Michael K. Sievert

Inhibition of plasma membrane monoamine transporters by β-ketoamphetamines

Methcathinone and methylone, the beta-ketone analogues of methamphetamine and 3,4-methylenedioxymethamphetamine (MDMA), respectively, were tested for neurotransmitter uptake inhibition in vitro. The beta-ketones were threefold less potent than the nonketo drugs at inhibiting platelet serotonin accumulation, with IC(50)s of 34.6+/-4.8 microM and 5.8+/-0.7 microM, respectively. Methcathinone and methylone were similar in potency to methamphetamine and MDMA at catecholamine transporters individually expressed in transfected glial cells. For dopamine uptake, IC(50)s were 0.36+/-0.06 microM and 0.82+/-0.17 microM, respectively; for noradrenaline uptake, IC(50) values were 0.51+/-0.10 microM and 1. 2+/-0.1 microM, respectively. In chromaffin granules, IC(50)s for serotonin accumulation were 112+/-8.0 microM for methcathinone and 166+/-12 microM for methylone, 10-fold higher than the respective values for methamphetamine and MDMA. Our results indicate that methcathinone and methylone potently inhibit plasma membrane catecholamine transporters but only weakly inhibit the vesicle transporter.

The Inhibitory γ Subunit of the Rod cGMP Phosphodiesterase Binds the Catalytic Subunits in an Extended Linear Structure

The unique feature of rod photoreceptor cGMP phosphodiesterase (PDE6) is the presence of inhibitory subunits (Pγ), which interact with the catalytic heterodimer (Pαβ) to regulate its activity. This uniqueness results in an extremely high sensitivity and sophisticated modulations of rod visual signaling where the Pγ/Pαβ interactions play a critical role. The quaternary organization of the αβγγ heterotetramer is poorly understood and contradictory patterns of interaction have been previously suggested. Here we provide evidence that supports a specific interaction, by systematically and differentially analyzing the Pγ-binding regions on Pα and Pβ through photolabel transfer from various Pγ positions throughout the entire molecule. The Pγ N-terminal Val16–Phe30 region was found to interact with the Pαβ GAFa domain, whereas its C terminus (Phe73–Ile87) interacted with the Pαβ catalytic domain. The interactions of Pγ with these two domains were bridged by its central Ser40–Phe50 region through interactions with GAFb and the linker between GAFb and the catalytic domain, indicating a linear and extended interaction between Pγ and Pαβ. Furthermore, a photocross-linked product αβγ(γ) was specifically generated by the double derivatized Pγ, in which one photoprobe was located in the polycationic region and the other in the C terminus. Taken together the evidence supports the conclusion that each Pγ molecule binds Pαβ in an extended linear interaction and may even interact with both Pα and Pβ simultaneously.

Peptide Mapping of the [125I]Iodoazidoketanserin and [125I]2-N-[(3′-Iodo-4′-azidophenyl)propionyl]tetrabenazine Binding Sites for the Synaptic Vesicle Monoamine Transporter

The full-length cDNA for the rat recombinant synaptic vesicle monoamine transporter (rVMAT2) containing a COOH-terminal polyhistidine epitope was engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed to high levels and purified to >95% homogeneity using immobilized Ni2+-affinity chromatography followed by lectin (concanavalin A) chromatography. Purified transporter was photolabeled using [125I]-7-azido-8-iodoketanserin ([125I]AZIK) and [125I]2-N-[(3′-iodo-4′-azidophenyl)propionyl]tetrabenazine ([125I]TBZ-AIPP). Both [125I]AZIK and [125I]TBZ-AIPP photoaffinity labeling of purified rVMAT2 were protectable by 10 μm tetrabenazine (TBZ), 10 μm 7-aminoketanserin, and 1 mm concentrations of the transporter substrates dopamine, norepinephrine, and serotonin. Radiolabeled peptides were generated using enzymatic and chemical methods, purified using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NH2-terminal microsequenced. Radiosequencing of [125I]AZIK-labeled rVMAT2 indicated derivatization of Lys-20 in the NH2 terminus, just prior to putative transmembrane domain 1 (TMD1). [125I]TBZ-AIPP derivatized a segment of rVMAT2 between Gly-408 and Cys-431 in TMD10 and 11. These data implicate juxtaposition of TMD1 and 10/11.

Affinities of dopamine analogs for monoamine granular and plasma membrane transporters: Implications for PET dopamine studies

Affinities of dopamine (DA) analogs to both granular and plasma membrane uptake transporters were measured in vitro by inhibition of [3H]DA uptake in bovine chromaffin granule ghosts and C6 glial cells transfected with cDNA for the rat presynaptic dopamine transporter, respectively. Five amines were studied: DA, 6-fluorodopamine (6FDA), m-tyramine (MTA), 6-fluoro-m-tyramine (6FMTA), and beta-fluoromethylene-m-tyramine (FMMTA). Direct uptake of 18F labeled 6FDA and 6FMTA was also measured in the chromaffin granule system and compared with [3H]DA uptake. Results show that the transporter affinities of 6FDA and MTA were similar to that of DA in both transport systems while affinities of 6FMTA and FMMTA were lower. Furthermore while the direct uptake of DA and FDA in chromaffin granules were essentially identical and significantly reserpine-inhibitable, the direct uptake of 6FMTA was about 15-fold less and only minimally sensitive to reserpine pretreatment. Thus, although vesicular protection and reuptake may influence the turnover of FDA in 6-fluoroDOPA studies, they are unlikely to be important determinants of the kinetics of the slowly clearing components in studies with either 6-fluoro-m-tyrosine (6FMT) or 6-fluoro-beta-fluoro-methylene-m-tyrosine (6FFMMT), the bioprecursors of 6FMTA and 6-fluoro-FMMTA, respectively. These results are consistent with the finding that the longterm component in 6FMT PET studies is 6-fluoro-hydroxyphenylacetic acid (6FHPAC), which can be explained by the lack of vesicular protection of 6FMTA from MAO oxidation.

Identification of the Substrate Binding Region of Vesicular Monoamine Transporter-2 (VMAT-2) Using Iodoaminoflisopolol as a Novel Photoprobe

Monoamines, such as serotonin, dopamine, and norepinephrine, are sequestered into synaptic vesicles by specific transporters (vesicular monoamine transporter-2; VMAT2) using energy from an electrochemical proton gradient across the vesicle membranes. Based on our previous studies using photoaffinity-labeling techniques in characterizing the VMAT2-specific ligands ketanserin and tetrabenazine, this study describes the synthesis and characterization of a fluorenone-based compound, iodoaminoflisopolol (IAmF), as a photoprobe to identify the substrate binding site(s) of VMAT2. Using vesicles prepared from rat VMAT2 containing recombinant baculovirus-infected Sf9 cells, we show the inhibition of [3H]5-hydroxytryptamine (5-HT) uptake and [3H]dihydrotetrabenazine (TBZOH) binding by aminoflisopolol and iodoaminoflisopolol. The interaction of [125I]IAmF with VMAT2 is highly dependent on the presence of ATP and an intact proton gradient. We report a simple and novel method to distinguish between a ligand and substrate using classic compounds such as [3H]5-HT and [3H]TBZOH by incubating the compound with the vesicles followed by washes with isotonic and hypotonic solutions. Using this method, we confirm the characterization of IAmF as a novel VMAT2 substrate. Sf9 vesicles expressing VMAT2 show reserpine- and tetrabenazine-protectable photolabeling by [125I]IAmF. [125I]IAmF photolabeling of recombinant VMAT2, expressed in SH-SY5Y cells with an engineered thrombin site between transmembranes 6 and 7, followed by thrombin digestion, retained photolabel in a 22-kDa fragment, indicating that iodoaminoflisopolol binds to the C-terminal half of the VMAT2 molecule. Thus, IAmF possesses a unique combination of VMAT2 substrate properties and a photoprobe and is, therefore, useful to identify the substrate binding site of the vesicular transporter.

Bacteriorhodopsin chimeras containing the third cytoplasmic loop of bovine rhodopsin activate transducin for GTP/GDP exchange

The mechanisms by which G‐protein‐coupled receptors (GPCRs) activate G‐proteins are not well understood due to the lack of atomic structures of GPCRs in an active form or in GPCR/G‐protein complexes. For study of GPCR/G‐protein interactions, we have generated a series of chimeras by replacing the third cytoplasmic loop of a scaffold protein bacteriorhodopsin (bR) with various lengths of cytoplasmic loop 3 of bovine rhodopsin (Rh), and one such chimera containing loop 3 of the human β2‐adrenergic receptor. The chimeras expressed in the archaeon Halobacterium salinarum formed purple membrane lattices thus facilitating robust protein purification. Retinal was correctly incorporated into the chimeras, as determined by spectrophotometry. A 2D crystal (lattice) was evidenced by circular dichroism analysis, and proper organization of homotrimers formed by the bR/Rh loop 3 chimera Rh3C was clearly illustrated by atomic force microscopy. Most interestingly, Rh3C (and Rh3G to a lesser extent) was functional in activation of GTPγ35S/GDP exchange of the transducin α subunit (Gαt) at a level 3.5‐fold higher than the basal exchange. This activation was inhibited by GDP and by a high‐affinity peptide analog of the Gαt C terminus, indicating specificity in the exchange reaction. Furthermore, a specific physical interaction between the chimera Rh3C loop 3 and the Gαt C terminus was demonstrated by cocentrifugation of transducin with Rh3C. This Gαt‐activating bR/Rh chimera is highly likely to be a useful tool for studying GPCR/G‐protein interactions.

Photoaffinity labeling of adenylyl cyclase

Photoaffinity is an effective tool to covalently tag amino acid residues that are in or near ligand-binding sites. The polypeptide chains that contribute to the binding site of a given receptor can be identified by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) if they are specifically labeled with a radioactive marker. Purification and subsequent N-terminal sequencing of these labeled peptides identify their location in the primary sequence of the receptor. Forskolin, a naturally occurring diterpene, has direct stimulatory effects on adenylyl cyclase. This chapter discusses the synthesis and use of two forskolin photoaffinity labels. N-13-(4-azido-3-[ 125 I]iodophenyl) propionyl]forskolin ( 125 I-6-AIPP-Fsk) and 125 I-7-AIPP-Fsk. Although both compounds bind to adenylyl cyclase, 125 I-6-AIPP-Fsk is a more effective photolabel for derivatization of adenylyl cyclase.