The I182 Region of K ir6.2 Is Closely Associated with Ligand Binding in K ATP Channel Inhibition by ATP

Abstract

The ATP-inhibited potassium (K ATP) channel is assembled from four inward rectifier potassium (K ir6.x) subunits and four sulfonylurea receptor (SURx) subunits. The inhibitory action of ATP is mediated by at least two distinct functional domains within the C-terminal cytoplasmic tail of K ir6.2. The G334D mutation of K ir6.2 virtually eliminates ATP-dependent gating with no effect on ligand-independent gating, suggesting a role in linkage of the site to the gate or in the ATP binding site, itself. The T171A mutation of K ir6.2 strongly disrupts both ATP-dependent and ligand-independent gating, suggesting a role for T171 in the gating step. A neighboring mutation, I182Q, virtually eliminates ATP inhibition, but its effect on ligand-independent gating remained unknown. We have now characterized both the K i values for inhibition by ATP and the ligand-independent gating kinetics of 15 substitutions at position 182. All substitutions decreased ATP-dependent inhibition gating as measured by the K i, many profoundly so, yet had little or no effect on ligand-independent gating kinetics. Thus, substitutions at position 182 are unlikely to act by disrupting inhibition gate movement. Our results indicate an indispensable role for I182 in a step of the ATP binding mechanism, the linkage mechanism coupling the ATP binding site to the inhibition gate, or both.