PH-Dependent Gating Mechanism of Kir2.1 Inward Rectifier K+ Channel Independent of Polyamine and Magnesium Block

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Kir2.1 inward rectifier K+ channel shows a strong inward rectification due to a voltage-dependent block of the channel pore by intracellular cations, such as polyamines and Mg2+. In this study, we conducted experiments using inside-out patch membranes and found that Kir2.1 channel exhibits an extremely slow, voltage-dependent gating that depends on the cytoplasmic pH in the acidic range. This gating seemed to be unrelated to the block induced by polyamines that remained trapped near the cytoplasmic pore or by Mg2+ or impurities of EDTA contaminated in the cytoplasmic solution. Acidification of the cytoplasmic solution did not markedly affect the polyamine block of the wild-type Kir2.1 channel, indicating that the acidic residues lining the Kir2.1 pore (e.g. D172, E224 and E299), whose negative charges are known to contribute to polyamine binding sites, were not neutralized at acidic pHs. Thus, these negative charges did not seem to confer the pH sensitivity of the gating. However, when Kir2.1 channels bearing a mutation at these residues were tested, neutralization of D172 in the transmembrane region abolished the pH-dependent gating. The findings suggest that the gating may be caused by a pore block by an unknown molecule, bearing a positive charge at acidic pHs.