Romanazzi T et al. (2021), Bile Acids Gate Dopamine Transporter Mediated C...

XB-ART-58788

Front Chem 2021 Dec 10;9:753990. doi: 10.3389/fchem.2021.753990.

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Bile Acids Gate Dopamine Transporter Mediated Currents.

Romanazzi T, Zanella D, Cheng MH, Smith B, Carter AM, Galli A, Bahar I, Bossi E.

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Bile acids (BAs) are molecules derived from cholesterol that are involved in dietary fat absorption. New evidence supports an additional role for BAs as regulators of brain function. Sterols such as cholesterol interact with monoamine transporters, including the dopamine (DA) transporter (DAT) which plays a key role in DA neurotransmission and reward. This study explores the interactions of the BA, obeticholic acid (OCA), with DAT and characterizes the regulation of DAT activity via both electrophysiology and molecular modeling. We expressed murine DAT (mDAT) in Xenopus laevis oocytes and confirmed its functionality. Next, we showed that OCA promotes a DAT-mediated inward current that is Na+-dependent and not regulated by intracellular calcium. The current induced by OCA was transient in nature, returning to baseline in the continued presence of the BA. OCA also transiently blocked the DAT-mediated Li+-leak current, a feature that parallels DA action and indicates direct binding to the transporter in the absence of Na+. Interestingly, OCA did not alter DA affinity nor the ability of DA to promote a DAT-mediated inward current, suggesting that the interaction of OCA with the transporter is non-competitive, regarding DA. Docking simulations performed for investigating the molecular mechanism of OCA action on DAT activity revealed two potential binding sites. First, in the absence of DA, OCA binds DAT through interactions with D421, a residue normally involved in coordinating the binding of the Na+ ion to the Na2 binding site (Borre et al., J. Biol. Chem., 2014, 289, 25764-25773; Cheng and Bahar, Structure, 2015, 23, 2171-2181). Furthermore, we uncover a separate binding site for OCA on DAT, of equal potential functional impact, that is coordinated by the DAT residues R445 and D436. Binding to that site may stabilize the inward-facing (IF) open state by preventing the re-formation of the IF-gating salt bridges, R60-D436 and R445-E428, that are required for DA transport. This study suggests that BAs may represent novel pharmacological tools to regulate DAT function, and possibly, associated behaviors.

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Species referenced: Xenopus laevis
Genes referenced: slc6a3

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	FIGURE 1. OCA generates an electrical current through the DAT. (A) Schematic representation of oocyte collection, cRNA synthesis and injection, and TEVC technique. (B) Representative traces of currents recorded by TEVC (Vh = −60 mV) from oocytes expressing mDAT, without or with hTGR5, uninjected, or expressing hTGR5 alone. The oocytes were perfused with 30 µM DA or 10 µM OCA. (C) Mean of the maximal DA-associated and OCA-induced currents (I nA ± SE of 14–47 oocytes, 6–11 batches) in oocytes expressing mDAT (C, left) or mDAT plus TGR5 (C, right). (D) Mean of the maximal OCA-induced currents in oocytes expressing the proteins indicated; one-way ANOVA F(2,74) = 36.13, p < 0.0001 followed by Tukey’s multiple comparison test (mDAT vs mDAT-hTGR5 p = 0.566, mDAT vs hTGR5 p < 0.0001). (E) Representative traces of currents recorded by TEVC (Vh = −60 mV) from oocytes tested with DA (30 µM) and OCA at the indicated concentrations. The current of each trace was normalized to the mean current of dopamine to reduce the variability between batches. (F) I/V relationships of DA (30 µM) and OCA (10 µM)-induced currents.
	FIGURE 2. OCA modulates the mDAT-mediated leak current. (A) Representative current traces recorded with TEVC (Vh = −60 mV) in oocytes expressing mDAT perfused with 30 µM DA or 10 µM OCA in ND98 or Li98 buffer. (B) Mean of maximal currents under conditions shown in A (I nA ± SE of 6–28 oocytes, 4 batches. One-way ANOVA F(4,94) = 42.29, p < 0.0001 followed by Bonferroni’s multiple comparison test (DA vs OCA p > 0.999, DA vs DA in Li+ p > 0.999, OCA vs OCA in Li+ p = 0.0004, DA in Li+ vs OCA in Li+ p < 0.0001). (C) Representative trace from oocytes expressing mDAT perfused with 30 µM DA or 10 µM OCA in ND98 or TMA98 buffer. (D) Mean of maximal currents under conditions shown in C (I nA ± SE of 6–15 oocytes, 3 batches. One-way ANOVA F(4,51) = 86.94, p < 0.0001 followed by Bonferroni’s multiple comparison test (DA vs OCA p = 0.359, DA vs DA in TMA+ p < 0.0001, OCA vs OCA in TMA+ p = 0.059, DA in TMA+ vs OCA in TMA+ p > 0.999).
	FIGURE 3. Intracellular calcium does not regulate OCA-induced currents. (A) Schematic representation of the EGTA injection technique. (B) Representative trace of current recorded by TEVC (Vh = −60 mV) in oocytes expressing mDAT and injected with 13 mM EGTA in intracellular solution 30 min before exposure to 30 µM DA or 10 µM OCA in ND98 buffer (top) and the mean of maximal transport-associated and OCA-induced currents (bottom) (I nA ± SE of 6–7 oocytes, 2 batches). (C) Mean of maximal OCA-induced currents in mDAT oocytes with or without injection of EGTA. Two-tailed Student’s t-test, p = 0.476.
	FIGURE 4. OCA does not alter the DA transport associated current. (A) Representative traces of the current recorded at increasing concentrations of DA in the absence or presence of 10 µM OCA. (B) Mean currents recorded from the traces in (A). Two-way ANOVA, Treatment F(1,142) = 0.51, p = 0.476 – DA concentration F(4,142) = 77.76, p < 0.0001 – Interaction F(4,142) = 0.24, p = 0.915. (C) Data from (A) were fitted to a Hill equation. (D) Imax, K0.5, and Hill coefficient obtained from fitting the data represented in (B) to the Hill equation.
	FIGURE 5. LCA induces a current that parallels the OCA-induced current. (A) Structure of OCA and LCA (B) Representative trace of current recorded with TEVC (Vh = −60 mV) in oocytes expressing mDAT and perfused with 30 µM DA or 10 µM LCA in ND98 buffer (top). Mean of maximal dopamine transport-associated and LCA-induced currents (I nA ± SE of 8 oocytes, 3 batches) (bottom). (C) Mean of maximal currents elicited by OCA or LCA in mDAT-expressing oocytes (n = 8; p = 0.22 by Student’s t-test).
	FIGURE 6. OCA binding to DAT depends on its protonation state and on DAT conformation. (A) Binding of negatively charged (left) and neutral (right) OCA (cyan) to hDAT in the OFo (top two panels) and IFo (middle and bottom panels) states. The binding energies to OFo hDAT are −6.1 kcal/mol (OCA(−)) and −9.9 kcal/mol (OCA(n)); and those to the IFo hDAT are −7.5 kcal/mol (OCA(−)) and −8.5 kcal/mol (OCA(n)). Interacting residues making atom-atom contacts closer than 4 Å with OCA are shown in panels (A–C). (B) Comparison with the known DA-binding pose. Alignment of the top binding poses of OCA(n) (green sticks), OCA(−) (orange sticks) (left panel) and the resolved DA (van der Waals (vdW) format) bound dDAT (PDB: 4XP1) (middle panel), and detailed view of DA-binding site and comparison of the non-overlapping spaces occupied by OCA (multiple binding poses) and DA (right panel). (C) Top 1 binding pose of OCA(n) (vdW format; −10.2 kcal/mol) to hDAT occluded (OCC) conformer (left panel), the resolved ibogaine-bound to hSERT in the OCC state (PDB:6DZV) (middle panel) and alignment, onto hDAT, of top 10 binding poses of OCA(n) (sticks in different colors; average = −9.1 ± 1.0 kcal/mol) and ibogaine bound to SERT (transparent vDW) (right panel). OCA, DA, and ibogaine are shown in vdW format in (A–C), with cyan, red, blue and white spheres representing the carbon, oxygen, nitrogen and hydrogen atoms, respectively. (D) Molecular structures of OCA in two protonation states.

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Adkins, Membrane mobility and microdomain association of the dopamine transporter studied with fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. 2007, Pubmed