Identification of the PIP2-binding site on Kir6.2 by molecular modelling and functional analysis

Abstract

ATP-sensitive potassium (K(ATP)) channels couple cell metabolism to electrical activity by regulating K(+) fluxes across the plasma membrane. Channel closure is facilitated by ATP, which binds to the pore-forming subunit (Kir6.2). Conversely, channel opening is potentiated by phosphoinositol bisphosphate (PIP(2)), which binds to Kir6.2 and reduces channel inhibition by ATP. Here, we use homology modelling and ligand docking to identify the PIP(2)-binding site on Kir6.2. The model is consistent with a large amount of functional data and was further tested by mutagenesis. The fatty acyl tails of PIP(2) lie within the membrane and the head group extends downwards to interact with residues in the N terminus (K39, N41, R54), transmembrane domains (K67) and C terminus (R176, R177, E179, R301) of Kir6.2. Our model suggests how PIP(2) increases channel opening and decreases ATP binding and channel inhibition. It is likely to be applicable to the PIP(2)-binding site of other Kir channels, as the residues identified are conserved and influence PIP(2) sensitivity in other Kir channel family members.

Figure 1

Models of the Kir6.2 tetramer viewed from side. (A) Surface electrostatic map of Kir6.2 model. The red colour corresponds to negatively charged regions and the blue colour to positively charged regions. The box placed over the central positively charged region defines the boundary within which the PIP₂ head group was allowed to dock. (B) Kir6.2 model (green) with the docked PIP₂ head group (IP₃, orange) as identified by the AUTODOCK programme. The grid defines the boundary within which the PIP₂ head group was allowed to dock. Two binding sites were observed (a) and (b): the lower one (b) was deemed not to be physiological (see text). (C) Kir6.2 model (green) placed in a POPC bilayer (the small yellow spheres represent phosphorus atoms) with one docked PIP₂ (red) positioned in its binding site. The acyl tails of PIP₂ extend to anchor it in the lipid bilayer.

Figure 2

Model of PIP₂-binding pocket of Kir6.2 highlighting the charged and polar residues that lie close to PIP₂. All residues lie on the same subunit except for R54.

Figure 3

(A) Surface representation of the complete Kir6.2 model with docked ATP (red) and PIP₂ (yellow) molecules in their respective binding sites. (B) Close-up of a single binding site with surface representations of ATP and PIP₂.

Figure 4

(A) Cα-RMSDs (for all residues) from the initial structure plotted versus time for the different simulations: Sim1, Kir6.2+4 PIP₂ (green); Sim2, Kir6.2+4 ATP (red); p Sim3, Kir6.2+4 ATP+4 PIP₂ (blue); Sim4, Kir6.2 (black). (B) Minimum distance between R201 α-phosphate (top), K185 β-phosphate (middle) and R50 γ-phosphate (bottom) compared between two different simulations, when ATP was present alone (Sim2, red) and when ATP was simultaneously present in its binding site along with PIP₂ (Sim1, black). R201–ATP and K185–ATP interactions are maintained throughout the simulation in model 2, but the R50–ATPγP interaction is broken midway during the simulation. In model 3, the interaction is broken early in the simulation.

Figure 5

Position of R50 in Sim2 (A) and in Sim3 (B). In Sim2, the R50–ATPγP interaction is maintained. However, in the presence of PIP₂ (Sim3), the R50–ATPγP interaction is broken and R50 is pulled towards 4P in the PIP₂ head group.

Figure 6

(A) above, K_ATP currents elicited by voltage ramps from −110 to +100 mV applied to an inside-out patch excised from a Xenopus oocyte expressing Kir6.2ΔC. below, Kir6.2ΔC-R176A currents recorded from an inside-out patch at a holding potential of −60 mV. The dotted line indicates the zero current level. Bars indicate addition of neomycin. (B) Mean relationship between neomycin concentration and K_ATP conductance (G), expressed relative to the conductance in the absence of neomycin (G₀) for Kir6.2ΔC (filled squares), Kir6.2ΔC-R54E (open circles), Kir6.2ΔC-R54L (open diamonds), Kir6.2ΔC-K67L (filled circles) or Kir6.2ΔC-R176A (open squares) channels. The curves are the best fit to equation 1. (C) K_ATP currents elicited by voltage ramps from −110 to +100 mV applied to an inside-out patch excised from a Xenopus oocyte expressing Kir6.2/SUR1 (above). Kir6.2-R176A/SUR1 currents recorded from an inside-out patch at a holding potential of −60 mV (below). The dotted line indicates the zero current level. Bars indicate addition of neomycin. (D) Mean relationship between neomycin concentration and K_ATP conductance (G), expressed relative to the conductance in the absence of neomycin (G₀) for SUR1 coexpressed with Kir6.2 (filled squares), Kir6.2-R54E (open circles), Kir6.2-R54L (open diamonds), Kir6.2-K67L (filled circles) or Kir6.2-R176A (open squares). The curves are the best fit to equation 1.

Figure 7

(A, B) Activation of wild-type and mutant K_ATP channels, as indicated, by 25 μM diC₈-PIP₂. The number of patches is indicated above the bar. Statistical significance against wild-type is indicated (Mann–Whitney U-test): P<0.05 (^*), P<0.01 (^**).