Allosteric Inhibition of Serotonin 5-HT Receptors by Zinc Ions.

The allosteric regulation of G protein-coupled receptors (GPCRs) is a well-known phenomenon, but there are only a few examples of allosteric modulation within the metabotropic serotonergic receptor family. Recently, we described zinc non-competitive interactions toward agonist binding at serotonin 5-HT receptors, in which biphasic effects, involving potentiation at sub-micromolar concentrations (10 μM) and inhibition at sub-millimolar concentrations (500 μM) of Zn in radioligand binding assays, were consistent with both the agonist and antagonist-like effects of zinc ions observed in in vivo studies. Here, we showed new data demonstrating zinc allosteric inhibition of both agonist and antagonist binding at human recombinant 5-HT receptors stably expressed in HEK293 cells as observed by radioligand binding studies as well as zinc neutral antagonism displayed by the concentration of 10 μM in the functional LANCE assay. The allosteric nature of the effect of Zn on 5-HT receptors was confirmed (1) in saturation studies in which zinc inhibited the binding of potent orthosteric 5-HT receptor radioligands, the agonist [H]5-CT, and the two antagonists [H]SB-269970 and [H]mesulergine, showing ceiling effect and differences in the magnitude of negative cooperativity (α = 0.15, 0.06, and 0.25, respectively); (2) in competition experiments in which 500 μM of zinc inhibited all radioligand displacements by non-labeled orthosteric ligands (5-CT, SB-269970, and clozapine), and the most significant reduction in affinity was observed for the 5-CT agonist (4.9-16.7-fold) compared with both antagonists (1.4-3.9-fold); and (3) in kinetic experiments in which 500 μM zinc increased the dissociation rate constants for [H]5-CT and [H]mesulergine but not for [H]SB-269970. Additionally, in the functional LANCE test using the constitutively active HEK293 cell line expressing the 5-HT receptor, 10 μM zinc had features of neutral antagonism and increased the EC value of the 5-CT agonist by a factor of 3.2. Overall, these results showed that zinc can act as a negative allosteric inhibitor of 5-HT receptors. Given that the inhibiting effects of low concentrations of zinc in the functional assay represent the most likely direction of zinc activity under physiological conditions, among numerous zinc-regulated proteins, the 5-HT receptor can be considered a serotonergic target for zinc modulation in the CNS.