine diamine 10. The slowing of polyamine block/unblock was partly relieved by raising PH from 7.2 to 9.0, which also slowed fast gating kinetics. These findings indicate that the fast flickery gate is located intracellularly with respect to the polyamine pore-plugging site near D172, thereby excluding the selectivity filter, and implicating the bundle crossing or more intracellular site as the gate. As additional proof, fast gating induced at the selectivity filter by disrupting P loop salt bridges in WT-E138D-E138D-WT tandem had no effect on polyamine block and unblock rates. The pH sensitivity of fast gating in E224 and E299 mutants was attributed to the protonation state of H226, since the double mutant E224Q/H226K induced fast gating which was pH insensitive. Moreover, introducing a negative charge in the 224-226 region was sufficient to prevent fast gating, since the double mutant E224Q/H226E rescued wild-type Kir2.1 slow gating. These observations implicate E224 and E299 as allosteric modulators of a fast gate, located at the bundle crossing or below in Kir2.1 channels. By suppressing fast gating, these negative charges facilitate polyamine block and unblock, which may be their physiologically important role.
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