Bile acids (BAs) are cholesterol derivatives primarily involved in dietary fat absorption; new evidence supports a possible additional role in regulating the reward system. Bile diversion, a bariatric surgery used to treat obesity, increases circulating BAs, and in mice reduces both food and cocaine reward-related behavior. Feeding mice addicted to cocaine with Obeticholic acid (OCA), a bile acid analogue, FDA-approved drug, and an agonist of the plasma membrane Takeda G protein-coupled receptor 5 (TGR5), induced the same effects as the bariatric surgery. The interactions of OCA with murine DAT (mDAT) heterologously expressed in X. laevis oocytes investigated by two-electrode voltage clamp showed that OCA promotes a DAT-mediated transient Na+-inward current, not dependent on the presence of hTGR5 and that OCA did not affect DA affinity and DAT kinetic parameters. Docking simulations highlighted possible binding sites for OCA on DAT suggesting that OCA could stabilize DAT in an occluded conformation (Romanazzi et al., 2021). Oocytes expressing mDAT exposed to a second OCA perfusion do not elicit OCA–inward transient current and only after DA perfusion, OCA-current was visible again. The modeling suggests that the OCA stabilizes DAT in an occluded conformation, the experiments let to hypothesize that the DA binding allows the displacement of OCA, and lets DAT complete the transport cycle. When the same experiment was repeated in the presence of other DAT substrates like norepinephrine (NE) and serotonin (5HT), agonists with lower apparent affinity, OCA elicits an inward transient current, but smaller in amplitude compared to the one recorded after DA perfusion. We hypothesize that this occurs because NE or 5HT results in a weaker displacement of the OCA, thus leaving some transporters bound to OCA, in the occluded conformation. Moreover, reducing DA concentration from 30 to 3µM (proximal to the K05), the second exposure to OCA-elicited a smaller current than the first, supporting the hypothesis that some transporters stay in occluded OCA-bind conformation, lowering the amplitude of the current elicited by the second OCA exposure. These experiments agree with the model: OCA binds and freezes DAT in an occluded conformation that can be recovered by the presence of the substrates. The lithocholic acid (LCA) a natural bile acid behaves similarly to OCA (Romanazzi et al., 2021). Exposing mDAT to LCA or OCA in sequence point out that the perfusion order changes the responses to the BA. OCA induces a current after LCA perfusion, but LCA does not after the application of OCA. These data suggest a stronger interaction of OCA with mDAT. The different apparent affinity could be caused by the R groups at positions 5 and 6 of the steroid-base ring, common to both BA. Modeling the interactions of R- groups with DAT residues may suggest possible determinants involved in the mechanism of action. These are preliminary results; the ongoing experiment will require to determining the apparent affinity and IC50 of BA in the presence of different substrates and in the presence of cocaine and amphetamine. Future work will be focused on the OCA binding site, studying DAT mutants suggested by docking experiments. Testing the BAs on other SLC6 transporters will shed a light on the specificity of the interaction.

Investigation of the interaction of bile acids with the dopamine transporter DAT

Tiziana Romanazzi;Angela Di Iacovo;Elena Bossi
2022-01-01

Abstract

Bile acids (BAs) are cholesterol derivatives primarily involved in dietary fat absorption; new evidence supports a possible additional role in regulating the reward system. Bile diversion, a bariatric surgery used to treat obesity, increases circulating BAs, and in mice reduces both food and cocaine reward-related behavior. Feeding mice addicted to cocaine with Obeticholic acid (OCA), a bile acid analogue, FDA-approved drug, and an agonist of the plasma membrane Takeda G protein-coupled receptor 5 (TGR5), induced the same effects as the bariatric surgery. The interactions of OCA with murine DAT (mDAT) heterologously expressed in X. laevis oocytes investigated by two-electrode voltage clamp showed that OCA promotes a DAT-mediated transient Na+-inward current, not dependent on the presence of hTGR5 and that OCA did not affect DA affinity and DAT kinetic parameters. Docking simulations highlighted possible binding sites for OCA on DAT suggesting that OCA could stabilize DAT in an occluded conformation (Romanazzi et al., 2021). Oocytes expressing mDAT exposed to a second OCA perfusion do not elicit OCA–inward transient current and only after DA perfusion, OCA-current was visible again. The modeling suggests that the OCA stabilizes DAT in an occluded conformation, the experiments let to hypothesize that the DA binding allows the displacement of OCA, and lets DAT complete the transport cycle. When the same experiment was repeated in the presence of other DAT substrates like norepinephrine (NE) and serotonin (5HT), agonists with lower apparent affinity, OCA elicits an inward transient current, but smaller in amplitude compared to the one recorded after DA perfusion. We hypothesize that this occurs because NE or 5HT results in a weaker displacement of the OCA, thus leaving some transporters bound to OCA, in the occluded conformation. Moreover, reducing DA concentration from 30 to 3µM (proximal to the K05), the second exposure to OCA-elicited a smaller current than the first, supporting the hypothesis that some transporters stay in occluded OCA-bind conformation, lowering the amplitude of the current elicited by the second OCA exposure. These experiments agree with the model: OCA binds and freezes DAT in an occluded conformation that can be recovered by the presence of the substrates. The lithocholic acid (LCA) a natural bile acid behaves similarly to OCA (Romanazzi et al., 2021). Exposing mDAT to LCA or OCA in sequence point out that the perfusion order changes the responses to the BA. OCA induces a current after LCA perfusion, but LCA does not after the application of OCA. These data suggest a stronger interaction of OCA with mDAT. The different apparent affinity could be caused by the R groups at positions 5 and 6 of the steroid-base ring, common to both BA. Modeling the interactions of R- groups with DAT residues may suggest possible determinants involved in the mechanism of action. These are preliminary results; the ongoing experiment will require to determining the apparent affinity and IC50 of BA in the presence of different substrates and in the presence of cocaine and amphetamine. Future work will be focused on the OCA binding site, studying DAT mutants suggested by docking experiments. Testing the BAs on other SLC6 transporters will shed a light on the specificity of the interaction.
2022
Romanazzi, Tiziana; Zanella, Daniele; DI IACOVO, Angela; Galli, Aurelio; Bossi, Elena
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11383/2146192
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