Acetylcholine is the first neurotransmitter discovered by humans and plays an important role in the chemical transmission of nerve impulses. Acetylcholine is the main neurotransmitter synthesized and utilized by cholinergic neurons. When acetylcholine is released from nerve endings, it can bind and activate acetylcholine receptors located on the presynaptic/postsynaptic membrane, induce neuronal excitation, mediate and regulate information transmission related to cognitive and motor processes in the brain. After acetylcholine completes signal transmission in the synaptic gap, acetylcholinesterase breaks it down into acetate and choline. Free choline is further taken up by the high affinity choline transporter CHT1 located on the presynaptic membrane, recovered to the presynaptic terminals, and once again participates in the synthesis and metabolic cycle of acetylcholine. Previous studies have shown that CHT1 mediated choline recovery is a rate limiting step in acetylcholine synthesis. Therefore, abnormal expression and dysfunction of CHT1 can induce many diseases, such as hereditary motor neuron disease, myasthenia syndrome, atherosclerosis, depression and Alzheimer's disease.
CHT1 is a member of the sodium ion/solute co transport family, which utilizes the electrochemical potential of intracellular and extracellular sodium ions to drive high affinity transmembrane transport of choline. HC-3 is a small molecule drug that selectively targets CHT1, competitively inhibiting CHT1 mediated choline transport, and is used as a neuromuscular blocker. However, the key sites for CHT1 to recognize choline are not yet clear, and the structural basis for its conformational changes and the inhibitory mechanism of small molecule HC-3 on transport activity need to be elucidated.
On April 8, Zhao Yan, the research team of the Institute of Biophysics, Chinese Academy of Sciences, published a research paper on Nature Structural&Molecular Biology under the title of transport mechanism of presynaptic high affinity chord uptake by CHT1. This study utilized single particle cryo electron microscopy technology to analyze the high-resolution structure of CHT1 in the outward HC-3 binding state, inward substrate free binding state, and inward choline binding state. Using experimental methods such as radioactive isotope tracing, substrate uptake experiments, and molecular dynamics simulations, the molecular model of CHT1 recognizing and transporting choline, as well as the mechanism of HC-3 inhibiting transport activity, were elucidated, providing a structural basis for the development of novel CHT1 targeted drugs.
In the outward structure of CHT1 that combines HC-3, HC-3 exhibits a rod-shaped shape and is inserted into the substrate binding pocket from the outside, locking the protein in an outward opening state. In the state of inward choline binding, the intracellular pocket of CHT1 opens inward, and choline remains stable by the tryptophan triad. In an inward substrate free binding state, conformational changes occur in the tryptophan triad pocket, exposing choline to the intracellular solvent side, leading to the release of choline from the substrate binding pocket.
In addition, intracellular short helix IH1 is also an important element that plays a role in the transport process of CHT1. In the outward oriented CHT1 structure, the IH1 helix participates in maintaining conformational stability. When the substrate binds to CHT1, CHT1 transitions from an outward state to an inward state. At this point, the IH1 helix is released, exposing the intracellular pocket of CHT1 and promoting transport. Further experiments have shown that the IH1 helix deficient mutant completely loses substrate transport activity and is unable to transition from an inward to an outward state.
The research work was supported by the scientific and technological innovation 2030- "brain science and brain like research" major project, the national key research and development plan, the National Natural Science Foundation of China and the Chinese Academy of Sciences strategic leading science and technology project.
Paper link
CHT1 binds to different ligands and enters different conformational states, as well as hypothetical substrate transport mechanisms
