Monoterpenoid indole alkaloids are a large (∼3000 members) and structurally diverse class of metabolites restricted to a limited number of plant families in the order Gentianales. Tabernanthe iboga or iboga (Apocynaceae) is native to western equatorial Africa and has been used in traditional medicine for centuries. Howard Lotsof is credited with bringing iboga to the attention of Western medicine through his accidental discovery that iboga can alleviate opioid withdrawal symptoms. Since this observation, iboga has been investigated for its use in the general management of addiction. We were interested in elucidating ibogaine biosynthesis to understand the unique reaction steps en route to ibogaine. Furthermore, because ibogaine is currently sourced from plant material, these studies may help improve the ibogaine supply chain through synthetic biology approaches. Here, we used next-generation sequencing to generate the first iboga transcriptome and leveraged homology-guided gene discovery to identify the penultimate hydroxylase and final O-methyltransferase steps in ibogaine biosynthesis, herein named ibogamine 10-hydroxylase (I10H) and noribogaine-10-O-methyltransferase (N10OMT). Heterologous expression in Saccharomyces cerevisiae (I10H) or Escherichia coli (N10OMT) and incubation with putative precursors, along with HPLC–MS analysis, confirmed the predicted activities of both enzymes. Moreover, high expression levels of their transcripts were detected in ibogaine-accumulating plant tissues. These discoveries coupled with our publicly available iboga transcriptome will contribute to additional gene discovery efforts and could lead to the stabilization of the global ibogaine supply chain and to the development of ibogaine as a treatment for addiction.
P2X receptors (nomenclature as agreed by the NC-IUPHAR Subcommittee on P2X Receptors [49, 146]) have a trimeric topology [118, 128, 144, 197] with two putative TM domains per P2X subunit, gating primarily Na+, K+ and Ca2+, exceptionally Cl-. The Nomenclature Subcommittee has recommended that for P2X receptors, structural criteria should be the initial basis for nomenclature where possible. X-ray crystallography indicates that functional P2X receptors are trimeric and three agonist molecules are required to bind to a single trimeric assembly in order to activate it [118, 144, 95, 103, 177]. Native receptors may occur as either homotrimers (e.g. P2X1 in smooth muscle) or heterotrimers (e.g. P2X2:P2X3 in the nodose ganglion [280], P2X1:P2X5 in mouse cortical astrocytes [162], and P2X2:P2X5 in mouse dorsal root ganglion, spinal cord and mid pons [53, 234]. P2X2, P2X4 and P2X7 receptor activation can lead to influx of large cationic molecules, such as NMDG+, Yo-Pro, ethidium or propidium iodide [211]. The permeability of the P2X7 receptor is modulated by the amount of cholesterol in the plasma membrane [193]. The hemi-channel pannexin-1 was initially implicated in the action of P2X7 [212], but not P2X2, receptors [41], but this interpretation is probably misleading [215]. Convincing evidence now supports the view that the activated P2X7 receptor is immediately permeable to large cationic molecules, but influx proceeds at a much slower pace than that of the small cations Na+, K+, and Ca2+ [66].
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