Molecular mechanism of the interaction between kaempferol and human nicotinic acetylcholine receptors.

Kaempferol has been investigated for its potential neuroprotective properties and its ability to modulate mood-regulating pathways through the regulation of neurotransmitter systems. Evidence suggests that it may inhibit the reuptake of selective neurotransmitters, thereby enhancing their synaptic availability. Here, using two-electrode voltage-clamp recordings in Xenopus oocytes expressing five human nicotinic acetylcholine receptor (nAChR) subtypes, we found that kaempferol selectively and potently inhibits α3β4 nAChRs, with weaker inhibition of α3β2 receptors and no effect on α7, α4β2, or α4β4 subtypes. This inhibition was reversible, voltage-independent, and non-competitive with acetylcholine, indicating an allosteric mechanism of action rather than direct competition at the orthosteric binding site. To identify the structural basis of this subtype selectivity, molecular docking of kaempferol onto the α3β4 nAChR structure was performed, and candidate binding residues at the α3-β4 interface were prioritized based on binding energy for site-directed mutagenesis. The results showed that the inhibitory effect of kaempferol was abolished in the α3 W55A and β4 R151A double mutant. These findings confirm that kaempferol selectively and effectively inhibits the α3β4 nAChR subtype via an allosteric site formed by residues W55 (α3) and R151 (β4), supporting the hypothesis that it interacts with a specific region of this receptor distinct from the acetylcholine-binding site.