Structure and function of multiple Ca ²⁺-binding sites in a K ⁺ channel regulator of K ⁺ conductance (RCK) domain

Regulator of K ⁺ conductance (RCK) domains control the activity of a variety of K ⁺ transporters and channels, including the human large conductance Ca ²⁺-activated K ⁺ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca ²⁺-gated K ⁺ 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 ²⁺. Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca ²⁺-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca ²⁺-binding sites, resulting in a stoichiometry of 24 Ca ²⁺ ions per channel, is consistent with the steep relation between [Ca ²⁺] and MthK channel activity. Comparison of Ca ²⁺-bound and unliganded RCK domains suggests a physical mechanism for Ca ²⁺-dependent conformational changes that underlie gating in this class of channels.