Identification of a Novel PIP2 Interaction Site and its Allosteric Regulation by the RCK1 Site Associated with Ca2+ Coordination in Slo1 Channels

Abstract

PIP2 is necessary for activation of Slo1 channels, but the molecular determinants mediating its effects are not known. Slo1 channel activity is regulated by Ca2+ and membrane voltage. Increased Ca2+ has been reported to enhance PIP2 sensitivity but the mechanism is not understood. Here we combined electrophysiology, mutagenesis and computational modeling methods to identify critical Slo1 interaction sites with PIP2 and study their relationship to Ca2+ regulation. We first confirmed that Ca2+ indeed increases the apparent affinity for PIP2. We mutated each of the two reported Ca2+ coordination sites, in the RCK1 and RCK2 domains, and found that only the RCK1 site affected PIP2 sensitivity. Further mutagenesis results suggested that the RCK1 Ca2+ coordination site also modulates PIP2 sensitivity independently of the presence of Ca2+. We have identified the positively charged residues K392/ R393 in the RCK1 domain, mutation of which greatly decreased PIP2 sensitivity of Slo1 channels. We found that RCK1 acidic residues involved in Ca2+ sensitivity together with a cluster of basic residues in close proximity all coupled with the PIP2 sensitive residues. We propose that Ca+ coordination by RCK1 acidic residues removes an inhibitory interaction with the PIP2 sensor, thus increasing channel activity.