Calcium and Pip2 Interplay Regulates BK Channel Activity via the RCK1 Gating Ring

Abstract

BK channels are high conductance K+ channels that are activated by voltage and intracellular Ca2+. Ca2+ binds to sites in the RCK2 and RCK1 domains of Slo1, the pore-forming subunit of BK channels, to enhance voltage-dependent gating of the channel. PIP2, a ubiquitous modulator of ion channel activity, enhances Ca2+-driven gating of BK channels but a molecular understanding of this interplay remains elusive. Here, we report that amino acid residues in the RCK2 calcium bowl that bind Ca2+ do not affect PIP2 sensitivity. In contrast, the RCK1 Ca2+ coordination site is intimately involved in PIP2 sensitivity. We identified two critical residues within the alphaB helix, K392 and R393, are directly involved in coordinating PIP2. In the absence of Ca2+, the alphaB helix interacts with the alphaA helix to decrease the apparent affinity of the channel to PIP2 and inhibit channel activity. Ca2+ binding to the RCK1 site engaging D367 of the KDRDD loop relieves the decrease in PIP2 apparent affinity and channel inhibition. Furthermore, mutation of residues in the KDRDD loop or the alphaA/alphaB residues that couple this region to the alphaB PIP2 regulatory site also relieve the decrease in the apparent affinity for PIP2 and current inhibition in the absence of Ca2+. In addition, we show that the PMA-induced current inhibition also exhibits a dependence on channel-PIP2 interactions that determine the extent of modulation. Together these results show that along with Ca2+ and voltage, PIP2 is a critical third factor providing integral control of BK channel activity.