Regulator of K þ conductance (RCK) domains control the activity of a variety of K þ transporters and channels, including the human large conductance Ca 2þ-activated K þ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca 2þ-gated K þ channel that has yielded structural insight toward mechanisms of RCK domain-controlled channel gating. In MthK, a gating ring of eight RCK domains regulates channel activation by Ca 2þ. Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca 2þ-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca 2þ-binding sites, resulting in a stoichiometry of 24 Ca 2þ ions per channel, is consistent with the steep relation between [Ca 2þ ] and MthK channel activity. Comparison of Ca 2þ-bound and unliganded RCK domains suggests a physical mechanism for Ca 2þ-dependent conformational changes that underlie gating in this class of channels. calcium | lipid bilayer | cooperativity R egulator of K þ conductance (RCK) domains are structurally conserved ligand-binding domains that control the activity of a diverse array of K þ channels and transporters (1-3). Many prokaryotic RCK domains contain a conserved sequence motif for binding of nucleotides (NAD þ or ATP) (4, 5). In some pro-karyotic and most of the known eukaryotic RCK-containing K þ channels, however, the nucleotide binding motif is absent, and these channels are modulated by cytoplasmic ions such as Na þ , H þ , or Ca 2þ (6-12). MthK is a prototypical RCK-containing K þ channel that has provided insight toward the structural basis of ion channel gating by RCK domains (2, 13, 14). In MthK, binding of Ca 2þ to an oc-tameric ring of RCK domains (the gating ring), which is tethered to the pore of the channel, leads to a series of conformational changes that facilitates channel opening and K þ conduction (2, 15, 16). Based on X-ray structures of the Ca 2þ-bound MthK channel and the unliganded MthK gating ring (17), it has been hypothesized that the principal Ca 2þ-dependent conformational change is initiated by the movement of a Glu side chain (E212) at a single Ca 2þ-binding site within each RCK domain (Fig. 1A; site 1, formed by D184, E210, and E212), followed by subsequent movement of a nearby Phe side chain (F232). However, the conformational changes in the immediate vicinity of site 1 are relatively subtle compared to apparent conformational changes in other regions of the RCK domains (17); thus the mechanism by which Ca 2þ binding at site 1 modulates channel gating is unclear. To gain insight toward mechanisms underlying Ca 2þ-dependent conformational changes in the MthK RCK domain, we probed MthK structure and function using electrophysiology and crystallography. Our results demonstrate that whereas site 1 contributes energetically to Ca 2þ-dependent gating, charge-neutrali-zation of the key Ca 2þ-coordinating residues at this site do not eliminate Ca 2þ...