Functional analysis of a structural model of the ATP-binding site of the KATP channel Kir6.2 subunit

Abstract

ATP-sensitive potassium (KATP) channels couple cell metabolism to electrical activity by regulating K+ flux across the plasma membrane. Channel closure is mediated by ATP, which binds to the pore-forming subunit (Kir6.2). Here we use homology modelling and ligand docking to construct a model of the Kir6.2 tetramer and identify the ATP-binding site. The model is consistent with a large amount of functional data and was further tested by mutagenesis. Ligand binding occurs at the interface between two subunits. The phosphate tail of ATP interacts with R201 and K185 in the C-terminus of one subunit, and with R50 in the N-terminus of another; the N6 atom of the adenine ring interacts with E179 and R301 in the same subunit. Mutation of residues lining the binding pocket reduced ATP-dependent channel inhibition. The model also suggests that interactions between the C-terminus of one subunit and the 'slide helix' of the adjacent subunit may be involved in ATP-dependent gating. Consistent with a role in gating, mutations in the slide helix bias the intrinsic channel conformation towards the open state.

Figure 1

(A) Alignment of Kir3.1, Kir6.2 and KirBac1.1 sequences. Regions boxed in red and black show residues in Kir3.1 and KirBac1.1, respectively, used to construct the model. (B, C) Model of the Kir6.2 tetramer viewed from the side (B) or above (C). (B) Residues are shown in ribbon format, with different colours representing individual subunits. (C) TMs are shown in backbone format and IC domains in ribbon format. Different colours indicate individual subunits, with dark and light shades representing the N and C domains, respectively.

Figure 2

Interactions between adjacent subunits. The C domain of one subunit is shown in grey and its N domain in yellow. The C domain of the adjacent subunit is shown in green. Residues are shown in cpk colours.

Figure 3

(A, B) Relationship between the N domain of one subunit (yellow) and the C domain of another (green), showing the relation of loops 1 and 2 to the slide helix. (C) Interactions between loops 1 and 2 and the C domain of one subunit and the slide helix of the adjacent subunit. (D) Web of predicted interactions between the C linker, loops 1 and 2 of the C domain of one subunit (yellow) and the slide helix of the adjacent subunit (blue). Coloured lines connect interacting residues, or indicate interactions with ATP.

Figure 4

(A) Side view of the ATP-binding site. For clarity, the TMs of only two subunits and the IC domains of two separate subunits are illustrated. ATP (yellow) is docked into its binding sites. (B) Kir6.2 tetramer, viewed from above, with the TMs removed (residues 64–177). ATP (yellow) is docked into its binding sites. The N domain is shown in ribbon format and the C domain in backbone format. Different colours represent individual subunits.

Figure 5

(A) Interactions between ATP and residues lining the ATP-binding pocket. The subunit origin (A or D) of the residue is indicated. Predicted hydrogen bonds are indicated by dashed lines and hydrophobic interactions by sunbursts. Residues are shown in cpk colours. (B) Space-filling model of the ATP-binding pocket. Different subunits are indicated in blue and pink. The electron shells of R50 and K185, and of ATP, are shown in transparency. (C) Close-up of the binding pocket with 8-azido-ATP placed in the same position as ATP. The dashed lines indicate where the azido group makes a steric clash with T180 and the ATP molecule itself.

Figure 6

(A) K_ATP currents elicited by voltage ramps from −110 to +100 mV to an inside-out patch excised from a Xenopus oocyte expressing Kir6.2ΔC or Kir6.2ΔC-E179M. The dotted line indicates the zero current level. (B) Mean relationship between [ATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (Gc) for Kir6.2ΔC with E179 mutated to N (, n=5), M (, n=5), L (, n=5) and Q (, n=7). The curves are the best fit to equation (1), using values for IC₅₀ and h given in Table I. The dashed line indicates the wild-type data.

Figure 7

(A) Kir6.2ΔC and Kir6.2ΔC-R201H currents evoked by voltage ramps from −110 to +100mV. (B) Mean relationship between [ATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c) for Kir6.2ΔC-R201C (n=5). The curve is the best fit to equation (1) using the values given in Table I. The dotted line indicates the wild-type data.

Figure 8

(A) Model of the ATP-binding pocket of Kir6.2 illustrating the proximity of F333 and Y330 to the phosphate tail of ATP. (B) Mean relationship between [ATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c) for Kir6.2-Y330L/SUR1-KA/KM (•, n=5), and Kir6.2-F333L/SUR1-KAKM (▪, n=6) channels. The smooth curves are the best fit to equation (1). For Kir6.2-Y330L, IC₅₀=16.9±1.9 μM, h=1.3±0.1. For Kir6.2-F333L, IC₅₀=514±93 μM, h=1.2±0.1. The dashed line indicates the control data (IC₅₀=17 μM, h=0.99; Gribble et al, 1997).

Figure 9

(A) Slide helix of Kir6.2, showing the location of V59. The slide helix of one subunit is shown in yellow and the TM domain of the adjacent subunit in green. (B) Kir6.2ΔC and Kir6.2ΔC-V59G currents evoked by voltage ramps from −110 to +100 mV in inside-out patches. (C) Mean relationship between [ATP] and K_ATP conductance (G), expressed relative to the conductance in the absence of nucleotide (G_c) for Kir6.2ΔC-V59G (n=6). The curve is the best fit to equation (1) using the values given in Table I. The dashed line indicates the wild-type data. (D) Single Kir6.2ΔC and Kir6.2ΔC-V59G channel currents recorded at –60 mV. The arrow indicates the zero current level.