Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1

Abstract

Mammalian sodium-coupled high-affinity choline transporter CHT1 uptakes choline in cholinergic neurons for acetylcholine synthesis and plays a critical role in cholinergic neurotransmission. Here, we present the high-resolution cryo-EM structures of human CHT1 in apo, substrate- and ion-bound, hemicholinium-3-inhibited, and ML352-inhibited states. These structures represent three distinct conformational states, elucidating the structural basis of the CHT1-mediated choline uptake mechanism. Three ion-binding sites, two for Na⁺ and one for Cl^-, are unambiguously defined in the structures, demonstrating that both ions are indispensable cofactors for high-affinity choline-binding and are likely transported together with the substrate in a 2:1:1 stoichiometry. The two inhibitor-bound CHT1 structures reveal two distinct inhibitory mechanisms and provide a potential structural platform for designing therapeutic drugs to manipulate cholinergic neuron activity. Combined with the functional analysis, this study provides a comprehensive view of the structural mechanisms underlying substrate specificity, substrate/ion co-transport, and drug inhibition of a physiologically important symporter.

Fig. 1. Radioactive choline uptake assay of human CHT1 expressed in HEK293 cells.

a Radioactive choline uptake of the CHT1-expressing HEK293 cells (wild-type, blue bar) as compared to the background radioactivity from the control cells transfected with the empty vector (EV, gray bar) 10 min after adding 0.1 mM choline with 10% of [³H]-choline. The inhibition of CHT1-mediated choline uptake was measured at 0.1 mM HC3 or 1 mM ML352. Data are mean ± SEM (n = 3 independent experiments). One-way ANOVA; ****P ≤ 0.0001. b Concentration-dependent choline uptake. Data points are mean ± SEM (n = 3 independent experiments) and fitted to the Michaelis–Menten equation with K_M = 3.86 ± 0.68 mM. c Na⁺ and Cl^–-dependent choline uptake. Data are mean ± SEM (n = 4 independent experiments) and are normalized against the radioactivity measurement with NaCl in the reaction solution. One-way ANOVA; ****P ≤ 0.0001. d, e Concentration-dependent inhibition of CHT1-mediated choline uptake by HC3 (d) and ML352 (e). Data points are mean ± SEM (n = 3 for HC3 and n = 3–6 for ML352) and fitted to the three-parameter dose–response curves (GraphPad Prism 9) with IC₅₀ of 4.98 ± 1.04 nM for HC3 and 168.6 ± 49.4 nM for ML352.

Fig. 2. The overall structure of the apo human CHT1 in inward-open conformation.

a Topology diagram of human CHT1. Segments of TM0, TMs 1–5, TMs 6–10, and TMs 11–12 are individually colored. b Side view of 3D reconstruction (left) and cartoon representation of the CHT1^apo structure with the four segments individually colored as the topology diagram in a. c Bottom view of the apo CHT1 from the intracellular side. The red dashed oval marks the entrance of the intracellular vestibule. d Side view of the cross-section of the surface-rendered CHT1^apo illustrates its inward-open conformation. The dotted circles and oval mark the locations of ions and substrate-binding sites.

Fig. 3. The substrate-bound CHT1^Chol structure.

a Bottom view of the CHT1^Chol structure with the bound ions and choline highlighted in red dashed boxes. b Zoomed-in view of choline-binding in CHT1. The density (blue mesh) for choline is contoured at 6 σ. Key choline-interacting residues are shown in sticks. The bound Cl^– and Na⁺ are also shown for reference. c The effect of mutagenesis at the substrate-binding site on choline uptake. Data are mean ± SEM (n = 4–6 independent experiments) and are normalized against the measurement from the wild-type CHT1. One-way ANOVA; ****P ≤ 0.0001. d Zoomed-in view of Cl^– binding in CHT1. The density (blue mesh) for the Cl^– ion is contoured at 4 σ. Key Cl^–-interacting residues are shown in sticks. The dotted lines mark the coordination between the Cl^– ion and the protein atoms. The inset provides an alternative view of the 63VGGGY67 region for enhanced clarity. e Zoomed-in view of Na⁺ binding at Na2 in CHT1. The density (blue mesh) for the Na⁺ ion is contoured at 4 σ. Key Na⁺ -interacting residues are shown in sticks. The dotted lines mark the coordination between the Na⁺ ion and the protein atoms. The surrounding residues for the Na3 site (red dotted circle) are also shown. f The effect of mutagenesis at Na2 and Na3 sites on choline uptake. Data are mean ± SEM (n = 3–8 independent experiments) and are normalized against the measurement from the wild-type CHT1. One-way ANOVA; ****P ≤ 0.0001.

Fig. 4. Conformational changes between the inward-open CHT1^apo and the substrate-bound CHT1^Chol.

a Structural comparison between CHT1^apo and CHT1^Chol viewed from the intracellular side. Only TM4 and TM8 are highlighted in color for clarity. Arrows mark the directions of the TM movements. The boxed insets provide zoomed-in views of the different sidechain positions of some Cl^– and substrate-interacting residues in these two structures. The orange arrow marks the flip of the W141 indole ring upon Cl^–/choline-binding. b Side view of the cross-section of the surface-rendered CHT1^Chol illustrates its inward-facing partially open conformation. The bound Na⁺ (at Na2), Cl^–, and choline are occluded from the intracellular solution. The dashed circle marks the solvent-exposed Na3 site.

Fig. 5. The structure of HC3-inhibited CHT1 in outward-open conformation.

a Side view of the cross-section of the surface-rendered CHT1^HC with the bound HC3 (yellow sticks). The dotted circles and oval mark the locations of the ions and substrate-binding sites. b Zoomed-in view of the protein–inhibitor interactions and cryo-EM density of HC3 in the contour level of 1.5 in ChimeraX. c Structural comparison between CHT1^Chol and CHT1^HC in side view. TM0 is removed from both structures for clarity. The three areas where the major conformational changes occur are boxed. d Side-by-side view of the structural difference at the N-terminal region of TM10 (area in box 1) between CHT1^Chol (left) and CHT1^HC (right). e Zoomed-in view of the conformational changes at the N-terminal region of TM5 (area in box 2) between CHT1^Chol (magenta) and CHT1^HC (blue). f Zoomed-in view of the conformational changes at TM4 and TM9 (area in box 3) between CHT1^Chol (magenta) and CHT1^HC (blue). Arrows mark the upward movement of the helices from CHT1^HC to CHT1^Chol. The hollow circles mark the C_α positions of L170 and A390 in the two structures and provide references for the one-turn helical movement between the two conformations.

Fig. 6. The structure of ML352-inhibited CHT1 in inward-open conformation.

a The structure of CHT1^ML with the bound ML352 inhibitor on the external surface of the transporter. The zoomed-in view of the surface-rendered CHT1^ML illustrates the space-filling binding of ML352. Cryo-EM density of ML352 in the contour level of 0.42 in ChimeraX. b Structural comparison between CHT1^apo and CHT1^ML illustrates subtle structural change at the EL6 loop between the two. c Zoomed-in view of the protein–inhibitor interactions.

Fig. 7. A working model for the Na⁺ and Cl^–-dependent choline transport in CHT1.

The orange arrows mark the conformational changes driven by the choline-binding from the extracellular side.