Abstract
Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca(2+)). Phosphatidylinositol 4,5-bisphosphate (PIP2), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca(2+)-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP2 regulation of this channel's activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca(2+) and PIP2 in regulating BK channel activity. In the absence of Ca(2+), RCK1 structural elements limit channel activation through a decrease in the channel's PIP2 apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca(2+) coordination site (Asp(367) or its flanking basic residues in the KDRDD loop) to the PIP2-interacting residues (Lys(392) and Arg(393)) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca(2+), the RCK1-inhibitory influence of channel-PIP2 interactions and channel activity is relieved by Ca(2+) engaging Asp(367). Our results demonstrate that, along with Ca(2+) and voltage, PIP2 is a third factor critical to the integral control of BK channel activity.
Highlights
PIP2 has been reported to enhance Ca2ϩ-driven gating, but the molecular determinants of this interplay are not known
These results suggest that PIP2 could serve in the role of allosterically coupling the cytosolic RCK1 structural elements (KDRDD loop, ␣A and ␣B helices) to the membranegating elements of the channel
We found that Ca2ϩ relieved a KDRDD loop inhibitory influence on channel activation by increasing the apparent affinity to PIP2
Summary
PIP2 has been reported to enhance Ca2ϩ-driven gating, but the molecular determinants of this interplay are not known. In the absence of [Ca2ϩ]i, it became clear that Asp367 as well as its two flanking basic residues, Lys366 and Arg368 (Fig. 1A), served to inhibit channel activation by decreasing the apparent affinity for PIP2 These results suggested that the KDRDD loop exerted an inhibitory effect on channel activation through PIP2. PIP2 docking simulations with the two available crystal structures and mutagenesis identified two basic residues in the ␣B helix, Lys392 and Arg393, as critical elements in the coordination of PIP2 (Fig. 1C, right) These results suggest that PIP2 could serve in the role of allosterically coupling the cytosolic RCK1 structural elements (KDRDD loop, ␣A and ␣B helices) to the membranegating elements of the channel
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