Ibogaine, a Noncompetitive Inhibitor of Serotonin Transport, Acts by Stabilizing the Cytoplasm-facing State of the Transporter

Abstract

Ibogaine, a hallucinogenic alkaloid with purported anti-addiction properties, inhibited serotonin transporter (SERT) noncompetitively by decreasing V max with little change in the K m for serotonin (5-HT). Ibogaine also inhibited binding to SERT of the cocaine analog 2␤-2-carbomethoxy-3-(4-[ 125 I]iodophenyl)tropane. However, inhibition of binding was competitive, increasing the apparent K D without much change in B max . Ibogaine increased the reactivity of cysteine residues positioned in the proposed cytoplasmic permeation pathway of SERT but not at nearby positions out of that pathway. In contrast, cysteines placed at positions in the extracellular permeation pathway reacted at slower rates in the presence of ibogaine. These results are consistent with the proposal that ibogaine binds to and stabilizes the state of SERT from which 5-HT dissociates to the cytoplasm, in contrast with cocaine, which stabilizes the state that binds extracellular 5-HT.Ibogaine is a hallucinogenic alkaloid found in the roots of the West African shrub Tabernanthe iboga. In Europe and North America, ibogaine has been promoted as a treatment for addiction, although clinical evidence has been difficult to obtain, partly due to its listing as a Schedule I controlled substance (1, 2) and partly due to concerns about toxicity (3). Ibogaine has affinity for 2 receptors, N-methyl-D-aspartate receptors, -opioid receptors, and serotonin and dopamine transporters (4 -7). Ibogaine is demethylated to 12-hydroxyibogamine (noribogaine), which has been reported to persist in the blood for over 24 h and to have even higher affinity for 5-HT transporters than ibogaine (8).The 5-hydroxytryptamine (5-HT) 3 transporter (SERT) is responsible for reuptake of 5-HT released from serotonergic neurons (9). This protein is the target for antidepressant drugs, such as imipramine and fluoxetine, and psychostimulants, such as cocaine and 3,4-methylenedioxymethamphetamine. SERT couples the entry of 5-HT into the cell to the entry of Na ϩ and Cl Ϫ and the exit of K ϩ (10). The transport of 5-HT has been envisaged as a two-step process, where 5-HT, Na ϩ , and Cl Ϫ are transported in a single step and K ϩ is transported in a second step (11). These transport steps are proposed to interconvert two states of the transporter: an extracellular state that binds extracellular substrates and a cytoplasmic state that releases those substrates to the cytoplasm (9).SERT belongs to a large family of Na ϩ -dependent transporters in prokaryotes and animals designated the SLC6 or NSS family. The high resolution x-ray structure of the prokaryotic leucine transporter LeuT (12) appears to be a good model for other members of the family, and homology models for SERT and other neurotransmitter transporters have been generated using structure-based alignments (13). The utility of LeuT as a model for SERT has recently been validated by the identification of the Cl Ϫ binding site in SERT through analysis of the LeuT structure (14). This structure shows an aqueous pathway leading from the ...

Mechanism for alternating access in neurotransmitter transporters

“…To better define the cytoplasmic pathway, we have examined the cytoplasmic accessibility of residues in helices 6b, 8 and 1a in SERT in addition to TM5 (20,21). Our results suggest that the cytoplasmic vestibule is formed by the topologically inverted helices corresponding to those that form the extracellular vestibule, which is consistent with the internal symmetry of the protein.…”

“…Asterisks indicate a significant difference from the control rate with MTS reagent alone (P Ͻ 0.05, paired Student's t test). TM5 data are from (20,21).…”

“…These reactive positions were proposed to contribute to the cytoplasmic substrate permeation pathway because their reactivity was modulated by substrates (serotonin, Na ϩ and Cl Ϫ ) and an inhibitor (cocaine) in a manner consistent with the effects of these ligands in the transport cycle (20). Moreover, ibogaine, an hallucinogenic alkaloid proposed to bind preferentially to the cytoplasm-facing state of SERT, induced a concerted conformational change leading to increased accessibility of the reactive positions in TM5 and decreased reactivity of residues in the extracellular permeation pathway (21).…”

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

“…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).…”

Big Lessons from Tiny Flies:<em> Drosophila Melanogaster </em>as a Model to Explore Dysfunction of Dopaminergic and Serotonergic Neurotransmitter Systems

“…The heterologously expressed mutant protein is trapped in the endoplasmic reticulum (ER), as a consequence dopamine uptake is abolished [45]. We tested two pharmacological approaches to restore the cell surface expression of this folding-deficient mutant: (i) relaxing the protein folding quality-control mechanisms in the ER by inhibiting HSP70 using pifithrin-µ [45,50]; (ii) pharmacochaperoning with noribogaine, which binds to and stabilizes monoamine transporters in the inward-facing state [51] The rationale of these two approaches is based on studies with SERT folding mutants [50, [52][53][54][55]. In fact, in transfected cells, both noribogaine and pifithrin-µ restored surface expression to and dopamine uptake by dDAT-G108Q and its human equivalent hDAT-G140Q [45].…”