PIP2 inhibits pore opening of the cyclic nucleotide-gated channel SthK

Abstract

The signaling lipid phosphatidylinositol-4,5-bisphosphate (PIP2) regulates many ion channels. It inhibits eukaryotic cyclic nucleotide-gated (CNG) channels while activating their relatives, the hyperpolarization-activated and cyclic nucleotide-modulated (HCN) channels. The prokaryotic SthK channel from Spirochaeta thermophila shares features with CNG and HCN channels and is an established model for this channel family. Here, we show SthK activity is inhibited by PIP2. A cryo-EM structure of SthK in nanodiscs reveals a PIP2-fitting density coordinated by arginine and lysine residues from the S4 helix and the C-linker, located between voltage-sensing and pore domains of adjacent subunits. Mutation of two arginine residues weakens PIP2 inhibition with the double mutant displaying insensitivity to PIP2. We propose that PIP2 inhibits SthK by gluing S4 and S6 together, stabilizing a resting channel conformation. The PIP2 binding site is partially conserved in CNG channels suggesting the possibility of a similar inhibition mechanism in the eukaryotic homologs.

Fig. 1. PIP2 inhibits SthK activity.

a Representative quenching kinetics from a Tl⁺ flux assay measuring SthK activity in proteo-liposomes with a lipid composition of 3:1 DOPC:POPG (shades of grey and black) and 3:1 DOPC:POPG + 2.5 % PIP2 (shades of blue). Individual traces are: without quencher and without cAMP (lightest grey and lightest blue, no signal change observed), with quencher but without cAMP (medium light grey and medium light blue, slow, minimal, linear fluorescence decline due to leakage of Tl⁺ across liposomal membrane) and with quencher and 200 µM cAMP (black and dark blue, exponential quenching kinetics reflecting channel activity upon activation). b Initial Tl⁺ flux rates (Eqs. (1) and (2)) from experiments as in (a), averaged values ± SEM are normalized to the flux rate without PIP2 (n = 3 independent repeats), individual data points are in grey. See “methods” for normalization procedure. c Representative single-channel recordings of SthK in the presence of 300 µM cAMP reconstituted into 3:1 DOPC:POPG (top) and 3:1 DOPC:POPG + 5 % PIP2 (bottom) at +100 mV and -100 mV. Closed levels are indicated by dashed lines. d Normalized all-amplitude histograms from single-channel recordings as in (c) (see “methods”), with PIP2 (blue) and without PIP2 (black), at +100 mV. e Single-channel current-voltage (I/V) relation and (f) single-channel open probability (Po) obtained from recordings as in (c). Individual data points (light colors) and averaged values ± s.d. (dark colors) are depicted (number of independent repeats n is specified next to each data point in the corresponding color).

Fig. 2. SthK structure in the presence of PIP2.

a Density map of SthK in 3:1 DOPC:POPG + 10 % PIP2 nanodiscs. Protein density is shown in grey, lipid densities in yellow and PIP2 density in blue. b Zoom of inner leaflet lipids shown in (a). A PIP2 molecule is modeled into the lipid density (blue mesh) in the inner leaflet. Yellow mesh is an additional lipid binding along the S6 helix, previously found to modulate SthK activity. c Model of the PIP2 binding site highlighting residues coordinating PIP2. d Electrostatic surface potential of the binding pocket with blue representing positive and red negative potential. e The modeled PIP2 density (left) is absent from other SthK structures in different lipid compositions at similar resolution and threshold (middle: EMD-25916, right: EMD-24670).

Fig. 3. Functional characterization of SthK with PIP2 binding site mutations.

Representative single-channel recordings for (a) SthK R120A, (b) SthK R124A, and (c) SthK R120A/R124A in the presence of 300 µM cAMP reconstituted into 3:1 DOPC:POPG (top traces) and 3:1 DOPC:POPG + 5 % PIP2 (bottom traces). Recordings at −100 mV and +100 mV are shown. Average Po ± s.d. of (d) SthK R120A, (e) SthK R124A and (f) SthK R120A/R124A at different voltages without PIP2 (squares) and in the presence of 5 % PIP2 (circles). The number of independent repeats, n, is indicated for each voltage in the plots. Individual data points are shown in grey. g Comparison of initial Tl⁺ flux rates of the different SthK variants. Averaged values ± SEM (n = 3 independent repeats) of measurements with different lipid compositions are normalized to the rate without PIP2 of the respective variant. Individual data points are shown in grey. h Normalized Po of single channel recordings at + 100 mV of different SthK variants in bilayers with or without 5 % PIP2. Averaged values ± s.d. are shown (number of independent repeats, n, for each condition is shown in the corresponding bars) as well as individual data points (grey). Statistical significance was assessed with a two-tailed unpaired t-test with a confidence level of 95 % resulting in P = 4 × 10⁻⁸ for WT SthK and P = 5 × 10⁻⁸ for SthK R120A. *** P < 0.001.

Fig. 4. Structural characterization of SthK with mutated PIP2 binding sites.

a Density map of SthK R120A in 3:1 DOPC:POPG + 10 % PIP2 nanodiscs. Protein density is shown in light blue, lipid densities in yellow and PIP2 density in blue. Zoom focuses on the lipid densities. b Density map of SthK R120A/R124A in 3:1 DOPC:POPG + 10 % PIP2 nanodiscs. Protein density is shown in light green and lipid densities in yellow. Zoom focuses on the lipid densities. c Comparison of lipid densities in cryo-EM maps of WT SthK (top), SthK R120A (middle), and SthK R120A/R124A (bottom). Density of PIP2 is shown in blue and of the additional lipid bound along the S6 helix in yellow.

Fig. 5. Putative mechanism of PIP2 inhibition.

a Alignment of the PIP2 inhibited structure (grey) with SthK in the open conformation (blue, PDB: 7TJ6). Movements of the helices associated with channel opening are depicted by yellow arrows. The C-linker undergoes an upward movement leading to an outward rotation of the S6 helix that can only be facilitated by outward movements of the lower S4-S5 helices. b PIP2 binding in the closed state (top) and superimposed on the open state (bottom). In the closed state, Arg residues on S4 electrostatically coordinate PIP2 (green dashes) while in the open state the distance is too far. In addition, steric clashes are observed between PIP2 and the C-linker (red traffic circles). c Alignment of the SthK PIP2 binding site with the structure of human CNGA1 (PDB: 7LFT). Positively charged residues responsible for PIP2 binding in SthK are labelled and three of them are conserved in CNGA1.