The inward rectifier potassium channel Kir2.1 is more sensitive to the weakly voltage-dependent block by extracellular Mg2+ (Mgo2+) than Kir2.2 and Kir2.3. We identified Glu125 in an extracellular loop before the pore region of Kir2.1 as a site responsible for this sensitivity to Mo2+ block, based on the observations that the Glu125Gln (E125Q) mutation strongly decreased the sensitivity, while a mutation to Glu at the corresponding sites of Kir2.2 and 2.3 led to an increase. The negative charge proved to be crucial since the Glu125Asp (E125D) mutant showed similar properties to the wild type (WT). A similar weakly voltage-dependent block was also caused by extracellular Ca2+ and La3+ in Kir2.1 WT but not in the E125Q mutant. The sensitivity to block by extracellular Ba2+ (Bao2+) was also decreased in the E125Q mutant, although the voltage dependency of half-inhibition concentration was not changed, as reported previously. We additionally observed that the speed of Bao2+ block and recovery was decelerated by the presence of Mgo2+ in WT, but not in the E125Q mutant. The sensitivity to the block by Mgo2+ was increased by lowering extracellular K+ (Ko+), suggesting a competitive interaction of Mgo2+ and Ko+. The single-channel conductance of the WT in 140 mM K+ was 39.6 pS (0 mM Mgo2+) and 11.5 pS (10 mM), while that of the E125Q mutant was 26.0 pS (0 mM) and 19.6 pS (10 mM). These results demonstrate that Mg2+ competes with K+ permeation in the WT and that E125 is required for efficient K+ permeation in the absence of Mgo2+. We conclude that E125 in an extracellular loop of Kir2.1 is a site which facilitates K+ permeation and entry of Ba2+ toward a deeper plugging site, and that Mgo2+ competes with Ko2+, and Bao2+ at this site.
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