Alpha-B Helix of RCK1 is a Major Transduction Pathway for Ca2+ Activation of BK Channels

Abstract

BK (Slo1) type K+ selective channels are activated by both depolarization and intracellular Ca2+. BK channels are comprised of an integral-membrane core consisting of four lateral voltage sensor domains surrounding a central pore-gate domain (S5-S6), and a large cytosolic domain (CTD) assembled from 8 RCK domains. The CTD (gating ring) is attached to the core through four short S6-RCK1 peptide linkers. Ca2+ binding to the CTD activates the channel. Crystal structures (Yuan et al. 2010, 2012) suggest that Ca2+ binding elevates the alpha-B helix in each RCK1 domain where it could push upwards against the core while simultaneously moving a rigid protein arm in RCK1 laterally and downward to pull on S6 through the S6-RCK1 linkers. Ca2+ activation thus may involve a synergistic push-pull mechanism. We examined the contribution of the alpha-B helix to this hypothetical mechanism by introducing several discrete mutations into the alpha-B helix. The mutation L390P in the alpha-B helix of mSlo1 greatly decreased Ca2+ activation. The crystal structure of the hSlo1 CTD with the L390P mutation indicated a discrete alteration of the alpha-B helix alone: Ca2+ still bound to the Ca2+ bowl and there were no other notable differences in the CTD structure. L390P decreased Ca2+ activation through both high affinity Ca2+ binding sites, and also increased the voltage required for activation in 0 Ca2+. Lengthening the RCK1-S6 linker of L390P by one residue in 0 Ca2+ decreased voltage activation, indicating that L390P did not remove all of the tension in the linker. Other alpha-B helix mutations were also consistent with push-pull activation. These observations suggest that an intact and rigid alpha-B helix plays a major role in transducing Ca2+ binding to pore opening in BK channels. Supported by R01 GM114694.