An iris diaphragm mechanism to gate a cyclic nucleotide-gated ion channel

Abstract

Cyclic nucleotide-gated (CNG) ion channels are non-selective cation channels key to signal transduction. The free energy difference of cyclic-nucleotide (cAMP/cGMP) binding/unbinding is translated into mechanical work to modulate the open/closed probability of the pore, i.e., gating. Despite the recent advances in structural determination of CNG channels, the conformational changes associated with gating remain unknown. Here we examine the conformational dynamics of a prokaryotic homolog of CNG channels, SthK, using high-speed atomic force microscopy (HS-AFM). HS-AFM of SthK in lipid bilayers shows that the CNBDs undergo dramatic conformational changes during the interconversion between the resting (apo and cGMP) and the activated (cAMP) states: the CNBDs approach the membrane and splay away from the 4-fold channel axis accompanied by a clockwise rotation with respect to the pore domain. We propose that these movements may be converted by the C-linker to pull the pore helices open in an iris diaphragm-like mechanism.

Fig. 1

SthK is a cyclic nucleotide-gated ion channel related to the CNG and the HCN subfamilies. a Cartoon of the topology of one subunit. The different functional domains are highlighted in colors: voltage sensor domain (VSD, red), pore domain (PD, blue), C-linker (CL, yellow), and cyclic nucleotide binding domain (CNBD, purple). b Phylogenetic tree of the CNGA (green) and HCN (blue) subfamilies. The prokaryotic CNG channels LliK and MloK1 (lacking the CL), of which the structures are known, are shown for comparison. c High-resolution cryo-EM structure of the SthK resting state (PDB 6CJQ) viewed from the extracellular (top), membrane plane (middle), and intracellular (bottom) side. The functional domains are color-coded as in a. Turrets are highlighted by arrowheads. For clarity, the VSD and PD domains of the protomer nearer to the viewer have been omitted in the membrane plane view

Fig. 2

Characterization of the SthK 2D-crystals in presence of cAMP. a HS-AFM image of a 2D-crystal viewed from the extracellular side. Each channel appears as a square-shaped tetramer. Each protruding turret of the pore domain within the channel tetramer is clearly resolved. Inset: The alternating packing of the channels is highlighted with dashed outlines (scale bar: 15 nm). b Four-fold symmetrized correlation average of a. The unit cell (dashed square, dimensions a = b = 11.6 nm, γ = 90°) comprises two tetramers (scale bar: 10 nm). c Left: Height profiles of SthK molecules along the dashed and solid lines in a are shown by the black and grey traces, respectively. Right: Height histogram of the two classes of molecules with 0.3 and 0.6 nm average protrusion heights. d Packing model of the 2D-crystal (PDB 6CJQ). e, f, g, h same as a, b, c, and d, but the 2D-crystal is imaged from the intracellular side exposing the CNBDs. Dashed circles in a and e highlight the alternating packing of the tetramers in the 2D-crystal. The stronger-protruding CNBDs on the intracellular side (e, f) are the less protruding ones from the extracellular side (a, b). White arrows in h indicate the CW rotation that the CNBDs should undergo upon cAMP-binding to match the HS-AFM data. The dotted white and cyan circles in h show the position of the protrusions in the data, which do not match the packing of the resting state SthK model. i Structural model with 25° CW rotated CNBDs matches the experimental data (compare with h). The white and cyan dashed circles in f highlight the two neighboring tetramers of high and low height, respectively. The 25° CW rotation of the CNBDs in the model brings the model CNBD back into the white and cyan dashed circles (i)

Fig. 3

Dynamics of ligand-induced conformational changes in SthK by real-time HS-AFM imaging. a HS-AFM time-lapse high-resolution image sequence of a SthK 2D-crystal initially in 0.1 mM cAMP and exposing CNBDs. Upon addition of 7 mM cGMP, SthK channels undergo a conformational change progressively from the borders to the center of the membrane patch (dotted while outline). Scale bar: 30 nm. b Representative electrophysiology traces from SthK channels in the presence of 0.1 mM cAMP (top and bottom traces), and 7 mM cGMP and 0.1 mM cAMP (middle trace). Activity from the same bilayer, perfused to different solutions is displayed to enable direct comparisons. C and O are closed and open channel levels, respectively. Three channels appear active, each with a Po of ~0.25 at 0.1 mM cAMP. c Kinetics of cAMP to cGMP conformational transitions of the 2D-crystal shown in a. d Height profiles along the blue (cAMP) and red (cGMP) dashed lines in a. e Distributions of the relative height differences of the very same molecules in the cGMP conformation and the higher (1) and lower (2) molecules in the cAMP conformation. f Histograms of the radial distance of the cAMP (blue) and cGMP (red) CNBDs from the central four-fold axis. g Left: High-resolution topography of a membrane containing well-ordered channels in both conformations. Right: Zoom into region outlined in g with overlaid red circles for the resting state channels and blue circles (upper molecules) and blue rhomboids (lower molecules) in the activated state. The crystal was imaged in the presence of 0.1 mM cAMP and was one of a handful of membranes that contained resting and activated state molecules simultaneously (scale bar: 10 nm). h High-resolution topographs during a cAMP to cGMP transition where the majority of the molecules are in the cAMP (left) and in the cGMP (right) conformation, respectively (scale bar: 10 nm). i Correlation average of the resting state 2D-crystal in g. Scale bar: 10 nm. j Packing model of the 2D-crystal using the SthK structure (PDB 6CJQ). The unit cell (dashed square in i and j, dimensions a = b = 8.1 nm, γ = 90°), comprises one tetramer

Fig. 4

Reversibility of cyclic nucleotide-dependent conformational changes. a HS-AFM time-lapse high-resolution image sequence showing CNBD conformational and 2D-lattice rearrangement dynamics of SthK upon cGMP injection (panels t = 12 min and t = 23 min) and after controlled cGMP removal and cAMP re-addition via a fluid exchange pumping system (panels t = 38 min and t = 71 min). Insets in t = 2 min, t = 23 min and t = 71 min are correlation averages (n = 22, n = 39, and n = 17 tetramers) of the corresponding frames. The molecular conformational changes lead to local membrane bending (bright area in the top part of the membrane). Scale bar: 30 nm. b cAMP (blue) and cGMP (red) concentrations as a function of time during the HS-AFM experiment. Dashed lines and adjacent labels indicate the times (bottom) and the cAMP (blue) / cGMP (red) concentrations when the images shown on top were acquired. Inset displays cGMP addition through pipetting (1) and cAMP buffer exchange (2)

Fig. 5

The conformational changes in SthK upon activation. a Left: SthK in the resting state (cartoon based on the high-resolution cryo-EM structure, PDB 6CJQ). Right: Model of SthK in the activated state: Upon activation, the CNBDs rotate by ~25° clockwise (when viewed from the intracellular side) and move by ~6 Å towards the membrane and by ~4 Å outwards from the four-fold axis (note, the activated state is a cartoon assembled using domains of the SthK structure repositioned according to the displacements found with HS-AFM). Key residues in and around S3 that are evidenced to undergo conformational changes are highlighted in white. b Close-up view onto the SthK C-linker (yellow) from the intracellular side in the resting state. The CNBDs are attached at the periphery to the C-linker disk with a radius ~3 nm from the central axis. Rotation of the CNBDs by ~25° clockwise induces a torque that is transmitted to the CNBD-S6 attachment. c Clockwise rotation of the C-linker will pull the right-handed S6 bundle-crossing open like a diaphragm. d Simple mechanical model where the C-linker is a type-2 lever with two attachment points to the S6 helix and to the CNBD. (S6) and (CNBD) indicate the respective attachment sites, r(S6) and r(CNBD) are the radial distance from the central axis, F(S6) and F(CNBD) the forces acting on the attachment sites, and ∆x(S6) and ∆x(C-linker) are the displacements, of the respective domains