Multiple Ca2+ Binding Sites in the RCK Domain Contribute to Gating in the MthK K+ Channel

Abstract

MthK is 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 that is steeply dependent on [Ca2+], with a Hill coefficient >8. 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. Charge-neutralizing mutations of individual Ca2+-coordinating sidechains at each of the three sites decrease Ca2+-sensitivity of the channel, shifting the EC50 for Ca2+-activation by up to 8.4-fold. Combined mutations at pairs of sites decreased the Ca2+-sensitivity of mutant channels in an approximately energetically additive manner, suggesting that Ca2+ binding at each site may act independent of one another to faciliate channel opening. Finally, a combined mutation at all three sites effectively eliminates Ca2+-dependent activation of the channel through very high [Ca2+]. 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, and crystal structures of the MthK RCK domain in the presence of Ca2+ suggest points at which subunits may physically interact to facilitate channel opening.