Structure and function of multiple Ca2+-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 Ca2+-activated K+ channel that is important for blood pressure regulation and control of neuronal firing, and MthK, a prokaryotic Ca2+-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 Ca2+. Here, using electrophysiology and X-ray crystallography, we show that each RCK domain contributes to three different regulatory Ca2+-binding sites, two of which are located at the interfaces between adjacent RCK domains. The additional Ca2+-binding sites, resulting in a stoichiometry of 24 Ca2+ ions per channel, is consistent with the steep relation between [Ca2+] and MthK channel activity. Comparison of Ca2+-bound and unliganded RCK domains suggests a physical mechanism for Ca2+-dependent conformational changes that underlie gating in this class of channels.