Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2018 Apr;55(4):2897-2910.
doi: 10.1007/s12035-017-0536-0. Epub 2017 Apr 28.

Allosteric Inhibition of Serotonin 5-HT 7 Receptors by Zinc Ions

Affiliations
Free PMC article

Allosteric Inhibition of Serotonin 5-HT 7 Receptors by Zinc Ions

Grzegorz Satała et al. Mol Neurobiol. .
Free PMC article

Abstract

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-HT1A receptors, in which biphasic effects, involving potentiation at sub-micromolar concentrations (10 μM) and inhibition at sub-millimolar concentrations (500 μM) of Zn2+ 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-HT7 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-HT7 receptors was confirmed (1) in saturation studies in which zinc inhibited the binding of potent orthosteric 5-HT7 receptor radioligands, the agonist [3H]5-CT, and the two antagonists [3H]SB-269970 and [3H]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 [3H]5-CT and [3H]mesulergine but not for [3H]SB-269970. Additionally, in the functional LANCE test using the constitutively active HEK293 cell line expressing the 5-HT7 receptor, 10 μM zinc had features of neutral antagonism and increased the EC50 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-HT7 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-HT7 receptor can be considered a serotonergic target for zinc modulation in the CNS.

Keywords: Allosteric modulation; Radioligand binding; Serotonin 5-HT7 receptor; Zn.

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Effects of monovalent and divalent cations (Na+, K+, Mg2+, Ca2+, and Zn2+) at concentrations of 500 μM and 5 mM on the binding of [3H]5-CT, [3H]SB-269970, and [3H]mesulergine to human recombinant 5-HT7 receptors expressed in HEK293 cells. The binding of radioligands in the absence of the tested cations is referred to as basal specific binding and was set to 100% (dotted line)
Fig. 2
Fig. 2
Effect of increasing concentrations of zinc ions on the binding of radioligands: a [3H]5-CT, b [3H]SB-269970, and c [3H]mesulergine to human recombinant 5-HT7 receptors expressed in HEK293 cells. The graphs in the right panel (which are diagnostic for negative allosteric modulation) show the non-linear regression analysis of the respective saturation data (left panel) according to Eq. (1)
Fig. 3
Fig. 3
Effect of zinc (500 μM) on the dissociation rates of a [3H] 5-CT, b [3H]SB-269970, and c [3H]mesulergine from 5-HT7 receptors expressed in HEK293 cells. In all cases, the dissociation was induced by the addition of a receptor-saturating concentration of serotonin (10 μM)
Fig. 4
Fig. 4
The cytotoxicity of zinc toward HEK293 cells was assessed in terms of a viability using the MTT assay and b mortality based on the release of lactate dehydrogenase (LDH). Cells were treated with different concentrations of zinc ranging from 10 to 500 μM. The results are presented as the means ± SD (n = 3), p < 0.05
Fig. 5
Fig. 5
Influence of zinc salts (chloride or hydroaspartate form) and sodium chloride on PerkinElmer LANCE Ultra working solution Eu-cAMP tracer and ULight-anti-cAMP
Fig. 6
Fig. 6
Effects of SB-269970 and mesulergine on unstimulated cAMP level in the 5-HT7 receptor expressed in HEK293 cells in the absence and presence of 10 μM of zinc ions, and zinc effects on spontaneous 5-HT7 receptor activity
Fig. 7
Fig. 7
Concentration-response curves of a agonist 5-CT, b antagonist SB-269970, and c antagonist mesulergine, determined in the absence (filled circle) and presence of 10 μM (grey square) of zinc ions in recombinant 5-HT7 receptor-expressing HEK293 cells

Similar articles

See all similar articles

Cited by 1 article

References

    1. May LT, Leach K, Sexton PM, Christopoulos A. Allosteric modulation of G protein–coupled receptors. Annu Rev Pharmacol Toxicol. 2007;47:1–51. doi: 10.1146/annurev.pharmtox.47.120505.105159. - DOI - PubMed
    1. Gentry PR, Sexton PM, Christopoulos A. Novel allosteric modulators of G protein-coupled receptors. J Biol Chem. 2015;290:19478–19488. doi: 10.1074/jbc.R115.662759. - DOI - PMC - PubMed
    1. Dohlman HG. Thematic minireview series: new directions in G protein-coupled receptor pharmacology. J Biol Chem. 2015;290:19469–19470. doi: 10.1074/jbc.R115.675728. - DOI - PMC - PubMed
    1. Christopoulos A, Kenakin T. G protein-coupled receptor allosterism and complexing. Pharmacol Rev. 2002;54:323–374. doi: 10.1124/pr.54.2.323. - DOI - PubMed
    1. van der Westhuizen ET, Valant C, Sexton PM, Christopoulos A. Endogenous allosteric modulators of G protein-coupled receptors. J Pharmacol Exp Ther. 2015;353:246–260. doi: 10.1124/jpet.114.221606. - DOI - PubMed

LinkOut - more resources

Feedback