Acetylcholine binding site in the vesicular acetylcholine transporter

Area of Science:

• Neuroscience
• Molecular Biology
• Biochemistry

Background:

• The vesicular acetylcholine transporter (VAChT) is essential for neurotransmission, responsible for packaging acetylcholine (ACh) into synaptic vesicles.
• Understanding the precise mechanisms of ACh binding and transport is critical for developing targeted therapeutics.

Purpose of the Study:

• To identify the specific binding site of acetylcholine (ACh) within the vesicular acetylcholine transporter (VAChT).
• To locate residues involved in the binding of the allosteric inhibitor vesamicol and in the kinetics of the transport cycle.
• To investigate the role of cation-pi interactions in ACh binding by mutating conserved aromatic residues.

Main Methods:

• Site-directed mutagenesis of conserved tryptophan, tyrosine, and phenylalanine residues in rat VAChT transmembrane domains.
• Expression and characterization of mutated VAChTs in PC12 cells.
• Determination of thermodynamic affinity for ACh and kinetic parameters for ACh transport using radioligand binding and transport assays.
• Analysis of microscopic kinetics to resolve binding and rate steps.

Main Results:

• Mutation of residue W331 significantly decreased ACh affinity, suggesting its role in cation-pi solvation of ACh.
• Other mutations affected ACh affinity and/or microscopic rate constants, indicating complex roles in binding and transport.
• Nine mutations successfully differentiated the ACh and vesamicol binding sites.
• Several mutations altered the rates of transmembrane reorientation steps, with some increasing transport rates.

Conclusions:

• The study identifies W331 as a key residue for ACh binding via cation-pi interactions in VAChT.
• Mutational analysis reveals complex roles for other residues in ACh binding, vesamicol inhibition, and transport kinetics.
• Microscopic kinetic analysis is essential for accurate interpretation of mutational effects on VAChT function.