Structure of a eukaryotic cyclic-nucleotide-gated channel

Abstract

Cyclic-nucleotide-gated channels are essential for vision and olfaction. They belong to the voltage-gated ion channel superfamily but their activities are controlled by intracellular cyclic nucleotides instead of transmembrane voltage. Here we report a 3.5-Å-resolution single-particle electron cryo-microscopy structure of a cyclic-nucleotide-gated channel from Caenorhabditis elegans in the cyclic guanosine monophosphate (cGMP)-bound open state. The channel has an unusual voltage-sensor-like domain, accounting for its deficient voltage dependence. A carboxy-terminal linker connecting S6 and the cyclic-nucleotide-binding domain interacts directly with both the voltage-sensor-like domain and the pore domain, forming a gating ring that couples conformational changes triggered by cyclic nucleotide binding to the gate. The selectivity filter is lined by the carboxylate side chains of a functionally important glutamate and three rings of backbone carbonyls. This structure provides a new framework for understanding mechanisms of ion permeation, gating and channelopathy of cyclic-nucleotide-gated channels and cyclic nucleotide modulation of related channels.

Extended Data Figure 1. Functional characterization of WT and mutant TAX-4 channels

a, Family of macroscopic currents recorded in an inside-out patch from a HEK 293T cell expressing WT TAX-4 channels. Currents were elicited by 10 μM cGMP, in response to voltage steps from −150 mV to +150 mV in 10-mV increments from a holding potential of 0 mV. b, Current-voltage (I-V) relationship from the same patch as in a. Currents were recorded first in the control bath solution (bath), then in the presence of 10μM cGMP, and then again in the control bath solution (washout). c, Averaged I‐V relationship of macroscopic currents from seven independent inside-out patch recordings from HEK 293T cells expressing WT TAX-4 channels. Currents were obtained as in a and b.For each patch, currents at different voltages were normalized by the absolute current value at -150 mV. In this and subsequent figures, error bars represent S.E.M. and are smaller than the symbols at some voltages. Notice the lack of voltage-dependent gating. d, Dose-response relationship of cGMP activation of WT TAX-4 channels at –100 mV. Currents were recorded in inside-out patches obtained from HEK 293T cell expressing WT TAX-4 channels. Data points represent mean ± S.E.M. of the indicated number of measurements. Solid curve represents fit to the Hill equation in the form of I(X)=Xⁿ/(Xⁿ+EC₅₀ⁿ), where I(X) is the normalized current, X the cGMP concentration, n the Hill coefficient, and EC₅₀ the cGMP concentration producing half maximal current. The fit yields an EC₅₀ of 0.16 μM and n of 1.7, which are very close to the values of 0.34 μM and 1.6 reported previously. e, Family of currents recorded in the whole-cell configuration from a HEK 293T cell expressing WT TAX-4 channels. Currents were elicited by 100 μM cGMP added in the recording pipette, in response to voltage steps from −100 mV to +150 mV in 10-mV increments from a holding potential of 0 mV. f-j, Averaged I-V relationships of whole-cell currents recorded from the indicated number of HEK 293T cell expressing WT TAX-4 channels and the indicated mutant TAX-4 channels. The recording pipette contained either no cGMP or 100 μM cGMP. The non-liner I-V relationship exhibited by cGMP-activated WT TAX-4 channels (f) is likely caused by block by an endogenous cytoplasmic molecule. In TAX-4_5A, R421, Q425, D429, K432 and D453 in helices A′B′ are mutated to alanine. In TAX-4_1G, TAX-4_2G and TAX-4_3G, one, two or three glycine residues are inserted between M417 and S418. k, Surface expression of WT and mutant TAX-4 channels in HEK 293T cells. The channels contain a GFP tag on its N-terminus and an HA tag in the extracellular linker between S1 and S2, except for GFP-TAX-4_WT, which does not contain the HA tag. Red fluorescence represents surface expression. Similar observations were made in >10 cells for each channel type (including GFP-TAX-4_1G-HA and GFP-TAX-4_2G-HA) in blind experiments.

Extended Data Figure 2. Amino acid sequence alignment of TAX-4, human CNGA1, bovine CNGA1, human HCN2 and the K_v1.2–2.1 chimera

Sequence alignment begins at D115 of TAX-4 and ends at L677. Green and yellow highlight identical and similar amino acids, respectively. Secondary structures are marked for TAX-4. The TAX-4 channel selectivity filter is boxed in blue. Amino acids in red in TAX-4 participate in intersubunit interactions between helices A′B′ and C′D′. Red triangles mark the amino acids involved in cGMP binding in TAX-4 and HCN2. Red dots mark the amino acids involved in forming the charge transfer center in the K_v1.2–2.1 chimera. Positions of S4 positive charges are boxed in red in the K_v1.2–2.1 chimera.

Extended Data Figure 3. Single-particle cryo-EM analysis of TAX-4

a, A representative motion-corrected micrograph of TAX-4 recorded using the K2 Summit camera. Typical particles are marked with yellow boxes. b, Fourier power spectrum of the micrograph shown in a with the Thon ring extending to 3Ǻ. c, Gallery of 2D class averages. d, Three enlarged views of representative 2D classes. e, Work flow of two-reference 3D classification. Structures produced by the final refinement with or without C4 imposed are given. Particle number is given below each reconstruction.

Extended Data Figure 4. 3D reconstruction and refinement of TAX-4

a, Two isosurface levels (low is grey and high is blue) of the final density map filtered to 5 Ǻ. The density contributed by amphipol is visible at the low isosurface level. b, Selected z-slice views of the unfiltered map in a at the corresponding layers indicated by the arrows. c, FSC curve of the final 3D reconstruction with C4 imposed (black) marked with a resolution of 3.5 Å corresponding to the FSC=0.143 cut-off criterion. FSC curve of the final 3D reconstruction without C4 imposed (red) marked with a resolution of 4.5 Å corresponding to the FSC=0.143 cut-off criterion. The FSC curve between the final reconstruction and the map calculated from the atom model (blue) shows a resolution of 3.8 Å according to the FSC=0.5 cut-off criterion. d, Euler angle distribution of all particles used in the final map reconstruction. Each orientation is represented by a cylinder, for which both the height and color (from blue to red) are proportional to the number of particles for that specific direction. e-i, Color-coded final 3D reconstruction of TAX-4 showing local resolutions. The tetramer (e) and four different orientations (f-i) of the protomer are shown, viewed parallel to the membrane. The orientation in f is the same as that in Fig. 1c.

Extended Data Figure 5. Validation of the TAX-4 model

a, 3D reconstruction and model refinement statistics. b, FSC curves for cross-validation of the final model. Blue: model versus the summed map. Red: model versus half 1 map (called ‘work’, used for model refinement). Black: model versus half 2 map (called ‘free’, not used for model refinement).

Extended Data Figure 6. Cryo-EM density maps and atomic models of selected key TAX-4 regions

Maps were low-pass filtered to 3.5 Å and amplified by a temperature factor of -160 Å, and were contoured at 3.0σ.

Extended Data Figure 7. Unique arrangement of the TAX-4 pore domain and voltage-sensor-like domain

a, Comparison of the arrangement of the pore domain (S5/P-loop/S6) and voltage-sensor domain (VSD, i.e., S1-S4/S4-S5 linker) or voltage-sensor-like domain (VSLD) of TAX-4 and the selected channels. The pore domain and VSD or VSLD of one subunit (for AtTPC1 and Ca_v1.1, one homologous repeat) is shown in green, and the pore domain of an adjacent subunit (or homologous repeat) is shown in red. In all comparison channels, the pore domain of the red subunit (or repeat) cross-interacts with the VSD or VSLD of the green subunit (or repeat). In TAX-4, however, the pore domain and VSLD of the green subunit form intrasubunit interactions. AtTPC1 is made up of two identical two-pore subunits. Each subunit contains two homologous 6-TM repeats, each of which has its own pore domain and VSD. AtTPC1(a) and AtTPC1(b) represent the two types of pore domain/VSD interactions present in AtTPC1 (ref). Ca_v1.1 contains 4 homologous 6-TM repeats, each of which has its own pore domain and VSD. Only one representative pore domain/VSD interaction is shown, with green and red representing repeat III and IV, respectively. The PDB codes of the comparison channels are: K_v1.2–2.1, 2R9R; Na_vAb, 3RVY; Na_vRh, 4DXW; Mlotik1, 3BEH; AtTPC1, 5E1J; TRPV1, 3J5P; TRPA1, 3J9P; Ca_v1.1, 3JBR; RyR1, 3J8H; InsP₃R, 3JAV. b, Superposition of all structures in a, aligned by S5 and S6 of the green subunit. For contrast, green is changed to indigo for TAX-4. c, Comparison of the tertiary and quaternary structures of TAX-4 and the K_v1.2–2.1 chimera, viewed from the extracellular side of the membrane. d, Interactions between the pore domain and VSLD of TAX-4, viewed parallel to the membrane (left) and from the extracellular side of the membrane (right).

Extended Data Figure 8. Structural and functional annotation of TAX-4

a, Amino acid sequence alignment of TAX-4 with human CNG channel subunits and TAX-2, a CNGB subunit in C. elegans. Secondary structures and selected functionally important amino acids are annotated for TAX-4. The selectivity filter is boxed in blue, and S4 positive charges are colored in blue. Residues boxed in purple are involved in ion-pair interactions between S4 and S2-S3. Residues boxed in red in S6 and the pore helix interact with residues in or immediately adjacent to the selectivity filter. Residues boxed in green participate in intersubunit interactions between helices A′B′ and C′D′. Residues boxed in orange are engaged in interactions between helices A′B′ of one subunit and S4/S4-S5 linker/S5 of an adjacent subunit. Positions of single-amino acid missense mutations that cause retinitis pigmentosa are highlighted in red in hCNGA1 and orange in hCNGB1, and those cause achromatopsia are highlighted in cyan in hCNGA3 and green in hCNGB3. The disease-causing mutations are listed and color-coded on both sides of the sequences. TAX-2 does not form functional homomeric channels but associates with TAX-4 to form functional heteromeric channels, both in heterologous expression systems and native cells. b, c, Mapping the disease-causing mutations listed in a on the TAX-4 protomer structure, shown in the same orientation here as in Fig. 1c.

Extended Data Figure 9. The TAX-4 channel selectivity filter

a, Electrostatic surface representation of TAX-4, viewed from the extracellular side of the membrane, showing a highly electronegative external entrance to the selectivity filter in the center. b, Comparison of the selectivity filter of TAX-4 and the selected Ca²⁺-conducting channels. For clarity, only two diagonally opposed subunits are shown. Ca_v1.1(a) and Ca_v1.1(b) represent repeats I/III and II/IV of Ca_v1.1, respectively. Notice the reoccurring utilization of a combination of negative side chains and backbone carbonyls to line the selectivity filter. The PDB codes of the comparison channels are: Ca_v1.1, 3JBR; RyR1, 3J8H; TRPV1, 3J5P; TRPA1, 3J9P. c, Superposition of the selectivity filter of TAX-4 and NaK2CNG-E (PDB code: 3K0G). Only two diagonally opposed subunits are shown. Purple spheres mark presumed ion binding sites in TAX-4.

Extended Data Figure 10. The TAX-4 channel gating ring

a, The gating ring, formed by helices A′B′C′D′ of the C-linker, is depicted in cylinder form and shown in the TAX-4 structure, viewed parallel to the membrane. b, The gating ring shown in isolation, viewed from the extracellular side of the membrane. c, A composite figure demonstrating that helix D′ of the gating ring must change its conformation in the unliganded state. The figure was generated in two steps: (1) The liganded structure of the TAX-4 C-linker/CNBD and the unliganded structure of the HCN2 C-linker/CNBD (PDB code: 2MPF) are aligned by the β strands of the CNBD, and only the indicated α helices are shown here for comparison. (2) Helices A, B, C and E′ of TAX-4 are aligned with those of HCN2, producing a hypothetical unliganded TAX-4 structure. The resulting helix D′, however, clashes with helix A, as shown in e. d, Space-filling model of the interface between helices A and D′ in the liganded TAX-4 structure, showing a snugly fit between these helices. e, Space-filling model of the interface between helices A and D′ in the hypothetical unliganded TAX-4 structure generated in c, showing clash between these helices, indicating that helix D′ must adopt a different conformation.

Figure 1. Architecture of TAX-4

a, Cryo-EM density map of TAX-4. Dashed lines mark the membrane boundaries. b, TAX-4 structure, viewed parallel to the membrane (left) and from the extracellular side (right). c, Structure of a TAX-4 protomer, viewed parallel to the membrane. Different regions (S1-S6, pore-loop, C-linker and CNBD) are illustrated in different colors. d, Intersubunit interface in the TAX-4 TMD. e, Superposition of the TMDs of TAX-4 and the K_v1.2–2.1 chimera (PDB code: 2R9R), aligned by S5 and S6.

Figure 2. An unusual voltage-sensor-like domain

a, Amino acid sequence alignment of S4 and neighboring regions of the indicated channel subunits. Positions of positive charges in TAX-4 and K_v1.2–2.1 chimera are marked. b, c, Comparison of S4 of TAX-4 and the K_v1.2–2.1 chimera, viewed parallel to the membrane and after a 25-degree clockwise rotation from Fig. 1e. Positions R0-R6 in c correspond to R287, Q290, R293, R296, R299, K302 and R305, respectively. d, Ion-pair interactions involving the indicated TAX-4 S4 positive charges.

Figure 3. The ion conduction pathway

a, The solvent-accessible pathway generated with the HOLE program, shown in the center. b, Pore-size profile generated with the HOLE program of different sections of the solvent-accessible pathway. The origin of the pore axis is set at the cytoplasmic end of S6. c, Close-up view of the selectivity filter. Only two diagonally opposed subunits are shown. The cryo-EM density map and the modeled amino acids are shown in mesh and sticks, respectively. Distances between the atoms are measured as the center-to-center distance of two diagonally opposed atoms. Purple spheres mark presumed ion binding sites. d, Protein packing around the selectivity filter. Amino acids in and immediately adjacent to the selectivity filter are colored in red.

Figure 4. The cyclic nucleotide binding domain

a, Structure of the cGMP-bound CNBD of TAX-4. b, Amino acids and interactions involved in the binding of cGMP, shown in a ball-and-stick model. c, Comparison of the structures of the cGMP-bound CNBD of TAX-4 and HCN2 (PDB code: 1Q3E), aligned by the β strands.

Figure 5. The C-linker

a, Superposition of the structures of the cGMP-bound C-linker/CNBD regions of TAX-4 and HCN2 (PDB code: 1Q3E), aligned by the β strands. b, Interactions between the C-linkers of two adjacent subunits. c, Interactions between the C-linker of one subunit and S4/S4-S5 linker/S5 of an adjacent subunit. For clarity, an ion-pair interaction between R300 in the S4-S5 linker and D119 in S1 is not illustrated. d, Comparison of the liganded structure of the TAX-4 C-linker/CNBD and the unliganded structure of the HCN2 C-linker/CNBD (PDB code: 2MPF). The structures are aligned by the β strands. Each arrow marks the positions of the same atom in the two structures.

Figure 6. Model for cyclic nucleotide gating

a, Schematic of the TAX-4 channel in the cGMP-bound and unbound states. In the liganded state (I), helices A′B′C′D′ of the C-linker form a compact, firmly bound gating ring, which is highlighted in color. A′B′ (green) and C′D′ (blue) of two neighboring subunits associate tightly, and A′B′ interact strongly with S4/S4-S5 linker/S5. These interactions, schematized in red, cause S6 to splay wide and the selectivity filter gate to open. In the unliganded state, the gating ring loosens or dissociates as the interactions between A′B′ and C′D′ and between A′B′ and S4/S4-S5 linker/S5 are either greatly weakened (II) or virtually abolished (III), causing S6 to constrict and the selectivity filter gate to close. For clarity, only two subunits are shown, but in (I) and (III), helices A′B′C′D′ of all four subunits are included to depict the gating ring. b, Whole-cell currents recorded at ‐100 mV from HEK 293T cells expressing WT TAX-4 and the indicated mutant channels, without or with 100 μM cGMP in the recording pipette. Number of measurements is indicated for each condition. Error bars represent S.E.M.