Orthosteric and Allosteric Activation of Human 5-HTA Receptors.

The serotonin type 3 receptor (5-HT) is a ligand-gated ion channel that converts the binding of the neurotransmitter serotonin (5-HT) into a transient cation current that mediates fast excitatory responses in peripheral and central nervous systems. Information regarding the activation and modulation of the human 5-HT type A receptor has been based only on macroscopic current measurements because of its low ion conductance. By constructing a high-conductance human 5-HTA receptor, we here revealed mechanistic information regarding the orthosteric activation by 5-HT and by the partial agonist tryptamine, and the allosteric activation by the terpenoids, carvacrol, and thymol. Terpenoids potentiated macroscopic currents elicited by the orthosteric agonist and directly elicited currents with slow-rising phases and submaximal amplitudes. At the single-channel level, activation by orthosteric and allosteric agonists appeared as openings in quick succession (bursts) that showed no ligand concentration dependence. Bursts were grouped into long-duration clusters in the presence of 5-HT and even longer in the presence of terpenoids, whereas they remained isolated in the presence of tryptamine. Kinetic analysis revealed that allosteric and orthosteric activation mechanisms can be described by the same scheme that includes transitions of the agonist-bound receptor to closed intermediate states before opening (priming). Reduced priming explained the partial agonism of tryptamine; however, equilibrium constants for gating and priming were similar for 5-HT and terpenoid activation. Thus, our kinetic analysis revealed that terpenoids are efficacious agonists for 5-HTA receptors. These findings not only extend our knowledge about the human 5-HTA molecular function but also provide novel insights into the mechanisms of action of allosteric ligands, which are of increasing interest as therapeutic drugs in all the superfamily.