Cooperative Regulation of Slack Channel by Na+, Cl− and PIP2

Abstract

Slack (or Slo2.2) channel activity is regulated by Na+ and Cl- ions. The Na+ coordination site of Slack channels bears similarity to the analogous site in Kir3 channels. Here, we show that the activity of Slack channels, like BK (or Slo1) and Slo3 channels, is also dependent on PIP2. We next ask: How are these multiple positive cooperative factors interact with each other to regulate Slack channel activity? We first examined the relationship of Na+ and PIP2 regulation. Unlike Kir3 channels, the D818N mutation, which decreases Na+ sensitivity greatly, showed no effect on PIP¬2 sensitivity. Manipulations that weaken channel-PIP2 interactions in Kir2.1 channels induce residence into subconductance states (Xie et al., 2008, J. Physiol. 586:1833). Unitary Slack channel activity rarely visited sub-conductance levels in the absence of Cl-. Yet, in the presence of Cl-, the Slack single channel conductance was shifted from 145 pS to 80∼100 pS, while the open probability of Slack channels was increased by 2-2.5 fold due to the cooperative regulation of Cl- with Na+. We are in the process of screening for potential Cl- binding site(s) in the cytoplasmic domain of Slack channels, utilizing a strategy similar to the one that enabled us to identify the D818 Na+ coordination site. We hypothesize a close cooperative relationship between PIP2 and Cl- regulation of Slack activity. We are working on investigating this cooperativity and developing a suitable model to explain this complex regulatory mechanism.