Structure of an open K_ATPchannel reveals tandem PIP₂binding sites mediating the Kir6.2 and SUR1 regulatory interface

Abstract

ATP-sensitive potassium (KATP) channels, composed of four pore-lining Kir6.2 subunits and four regulatory sulfonylurea receptor 1 (SUR1) subunits, control insulin secretion in pancreatic β-cells. KATPchannel opening is stimulated by PIP2and inhibited by ATP. Mutations that increase channel opening by PIP2reduce ATP inhibition and cause neonatal diabetes. Although considerable evidence has indicated PIP2in KATPchannel function, previously solved open-channel structures have lacked bound PIP2, and mechanisms by which PIP2regulates KATPchannels remain unresolved. Here, we report cryoEM structure of a KATPchannel harboring the neonatal diabetes mutation Kir6.2-Q52R, bound to PIP2in open conformation. The structure reveals two adjacent PIP2molecules between SUR1 and Kir6.2. The first PIP2binding site is conserved among PIP2-gated Kir channels. The second site forms uniquely in KATPat the interface of Kir6.2 and SUR1. Functional studies demonstrate both binding sites determine channel activity. Kir6.2 pore opening is associated with a twist of the Kir6.2 cytoplasmic domain and a rotation of the N-terminal transmembrane domain of SUR1, which widens the inhibitory ATP binding pocket to disfavor ATP binding. The open conformation is particularly stabilized by the Kir6.2-Q52R residue through cation-π bonding with SUR1-W51. Together, these results uncover the cooperation between SUR1 and Kir6.2 in PIP2binding and gating, explain the antagonistic regulation of KATPchannels by PIP2and ATP, and provide the mechanism by which Kir6.2-Q52R stabilizes an open channel to cause neonatal diabetes.

Dynamic duo: Kir6 and SUR in K ATP channel structure and function

“…These include the structure of a human channel consisting of SUR1 and a mutant Kir6.2 harboring a pore mutation C166S known to stabilize channel opening as well as a G334D mutation that prevents ATP binding at the inhibition site (denoted by Kir6.2 C166S, G334D ), and the structure of a rodent SUR1-Kir6.2 fusion protein channel containing a Kir6.2 mutation H175K (Kir6.2 H175K ), which was incorporated to enhance PIP 2 binding. Recently, the cryoEM structure of a PIP 2 -bound open K ATP channel was reported in a preprint by Driggers et al [ 21 ]. Using a Kir6.2 variant harboring the neonatal diabetes-causing mutation Q52R (Kir6.2 Q52R ), which has been shown to increase channel open probability and decrease channel sensitivity to ATP inhibition in a SUR1-dependent manner [ 59 ], and by pre-incubating membranes expressing SUR1/Kir6.2 Q52R channels with natural long-chain PIP 2 , which provides more hydrophobic anchoring compared to synthetic short-chain PIP 2 , the authors observed, surprisingly, that not one but two adjacent well-resolved PIP 2 molecules occupy intramembrane positions between the Kir6.2 TMDs and SUR1-TMD0 ( Figure 3(a, b) ).…”

“…Panels (a) and (b) are taken from Figure 2 , and panel (c) from Figure 4 of bioRxiv preprint by Driggers et al. (reference [ 21 ]).…”

“…The second, adjacent PIP 2 binding site is formed by the transmembrane interface between the Kir6.2 TMDs and SUR1-TMD0, and is unique to the K ATP channel. Perturbation of either binding site by mutating key residues coordinating PIP 2 binding based on the structure markedly reduced channel open probability in electrophysiological studies under ambient membrane PIP 2 conditions [ 21 ], indicating both PIP 2 binding sites are functionally important for regulating K ATP channel activity in native membranes. The direct involvement of SUR1 in PIP 2 binding accounts for the previously observed increase in PIP 2 sensitivity conferred by SUR1 on Kir6.2 [ 36 , 39 , 40 ].…”

“…The PIP 2 -bound open SUR1/Kir6.2 Q52R structure also revealed a cation-π bonding between Kir6.2-Q52R and SUR1-W51. Disrupting this interaction by mutating SUR1-W51 to cysteine compromised the ability of Kir6.2-Q52R to increase channel open probability and reduce channel sensitivity to ATP inhibition [ 21 ]. These observations indicate that cation-π interaction between Kir6.2-Q52R and SUR1-W51 stabilizes the channel in an open conformation, providing a molecular explanation for how Kir6.2-Q52R causes gain-of-function channel defect in a SUR1-dependent manner [ 59 ].…”

“…PIP 2 and ATP functionally compete to open and close K ATP channels, respectively [ 36 , 61 ]. Comparison of the PIP 2 -bound open structure and the ATP-bound closed structure reveals that PIP 2 binding disrupts ATP binding at two levels, first by directly competing for shared binding residues and second by causing a conformational change that disfavors ATP binding [ 21 ] ( Figure 3c ). Specifically, Kir6.2-K39, which stabilizes ATP binding, is also involved in PIP 2 binding at the novel site ( Figure 3b ).…”