Structural rearrangements underlying ligand-gating in Kir channels

Abstract

Inward rectifier potassium (Kir) channels are physiologically regulated by a wide range of ligands that all act on a common gate, although structural details of gating are unclear. Here we show, using small molecule fluorescent probes attached to introduced cysteines, the molecular motions associated with gating of KirBac1.1 channels. The accessibility of the probes indicates a major barrier to fluorophore entry to the inner cavity. Changes in fluorescence resonance energy transfer between fluorophores, attached to KirBac1.1 tetramers, show that phosphatidylinositol-4,5-bisphosphate-induced closure involves tilting and rotational motions of secondary structural elements of the cytoplasmic domain that couple ligand binding to a narrowing of the cytoplasmic vestibule. The observed ligand-dependent conformational changes in KirBac1.1 provide a general model for ligand-induced Kir channel gating at the molecular level.

Fig. 1. Accessibility of KirBac1.1 channel pore lining residues

Time course (a, b) and 10 min time point data (c, d, boxed in a, b) of Alexa-Fluor 488 C5 maleimide incorporation (F, a.u.) of cysteine-substituted KirBac1.1 mutants in the presence or absence of 10 µg/ml diC8-PIP₂ (mean ± S.E., n=3 in each case, error bars are smaller than symbol in most cases) (e) Ribbon diagram indicating accessibility of AF-488 to substituted cysteine residues. Alpha carbons of tested residues in this and subsequent figures are highlighted by spheres, with inaccessible residues colored red, limited accessible (147 and 149) purple and highly accessible blue.

Fig. 2. Functional analysis of fluorophore-labeled KirBac1.1 cysteine-substituted mutants

Fluorophore-labeled KirBac1.1 mutants were reconstituted into liposomes (POPE:POPG=3:1) with or without 1.25% PIP₂ at protein/lipid ratio of 1:100 (w/w). The intraliposome buffer was 10 mM HEPES, 450 mM KCl and 4 mM NMDG, pH7.5, and the extraliposome buffer was 10 mM HEPES, 50 mM KCl, 400 mM sorbitol and 4 mM NMDG, pH7.5. ⁸⁶Rb⁺ uptake was measured at 15 min and normalized against the maximal ⁸⁶Rb⁺ uptake in the presence of valinomycin (Rb uptake). ⁸⁶Rb⁺ uptake of fluorophore-labeled mutants is shown as ⁸⁶Rb⁺ flux relative to wild type (mean±S.E, n=3 in each case). Background level of ⁸⁶Rb⁺ uptake (in liposomes with no protein) is marked by a red dashed line.

Fig. 3. FRET measurements reveal movements of individual residues during PIP₂ induced closure

(a) Representative time course of FRET measurements by proteinase K-mediated donor dequenching. KirBac1.1 R151C and T264C tetramers were labeled by A/D mixtures, then reconstituted into liposomes (POPE:POPG=3:1). Proteinase K (0.08U/well) was added after 8 repeated readings (F_o) (T=5 min); Alexa-Fluor-546 emission (F, a.u.) was monitored until emission reached maximum (F_max). (b) Cα-Cα distance between adjacent subunits of labeled residues predicted by FRET (mean±S.E, n=6–9 in each case) versus those present in the KirBac1.1 (2WLL) crystal structure. R and p values of correlation are 0.51 (p<0.010), 0.54 (p<0.006) for Cα-Cα distances calculated from measured FRET efficiencies in the absence (control) and presence of 1.25% PIP₂, respectively.

Fig. 4. Mapping FRET changes of individual residues to KirBac1.1 crystal structure suggest specific motions during gating

Changes of apparent FRET efficiencies of KirBac1.1 cysteine mutants in the large β-sheet (βI, panel a and b, green) and small β-sheets (βII and associated loops, panel c and d, green) in presence versus absence of PIP₂ (ΔE_PIP2, mean±S.E., n=6–9 in each case). Cα of the labeled residue is highlighted by spheres; residues demonstrating inward motion in the presence of PIP₂ are colored blue, those demonstrating outward motion are colored red; the pore axis of KirBac1.1 is marked by dashed black line; amino acid residues in panels b and d are listed from top to bottom, along the axis indicated by a solid blue line.

Fig. 5. ‘Cartoon’ model of ligand-gating of Kir channels

Views of closed (1P7B, gray) and ‘open’ (red) models of KirBac1.1 in (left) and from below (right) the plane of the membrane. Opening requires (i) outward twisting and tilting of βI, and (ii) outward motion of βII and associated short helices. For clarity, only two subunits are shown in each view.