RCK is a cytoplasmic regulatory domain of calcium-gated potassium channels. Binding of Ca2+ by RCK leads to channel activation through a series of yet unknown conformational changes. Structures of the K+ channel, MthK, and its cytoplasmic RCK domain revealed two binding sites for Ca2+ per dimer. We determined the crystal structure of RCK in complex with Cd2+ at 2.2 angstrom resolution. Cd2+ activates MthK more efficiently, and binds at the same binding sites for Ca2+ but with reduced coordination number. Two additional binding sites for Cd2+ are found per dimer: one on the main Rossman-fold lobe, and the other on the small lobe of RCK. Using patch-clamp experiments, we demonstrate that Cd2+ binding to these novel sites enhances activation by Cd2+ and not by Ca2+. The structure reveals a large negatively charged surface patch in the proximity of the Ca2+/Cd2+ binding sites, charge neutralization of which appears to promote the channel open state. (C) 2010 Elsevier Inc. All rights reserved.