The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters

Bulling, Simon; Schicker, Klaus; Zhang, Yuan‐Wei; Steinkellner, Thomas; Stockner, Thomas; Gruber, Christian W.; Boehm, Stefan; Freissmuth, Michael; Rudnick, Gary; Sitte, Harald H.; Sandtner, Walter

doi:10.1074/jbc.m112.343681

Cited by 108 publications

(126 citation statements)

References 51 publications

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“…Such a selective inhibition is also a very rare property, and has been described, to our knowledge, for only three other transporters. One such example is the plasma membrane serotonin transporter SERT, where the noncompetitive inhibitor ibogaine stabilizes a cytoplasm-facing conformation, whereas other competitive inhibitors, such as cocaine, bind at the substrate-binding site in the cytoplasm-closed state (41,42). The other two examples are the ADP:ATP translocator protein (43) and the human erythrocyte glucose transporter (44).…”

Section: Discussionmentioning

confidence: 99%

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Yaffe

Vergara-Jaque

Forrest

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Neurotransporters located in synaptic vesicles are essential for communication between nerve cells in a process mediated by neurotransmitters. Vesicular monoamine transporter (VMAT), a member of the largest superfamily of transporters, mediates transport of monoamines to synaptic vesicles and storage organelles in a process that involves exchange of two H + per substrate. VMAT transport is inhibited by the competitive inhibitor reserpine, a second-line agent to treat hypertension, and by the noncompetitive inhibitor tetrabenazine, presently in use for symptomatic treatment of hyperkinetic disorders. During the transport cycle, VMAT is expected to occupy at least three different conformations: cytoplasm-facing, occluded, and lumen-facing. The lumen-to cytoplasm-facing transition, facilitated by protonation of at least one of the essential membrane-embedded carboxyls, generates a binding site for reserpine. Here we have identified residues in the cytoplasmic gate and show that mutations that disrupt the interactions in this gate also shift the equilibrium toward the cytoplasm-facing conformation, emulating the effect of protonation. These experiments provide significant insight into the role of proton translocation in the conformational dynamics of a mammalian H + -coupled antiporter, and also identify key aspects of the mode of action and binding of two potent inhibitors of VMAT2: reserpine binds the cytoplasm-facing conformation, and tetrabenazine binds the lumen-facing conformation.neurotransmitter transporter | ion coupling | reserpine | tetrabenazine

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Section: Discussionmentioning

confidence: 99%

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Yaffe

Vergara-Jaque

Forrest

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…3). In contrast, ibogaine, a noncompetitive SERT inhibitor that stabilizes inward-open conformations (17,26,27), dramatically increased Thr276 phosphorylation in the presence of Na + (Fig. 3).…”

Section: -Brmentioning

confidence: 97%

“…Cocaine and ibogaine compete for sites on SERT that are mutually exclusive (17,26,27). This competition is also apparent in the opposing effects of these drugs on PKG-induced SERT phosphorylation in vitro.…”

Section: -Brmentioning

confidence: 97%

“…3). 5-HT, the substrate for SERT, stimulated conversion PKG activity against the peptide substrate (RKRSRAE) was determined in the presence of the indicated concentrations of various agents as described previously (15) of the transporter to inward-open conformations (19,27,28) and also stimulated phosphorylation, although not as strongly as ibogaine (Fig. 3).…”

Section: -Brmentioning

confidence: 99%

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Control of serotonin transporter phosphorylation by conformational state

Zhang

Turk

Rudnick

2016

Proc. Natl. Acad. Sci. U.S.A.

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Serotonin transporter (SERT) is responsible for reuptake and recycling of 5-hydroxytryptamine (5-HT; serotonin) after its exocytotic release during neurotransmission. Mutations in human SERT are associated with psychiatric disorders and autism. Some of these mutations affect the regulation of SERT activity by cGMPdependent phosphorylation. Here we provide direct evidence that this phosphorylation occurs at Thr276, predicted to lie near the cytoplasmic end of transmembrane helix 5 (TM5). Using membranes from HeLa cells expressing SERT and intact rat basophilic leukemia cells, we show that agents such as Na + and cocaine that stabilize outward-open conformations of SERT decreased phosphorylation and agents that stabilize inward-open conformations (e.g., 5-HT, ibogaine) increased phosphorylation. The opposing effects of the inhibitors cocaine and ibogaine were each reversed by an excess of the other inhibitor. Inhibition of phosphorylation by Na + and stimulation by ibogaine occurred at concentrations that induced outward opening and inward opening, respectively, as measured by the accessibility of cysteine residues in the extracellular and cytoplasmic permeation pathways, respectively. The results are consistent with a mechanism of SERT regulation that is activated by the transport of 5-HT, which increases the level of inward-open SERT and may lead to unwinding of the TM5 helix to allow phosphorylation.(1) reported a rare missense mutation in the coding region of serotonin transporter (SERT) in two unrelated families. Within these families, individuals with the mutation (Ile425Val, or I425V) were more strongly affected with several psychiatric disorders, including obsessive-compulsive disorder and autism spectrum disorders. Subsequently, additional families with this mutation and others in SERT have been identified (2-8).In transfected cells, the I425V mutation led to enhanced 5-HT transport relative to WT SERT (5, 9). The enhanced transport was similar to the up-regulation of 5-HT transport that had been observed in rat basophilic leukemia (RBL-2H3) cells on stimulation of adenosine A3 receptors with subsequent NO formation and cGMP production (10). Results with heterologously expressed SERT suggest that the I425V mutation interferes with this pathway either by directly increasing basal SERT phosphorylation or by inhibiting SERT dephosphorylation (11).Mutation of serine and threonine residues on the cytoplasmic surface of SERT led to the identification of Thr276 as a potential cGMP-dependent phosphorylation site (12). From homology models based on prokaryotic and eukaryotic homologs, this residue is located near the cytoplasmic end of transmembrane helix 5 (TM5), a location that agrees with cysteine scanning accessibility studies (13) that also have shown this position to be poorly accessible to aqueous reagents. The unlikely location of a phosphorylation site in a transmembrane helix, and the inaccessibility of this position, cast some doubt on the proposal that Thr276 is the site of cGMP-stimulated phosphorylat...

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“…steady-state current without affecting the peak current (Erreger et al, 2008;Bulling et al, 2012;Schicker et al, 2012). Here, we take advantage of this predicted suppression to isolate the peak current (compare Fig.…”

Section: Binding Kinetics Of Monoamine Transporter Inhibitorsmentioning

confidence: 99%

Ligand Selectivity among the Dopamine and Serotonin Transporters Specified by the Forward Binding Reaction

et al. 2015

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The membrane transporters for the monoamines serotonin (SERT) and dopamine (DAT) are prominent targets of various psychoactive substances, including competitive inhibitors, such as tricyclic antidepressants, methylphenidate, and cocaine. Upon rapid application of a substrate, SERT and DAT display an inwardly directed current comprised of a peak and a steady-state component. Binding of a competitive inhibitor to the transporter leads to reduction of the peak current amplitude because occupancy of the transporter by an inhibitor prevents the induction of the peak current by the substrate. We show that the inhibitory effect on the peak current can be used to study the association rate constant (k on ), dissociation rate constant (k off ), and equilibrium dissociation constant (K D ) of chemically distinct SERT and DAT inhibitors, with high temporal precision and without the need of high-affinity radioligands as surrogates. We exemplify our approach by measuring the kinetics of cocaine, methylphenidate, and desipramine binding to SERT and DAT. Our analysis revealed that the selectivity of methylphenidate and desipramine for DAT and SERT, respectively, can be accounted for by their rate of association and not by the residence time in their respective binding sites.

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The Mechanistic Basis for Noncompetitive Ibogaine Inhibition of Serotonin and Dopamine Transporters

Cited by 108 publications

References 51 publications

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

Control of serotonin transporter phosphorylation by conformational state

Ligand Selectivity among the Dopamine and Serotonin Transporters Specified by the Forward Binding Reaction

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