Transient low-affinity agonist binding to Torpedo postsynaptic membranes resolved by using sequential mixing...

Area of Science:

• Biochemistry
• Molecular Biology
• Neuroscience

Background:

• Nicotinic acetylcholine receptors (nAChRs) are crucial for neurotransmission.
• Understanding nAChR ligand binding is key to elucidating receptor function and dysfunction.
• Agonist binding induces conformational changes and affinity state transitions in nAChRs.

Purpose of the Study:

• To characterize the binding kinetics of a fluorescent agonist, Dns-C6-Cho, to low- and high-affinity states of nAChRs.
• To investigate agonist-induced affinity state conversion in nAChRs.
• To correlate agonist dissociation rates with receptor desensitization states.

Main Methods:

• Sequential mixing stopped-flow fluorescence spectroscopy was employed.
• Receptor membranes were preincubated with Dns-C6-Cho followed by chemical dilution.
• Fluorescence decay kinetics were analyzed to determine dissociation rates and binding affinities.

Main Results:

• Distinct fast and slow fluorescence decays indicated dissociation from low- (140 s⁻¹) and high-affinity (0.1 s⁻¹) nAChR states.
• Agonist concentration and preincubation time modulated the interconversion between low- and high-affinity states.
• The dissociation rate of the fast desensitized state was found to be similar to the slow desensitized state.
• Low-affinity binding dissociation constant (KD) was determined to be approximately 1.1-1.4 µM.
• Fluorescence enhancement suggested conformational isomerization in Dns-C6-Cho-inhibited nAChRs.

Conclusions:

• Dns-C6-Cho binds to both low- and high-affinity states of nAChRs, supporting affinity state conversion models.
• Agonist-induced high-affinity state formation correlates with ion flux desensitization.
• The study provides quantitative kinetic data on nAChR-ligand interactions and conformational changes.