The gate at the pore-forming domain of potassium channels is allosterically controlled by a stimulus-sensing domain. Using Cd2+ as a probe, we examined the structural elements responsible for gating in an inward-rectifier K+ channel (Kir3.2). One of four endogenous cysteines facing the cytoplasm contributes to a high-affinity site for inhibition by internal Cd2+. Crystal structure of its cytoplasmic domain in complex with Cd2+ reveals that octahedral coordination geometry supports the high-affinity binding. This mode of action causes the tethering of the N-terminus to CD loop in the stimulus-sensing domain, suggesting that their conformational changes participate in gating and Cd2+ inhibits Kir3.2 by trapping the conformation in the closed state like "inverse agonist". (C) 2011 Elsevier Inc. All rights reserved.