CryoEM structure of PIP2-bound, open SUR1/Kir6.2Q52R KATP channel.

(A) The 2.9 Å cryoEM map (the micelle not shown), from C4 non-uniform refinement of 14,115 particles, shown from the side and bottom (cytoplasmic view). (B) Structural model of the SUR1/Kir6.2Q52R KATP channel in complex with PIP2. Only two Kir6.2 and two SUR1 subunits are shown for clarity. The potassium ion selectivity filter is shown as sticks with oxygens colored red, the K+ conduction pathway is shown as magenta mesh and PIP2 as sticks with cyan carbons. A view rotated 90° shown from the bottom (cytoplasmic view) displays all four Kir6.2 subunits and all four SUR1 subunits. (C) Structural model of a closed WT SUR1/Kir6.2 KATP channel in complex with ATP and gliblenclamide (Glib) (PDB ID 6BAA), shown for comparison and rendered similar to (B), except for the Kir6.2 subunits which are colored grey.

Two PIP2 binding sites at the interface of Kir6.2 and SUR1.

(A) CryoEM map features of two PIP2 molecules colored in magenta and cyan (0.08V, 4.0σ contour), respectively. (B) Structural model of the PIP2 binding pocket (red boxed region in (A)) viewed from the side. Residues from both Kir6.2 (adjacent subunit denoted “b”) and SUR1 (blue outline) surrounding bound PIP2 molecules are labeled. (C) Structural model of the PIP2 binding pocket view from the top (extracellular side). (D) Representative inside-out patch-clamp recordings (−50mV, inward currents shown as upward deflections) of various channels: WT, SUR1-K134A (the second PIP2 binding site mutation)/Kir6.2, SUR1/Kir6.2-R176A (the first PIP2 binding site mutation), and SUR1-K134A/Kir6.2-R176A double mutant, exposed alternately in 5 μM PIP2, 0.1 mM ATP, and 1 mM ATP as indicated by the bars above the recordings. (E) Group data of recordings shown in (D) showing maximum fold-increase in currents after PIP2 exposure. Each bar represents mean ± SEM of 5-9 patches. *Statistical significance between WT and each mutant, by two-tailed Student’s t-test, p < 0.005.

Comparison of the inner helix gate in the open and closed KATP channel structures.

(A) Structure of Kir6.2Q52R in a PIP2-bound open state. Bound PIP2 are shown as green and red sticks and spheres, and the side chains of inner helix gating residues L164 and F168 at the helix bundle crossing are shown as yellow sticks and labeled in red. The pore for the ion pathway (purple mesh) is constricted at the selectivity filter (SF) but open at the inner helix gate (3.3 Å). The transmembrane α-helices (M1 and M2), the interfacial α-helices (IFH) and the pore α-helices (PH) are labeled. (B) Kir6.2 in an ATP-bound closed state (PDB ID 6BAA) with similar labels as panel A for comparison. (C) Pore radii for ion conduction pathway plotted against the distance from the extracellular opening, with the location of the selectivity filter (residues 130-133), the inner helix gate (residues L164 and F168), and the G-loop gate (residues 294-297) shown. The pore radii were calculated using the program HOLE implemented in coot and viewed using a MOLE rendering in PyMOL.

SUR1 and Kir6.2 cytoplasm-plasma membrane interface in open and closed KATP channel conformations.

(A) An overlay of the SUR1-Kir6.2 cytoplasm-plasma membrane interface between open PIP2 (cyan and magenta sticks)-bound SUR1/Kir6.2Q52R (SUR1 in teal and Kir6.2 in yellow) KATP channel and closed ATP-(orange phosphate and grey carbon sticks) and repaglinide (out of view)-bound WT(SUR1 in pink and Kir6.2 in grey) KATP channel (PDB ID 7TYS) viewed from the extracellular side. Reorientation of side chains of the Kir6.2 inner helix gate residue F168 and M1 residue F60 in the two conformations is shown. A 6.4° clockwise rotation of the Kir6.2-CTD is indicated by the magenta curved arrow (with D323 Cα in each structure marked as spheres). Reorientation of the side chain of W51 at the bottom of TM1 of SUR1-TMD0 is evident. SUR1-L0, marked by the ATP-binding residue K205 and an adjacent residue E203, moves away from the ATP binding pocket (magenta arrow) in the open conformation. (B) A side view of the interface in the open conformation. SUR1-intracellular loop 1 (ICL1) forms a β-sheet with Kir6.2-βA just before the interfacial helix (IFH). Remodeling of residues involved in ATP binding and gating compared to the closed conformation in (C), including Kir6.2-R50, R54, E51 and K39(b) (from the adjacent subunit chain b) as well as SUR1-E203 and K205 in L0 are shown. Most notably, the Kir6.2 mutant residue Q52R has its side chain reoriented to interact with the side chain of SUR1-W51 which also has its side chain reoriented from the closed conformation. Color scheme same as (A). (C) The same side view as in (B) but of the closed conformation. In all panels, SUR1 residue labels are outlined in blue.

Structural changes between open- and closed-forms of the KATP channel at the SUR1 and Kir6.2 TM interface.

(A) An overlay of the SUR1-Kir6.2 TM interface between open SUR1/Kir6.2Q52R KATP channel and the closed KATP channel (PDB ID 6BAA) showing the outward movement of the cytoplasmic half of the SUR1-TMD0. In the open structure, SUR1 is shown in teal and Kir6.2 in yellow. The closed structure is shown in grey. (B) Close-up view of the open structure in the boxed region in (A). CryoEM map (blue mesh, 0.17V/8.5σ map contour) is superposed with the structural model. For clarity, PIP2 is omitted. (C) An overlay of the open with the closed KATP channel structures in the boxed region in (A) (same color scheme) showing significant changes in residues of SUR1-TM1 at the inner leaflet half of the plasma membrane.

Kir6.2-Q52R interacts with SUR1-W51 to reduce channel sensitivity to ATP inhibition and enhance channel activity.

(A) A close-up view of the interaction between Kir6.2-Q52R and SUR1-W51 with cryoEM map (blue mesh, 0.17V/8.5σ contour) superposed with the structural model. (B) Representative inside-out patch-clamp recordings (−50mV, inward currents shown as upward deflections) of the WT-SUR1/Kir6.2-Q52R (Q52R) channels and SUR1-W51C/Kir6.2-Q52R (W51C+Q52R) channels exposed to differing concentrations of ATP as indicated by the bars above the recordings. (C) ATP dose response of WT, SUR1-W51C/Kir6.2, SUR1/Kir6.2-Q52R (Q52R), and SUR1-W51C/Kir6.2-Q52R (Q52R+W51C) channels. Curves were obtained by fitting the data points (mean ± SEM of 3-5 patches) to the Hill equation (see Methods). (D) Rb+ efflux of various channels expressed in COSm6 cells. Untransfected cells were included to show background efflux. Only SUR1/Kir6.2Q52R channels show significant activity above WT (p < 0.05, n=5; one-way ANOVA with Tukey post hoc test).

KATP channel disease mutations near the PIP2 binding pocket.

Residues with variants that have been reported to cause neonatal diabetes/DEND syndrome are shown as green spheres and congenital hyperinsulinism shown as orange spheres. Neonatal diabetes/DEND syndrome mutations include SUR1-P45L, I49F, F132L/V, L135P, and Kir6.2-K39R, E51A/G, Q52L/R, G53D/N/R/S/R/V, V59A/G/M, W68C/G/L/R, K170N/R/T, E179A/K. Congenital hyperinsulinism mutations include SUR1-I46T, P133R and L135V, and Kir6.2-R54C, L56G, K67D, R176H, and R177W.

CryoEM data processing workflow.

The total number of particles yielding the 2.9 Å C4 Non-uniform refinement reconstruction is 14,115 (FSC auto mask for GSFSC calculation).

Fourier Shell Correlation (FSC) curves and local resolution for the C4 non-uniform reconstruction of 14,115 particles.

(A) FSC curves between two independent half-maps calculated within a mask for the full particle including micelle (green), a mask for full KATP channel without the micelle (pink), and a mask created by cryoSPARC auto-mask tightening that excludes NBD2 (blue). FSC curves for model:map (unmasked) for the full KATP channel model and map (PDB ID 8TI2; EMD-41278), and for the KATP channel without NBD2 (PDB ID 8TI1). (B) Images of the masks used for map:map FSC calculations shown from the cytoplasmic view. (C) The local resolution estimate for the reconstructed map at 4 σ (0.08 V) contour, with micelle density not shown, in side view (left), top view (middle) and bottom view (right). No local filtering or local sharpening was used for visualization. Local resolution estimates were calculated in cryoSPARC and visualized in ChimeraX. FSC curves were calculated in PHENIX and cryoSPARC.

CryoEM lipid density fitting.

(A) CryoEM map reconstructed from symmetry expansion of the ∼14k particles followed by local refinement with a focused mask of Kir6.2 tetramer plus one SUR1, with density modification in Phenix and map segmentation in Chimera, shows the cryoEM densities of SUR1 and the Kir6.2 tetramer colored blue and yellow, respectively. Two PIP2 molecules are shown in magenta and cyan, and other density including lipid density shown in grey (0.08V/4σ contour). (B) Isolated lipid density located between SUR1-TMD0 and SUR1-TMD1 fit with a model of phosphatidylserine (PS, grey carbons, 0.08V/4σ contour) and isolated lipid density at each of the two PIP2 binding sites located between SUR1-TMD0 and Kir6.2 shown fit with model of the first-(cyan carbons) and second-(magenta carbons) (C18:0/C20:4) PI(4,5)P2. (C) Rotation of the map 180° relative to panel A reveals other densities (grey, 0.08V/4.0σ contour) corresponding to two adjacent lipids primarily associated with the TMD0 domain. (D) Adjacent lipid densities can be well fit as a phosphatidylserine (PS) and phosphatidylethanolamine (PE), with the amine group of PE hydrogen bonding to the phosphate group of PS. On the right, density for each of these adjacent lipid molecules is isolated to show the fit of the lipid model into the lipid’s corresponding density. These five lipid densities (20 lipids for full-channel) that are captured in the micelle are closely associated with the KATP channel and are sufficiently resolved to allow modeling.

Comparison of the SUR1-ABC core structure in the PIP2-bound SUR1/Kir6.2Q52R open channel (A) and the repaglinide (RPG)- and ATP-bound SUR1/Kir6.2 closed channel (B).

In the closed channel structure shown in (B) (EMD-26193, PDB ID 7TYS) a cryoEM density (map filtered to 6 Å and contoured to 0.5 σ) corresponding to Kir6.2-KNtp (grey stick model) next to the bound RPG is clearly visible. In contrast, in the open structure shown in (A) no comparable peptide-like cryoEM density is observed (map also filtered to 6 Å and contoured to 0.5σ), consistent with the absence of the Kir6.2-Ntp or a highly flexible Kir6.2-Ntp. Note the density protruding from SUR1-TMD2 TM helix likely corresponds to W1297 with some R1145 contribution.

Conformational dynamicsof the SUR1 subunit revealed by CryoSPARC 3D classification.

Symmetry expansion of the 14,115 particles gave 56,460 particles, which were subjected to 3D classification with a focused mask of Kir6.2 tetramer plus one SUR1. Three unique NBD-separated SUR1 positions were resolved. (A) Overlay of side view maps of class 1 (3.0 Å resolution, 23,454 particles, red map), class 2 (3.3 Å resolution, 16,863 particles, yellow map) and class 3 (3.4 Å resolution, 13604 particles, blue map) reconstructed from a local non-uniform refinement with density modification in Phenix, shows the variability in SUR1 including the transmembrane regions of TMD1 and TMD2, with a common Kir6.2 tetramer position. (B) View from the cytoplasmic side highlights the difference in the SUR1-NBD positions, with mobility seen in both SUR1-NBD1 and SUR1-NBD2.

The KATP channel closed conformation clashes with the novel second PIP 2 molecule, but both the open and closed conformations could accommodate the conserved first PIP 2.

(A,B) The open KATP channel with the conserved first PIP2 (cyan carbons) and novel second PIP2 (magenta carbons) shown interacting with Kir6.2 subunits (pink, green and blue surface) and the SUR1 subunit (orange surface), with the two panels rotated ∼15° to show the surface of each PIP2 site. (C,D) The closed KATP channel (PDB ID 7TYS, same surface colors as A,B) aligned with the PIP 2 molecules of the open channel shows that the closed KATP channel accommodates the conserved first PIP2 (cyan carbons) but clashes with the novel second PIP2 (magenta carbons).

Model statistics of the PIP2-bound SUR1/Kir6.2Q52R structure