Identification of a site involved in the block by extracellular Mg2+ and Ba2+ as well as permeation of K+ in the Kir2.1 K+ channel

Abstract

The inward rectifier potassium channel Kir2.1 is more sensitive to the weakly voltage-dependent block by extracellular Mg²⁺ () 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 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⁺ () 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 block and recovery was decelerated by the presence of in WT, but not in the E125Q mutant. The sensitivity to the block by was increased by lowering extracellular K⁺ (), suggesting a competitive interaction of and . The single-channel conductance of the WT in 140 mm K⁺ was 39.6 pS (0 mm ) 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 . 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 competes with and at this site.