N-terminus regulation of VMAT2 mediates methamphetamine-stimulated efflux.

The 20 amino acid (AA) N-terminus of the vesicular monoamine transporter 2 (VMAT2) was examined as a regulator of VMAT2 function. Removal of the first 16 or 19 AAs of the N-terminus resulted in a molecule with reduced ability to sequester [(3)H]-5HT. A glutathione-S-transferase-construct of the N-terminus underwent phosphorylation in the presence of PKC at serines 15 and 18. These putative phosphorylation sites were examined for effects on function. Phospho-mimetic substitution of serines 15 and 18 with aspartate in the full-length VMAT2 resulted in reduced [(3)H]-5HT sequestration and reduced methamphetamine (METH)-stimulated efflux of preloaded [(3)H]-5HT. In contrast, mutation of serines 15 and 18 to alanines maintained intact net substrate sequestration but eliminated METH-stimulated efflux of pre-accumulated [(3)H]-5HT. In summary, these data suggest a model in which the VMAT2 N-terminus regulates monoamine sequestration.

Lack of sigma-1 receptor exacerbates ALS progression in mice.

The function of the sigma-1 receptor (S1R) has been implicated in modulating the activity of various ion channels. In the CNS S1R is enriched in cholinergic postsynaptic densities in spinal cord motoneurons (MNs). Mutations in S1R have been found in familial cases of amyotrophic lateral sclerosis (ALS). In this study we show that a knockout of S1R in the SOD1*G93A mouse model of ALS significantly reduces longevity (end stage). Electrophysiological experiments demonstrate that MN of mice lacking S1R exhibit increased excitability. Taken together the data suggest the S1R acts as a brake on excitability, an effect that might enhance longevity in an ALS mouse model.

Development of the sigma-1 receptor in C-terminals of motoneurons and colocalization with the N,N'-dimethyltryptamine forming enzyme, indole-N-methyl transferase.

The function of the sigma-1 receptor (S1R) has been linked to modulating the activities of ion channels and G-protein-coupled receptors (GPCR). In the CNS, the S1R is expressed ubiquitously but is enriched in mouse motoneurons (MN), where it is localized to subsurface cisternae of cholinergic postsynaptic densities, also known as C-terminals. We found that S1R is enriched in mouse spinal MN at late stages of embryonic development when it is first visualized in the endoplasmic reticulum. S1Rs appear to concentrate at C-terminals of mouse MN only on the second week of postnatal development. We found that indole-N-methyl transferase (INMT), an enzyme that converts tryptamine into the sigma-1 ligand dimethyltryptamine (DMT), is also localized to postsynaptic sites of C-terminals in close proximity to the S1R. This close association of INMT and S1Rs suggest that DMT is synthesized locally to effectively activate S1R in MN.

The sigma-1 receptor is enriched in postsynaptic sites of C-terminals in mouse motoneurons. An anatomical and behavioral study.

The sigma-1 receptor regulates various ion channel activity and possesses protein chaperone function. Using an antibody against the full sequence of the sigma-1 receptor we detected immunostaining in wild type but not in knockout mice. The receptor was found primarily in motoneurons localized to the brainstem and spinal cord. At the subcellular level the receptor is restricted to large cholinergic postsynaptic densities on the soma of motoneurons and is colocalized with the Kv2.1 potassium channel and the muscarinic type 2 cholinergic receptor. Ultrastructural analysis of the neurons indicates that the immunostained receptor is located close but separate from the plasma membrane, possibly in subsurface cisternae formed from the endoplasmic reticulum (ER), which are a prominent feature of cholinergic postsynaptic densities. Behavioral testing on a rotorod revealed that Sigma-1 receptor knockout mice remained on the rotorod for significantly less time (a shorter latency period) compared to the wild type mice. Together these data indicate that the sigma-1 receptor may play a role in the regulation of motor behavior.

Stereospecific inhibition of monoamine uptake transporters by meta-hydroxyephedrine isomers.

Meta-hydroxyephedrine (HED) comprises four stereoisomers consisting of two enantiomeric pairs related to ephedrine and pseudoephedrine. HED is transported into adrenergic neurons and radiolabeled HED has been employed in positron emission tomography (PET) to image adrenergic neurons in vivo. To extend structure-activity analyses of binding sites within monoamine transporters and to determine which stereoisomer displayed the best selectivity for PET imaging applications, we tested the HED compounds for their abilities to inhibit [(3)H]neurotransmitter uptake into platelets, transfected cells, and chromaffin vesicles. We hypothesized that the HED compounds would be most potent at the norepinephrine transporter (NET) compared to the serotonin or dopamine transporters and that the 1R diastereomers would be more effective than 1S diastereomers. Supporting the hypotheses, all stereoisomers were most potent at the NET and the 1R,2S stereoisomer was the most potent inhibitor overall. However, the 1S,2R isomer may be preferred for PET applications because of better selectivity among the transporters and reduced neuronal recycling.

Mutagenesis and derivatization of human vesicle monoamine transporter 2 (VMAT2) cysteines identifies transporter domains involved in tetrabenazine binding and substrate transport.

The vesicle monoamine transporter (VMAT2) concentrates monoamine neurotransmitter into synaptic vesicles. Photoaffinity labeling, chimera analysis, and mutagenesis have identified functionally important amino acids and provided some information regarding structure and ligand binding sites. To extend these studies, we engineered functional human VMAT2 constructs with reduced numbers of cysteines. Subsets of cysteines were discovered, which restore function to an inactive cysteine-less human VMAT2. Replacement of three transmembrane (TM) cysteines together (net removal/replacement of three atoms) significantly enhanced monoamine transport. Cysteine modification studies involving single and combination cysteine mutants with methanethiosulfonate ethylamine revealed that [(3)H]dihydrotetrabenazine binding is > 90% inhibited by modification of two sets of cysteines. The primary target (responsible for approximately 80% of inhibition) is Cys(439) in TM 11. The secondary target (responsible for approximately 20% of inhibition) is one or more of the four non-TM cysteines. [(3)H]Dihydrotetrabenazine protects against modification of Cys(439) by a 10,000-fold molar excess of methanethiosulfonate ethylamine, demonstrating that Cys(439) is either at the tetrabenazine binding site, or conformationally linked to tetrabenazine binding. Supporting a direct effect, the position of tetrabenazine-protectable Cys 439 is consistent with previous mutagenesis, chimera, and photoaffinity labeling data, demonstrating involvement of TM 10-12 in a tetrabenazine binding domain.

Tyr199 in transmembrane domain 5 of the beta2-adrenergic receptor interacts directly with the pharmacophore of a unique fluorenone-based antagonist.

Mutagenesis of the beta2-adrenergic receptor (beta2AR) has suggested that amino acids in transmembrane domain 5 (TMD 5) play an important role in the interaction of the receptor with the catechol end of adrenergic agonists. However, little direct biochemical evidence for the interaction of any beta2AR agonist or antagonist with TMD 5 has been reported. To identify receptor amino acids that contribute to the beta2AR antagonist binding site, we identified the precise amino acid photoinsertion site of a novel carazolol-like fluorenone antagonist photoaffinity label, [125I]iodoaminoflisopolol ([125I]IAmF). A unique property of this photolabel is that the photoreactive centre is also the binding pharmacophore, which corresponds to the catechol end of related beta2AR agonists. [125I]IAmF specifically photolabels membrane-bound and purified beta2AR from a baculovirus/Spodoptera frugiperda (fall armyworm) ('Sf9') expression system. When the photolabelled beta2AR was cleaved by trypsin or Factor Xa, 30 kDa labelled peptides were generated. On the basis of concanavalin A binding and amino acid sequencing, these contain the N-terminus of the beta2AR, including TMDs 1-5. Further cleavage of the 30 kDa peptides with endoproteinase Lys-C generated a 4 kDa labelled peptide with an N-terminal amino acid sequence between TMDs 4 and 5. Radiosequencing of this peptide demonstrated that the precise [125I]IAmF photoinsertion site was Tyr(199) in TMD 5. Since the photoreactive centre and the binding pharmacophore of IAmF are the same, these data demonstrate that Tyr(199) interacts with the planar fluorenone moiety of a carazolol-like beta2AR antagonist, and contributes significant new information regarding the binding site for beta2AR antagonists.

A high-affinity fluorenone-based beta 2-adrenergic receptor antagonist with a photoactivatable pharmacophore.

To develop molecules capable of directly probing the catechol binding region of the beta(2)-adrenergic receptor (beta(2)AR), novel benzophenone- and fluorenone-based beta(2)AR antagonists were prepared as potential photoaffinity probes. While the benzophenone-containing ligands bound with relatively modest affinity, one of the fluorenone-based compounds, 4-(2-hydroxy-3-isopropylaminopropoxy)-7-amino-6-iodofluorenone+ ++ (iodoaminoflisopolol, IAmF), showed very high affinity for the beta(2)AR, inhibiting [(125)I]ICYP binding with an apparent K(i) of approximately 1 x 10(-)(9) M. In comparison to the benzophenone ligands, the fluorenone ligands have one additional carbon-carbon bond that creates a planar unsaturated ring system and leads to a large increase in receptor binding affinity. Unlike previous beta(2)AR photoaffinity ligands, an attractive and unique feature of the fluorenone derivative IAmF is that the large planar unsaturated ring (believed to correspond to the catechol end of other beta(2)AR ligands) serves as both the binding pharmacophore and the photoreaction center for this molecule. With this potential for directly probing the catechol binding region of the beta(2)AR, we synthesized and tested IAmF in carrier-free radioiodinated form ([(125)I]IAmF). When photoreduction was conducted at 350 nm for 20 min, [(125)I]IAmF was able to produce cross-linked products in both triethylamine and methanol, with a reactivity pattern similar to that found in benzophenone photochemistry. As a final test of suitability as a photoaffinity label, specific labeling of the beta(2)AR in membranes (protectable by 10 microM alprenolol) was demonstrated. [(125)I]IAmF represents a new class of beta(2)AR photoaffinity labels that can directly probe the catechol-analogous antagonist pharmacophore binding site in the beta(2)AR ligand binding pocket.

Membrane-delimited coupling between sigma receptors and K+ channels in rat neurohypophysial terminals requires neither G-protein nor ATP.

Receptor-mediated modulation of ion channels generally involves G-proteins, phosphorylation, or both in combination. The sigma receptor, which modulates voltage-gated K+ channels, is a novel protein with no homology to other receptors known to modulate ion channels. In the present study patch clamp and photolabelling techniques were used to investigate the mechanism by which sigma receptors modulate K+ channels in peptidergic nerve terminals. The sigma receptor photoprobe iodoazidococaine labelled a protein with the same molecular mass (26 kDa) as the sigma receptor protein identified by cloning. The sigma receptor ligands pentazocine and SKF10047 modulated K+ channels, despite intra-terminal perfusion with GTP-free solutions, a G-protein inhibitor (GDPbetaS), a G-protein activator (GTPgammaS) or a non-hydrolysable ATP analogue (AMPPcP). Channels in excised outside-out patches were modulated by ligand, indicating that soluble cytoplasmic factors are not required. In contrast, channels within cell-attached patches were not modulated by ligand outside a patch, indicating that receptors and channels must be in close proximity for functional interactions. Channels expressed in oocytes without receptors were unresponsive to sigma receptor agonists, ruling out inhibition through a direct drug interaction with channels. These experiments indicate that sigma receptor-mediated signal transduction is membrane delimited, and requires neither G-protein activation nor protein phosphorylation. This novel transduction mechanism is mediated by membrane proteins in close proximity, possibly through direct interactions between the receptor and channel. This would allow for more rapid signal transduction than other ion channel modulation mechanisms, which in the present case of neurohypophysial nerve terminals would lead to the enhancement of neuropeptide release.

Inhibition of plasma membrane monoamine transporters by beta-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.

Probing the salmeterol binding site on the beta 2-adrenergic receptor using a novel photoaffinity ligand, [(125)I]iodoazidosalmeterol.

Salmeterol is a long-acting beta2-adrenergic receptor (beta 2AR) agonist used clinically to treat asthma. In addition to binding at the active agonist site, it has been proposed that salmeterol also binds with very high affinity at a second site, termed the "exosite", and that this exosite contributes to the long duration of action of salmeterol. To determine the position of the phenyl ring of the aralkyloxyalkyl side chain of salmeterol in the beta 2AR binding site, we designed and synthesized the agonist photoaffinity label [(125)I]iodoazidosalmeterol ([125I]IAS). In direct adenylyl cyclase activation, in effects on adenylyl cyclase after pretreatment of intact cells, and in guinea pig tracheal relaxation assays, IAS and the parent drug salmeterol behave essentially the same. Significantly, the photoreactive azide of IAS is positioned on the phenyl ring at the end of the molecule which is thought to be involved in exosite binding. Carrier-free radioiodinated [125I]IAS was used to photolabel epitope-tagged human beta 2AR in membranes prepared from stably transfected HEK 293 cells. Labeling with [(125)I]IAS was blocked by 10 microM (-)-alprenolol and inhibited by addition of GTP gamma S, and [125I]IAS migrated at the same position on an SDS-PAGE gel as the beta 2AR labeled by the antagonist photoaffinity label [125I]iodoazidobenzylpindolol ([125I]IABP). The labeled receptor was purified on a nickel affinity column and cleaved with factor Xa protease at a specific sequence in the large loop between transmembrane segments 5 and 6, yielding two peptides. While the control antagonist photoaffinity label [125I]IABP labeled both the large N-terminal fragment [containing transmembranes (TMs) 1-5] and the smaller C-terminal fragment (containing TMs 6 and 7), essentially all of the [125I]IAS labeling was on the smaller C-terminal peptide containing TMs 6 and 7. This direct biochemical evidence demonstrates that when salmeterol binds to the receptor, its hydrophobic aryloxyalkyl tail is positioned near TM 6 and/or TM 7. A model of IAS binding to the beta 2AR is proposed.

Sigma receptor photolabeling and sigma receptor-mediated modulation of potassium channels in tumor cells.

Recent work has indicated that sigma receptor ligands can modulate potassium channels. However, the only sigma receptor characterized at the molecular level has a novel structure unlike any other receptor known to modulate ion channels. This 26-kDa protein has a hydropathy profile suggestive of a single membrane-spanning domain, with no apparent regions capable of G-protein activation or protein phosphorylation. In the present study patch clamp techniques and photoaffinity labeling were used in DMS-114 cells (a tumor cell line known to express sigma receptors) to investigate the role of the 26-kDa protein in ion channel modulation and probe the mechanism of signal transduction. The sigma receptor ligands N-allylnormetazocine (SKF10047), ditolylguanidine, and (+/-)-2-(N-phenylethyl-N-propyl)-amino-5-hydroxytetralin all inhibited voltage-activated potassium current (IK). Iodoazidococaine (IAC), a high affinity sigma receptor photoprobe, produced a similar inhibition in IK, and when cell homogenates were illuminated in the presence of IAC, a protein with a molecular mass of 26 kDa was covalently labeled. Photolabeling of this protein by IAC was inhibited by SKF10047 with half-maximal effect at 7 microM. SKF10047 also inhibited IK with a similar EC50 (14 microM). Thus, physiological responses to sigma receptor ligands are mediated by a protein with the same molecular weight as the cloned sigma receptor. This indicates that ion channel modulation is indeed mediated by this novel protein. Physiological responses were the same when cells were perfused internally with either guanosine 5'-O-(2-thiodiphosphate) or GTP, indicating that signal transduction is independent of G-proteins. These results demonstrate that ion channels can be modulated by a receptor that does not have seven membrane-spanning domains and does not employ G-proteins. Sigma receptors thus modulate ion channels by a novel transduction mechanism.

Interaction sites of the C-terminal region of the cGMP phosphodiesterase inhibitory subunit with the GDP-bound transducin alpha-subunit.

In the present report, the region of interaction between the GDP-bound alpha-subunit of transducin (alphat.GTP) and the cGMP phosphodiesterase inhibitory gamma-subunit (Pgamma) has been studied. It is widely accepted that the alphat.GTP is the active form of transducin and that the GDP-bound transducin alpha-subunit (alphat. GDP) is the inactive form. We have reported previously that the binding region of the C-terminal of Pgamma on alphat.GTP is in a region between the exposed face of the alpha3 and alpha4 helices of alphat.GTP [Liu, Arshavsky and Ruoho (1996) J. Biol. Chem. 271, 26900-26907]. We now report that N-[(3-[125I]iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP)-derivatized Pgamma (at Cys-68) reversibly undergoes a unique disulphide exchange of the radioiodinated moiety N-(3-[125I]iodo-4-azidophenylpropionamido)cysteine ([125I]APC) from Cys-68 of Pgamma to alphat.GDP but not to the guanosine 5'-(gamma-thio)-triphosphate (GTP[S])-bound transducin alpha-subunit (alphat-GTP[S]). The specificity of the interaction was demonstrated by the fact that exchange was protected by the functionally active Cys-68-->Ala Pgamma mutant, and by pretreatment of the alphat.GDP with the betagamma-subunit of transducin. Chemical cleavage and amino acid sequencing demonstrated that the [125I]ACTP-derived Pgamma specifically transferred the [125I]APC group to Cys-250 and Cys-210 of alphat.GDP. These data indicate that the C-terminal region (especially Cys-68-Trp-70) of Pgamma interacts with alphat. GDP on the exposed interface between alpha2/beta4 and alpha3/beta5 of the alpha-subunit of transducin. Disulphide exchange was also observed with the alpha-subunit of holotransducin but this was only approx. 60% of that of pure alphat.GDP. The variation in the binding pattern between alphat.GDP and alphat.GTP with the C-terminal region of Pgamma may contribute to the functional difference between the GDP- and GTP-bound states.

High-efficiency expression and characterization of the synaptic-vesicle monoamine transporter from baculovirus-infected insect cells.

The full-length cDNA for the rat synaptic-vesicle monoamine transporter (VMAT2) containing a C-terminal polyhistidine epitope has been engineered into baculovirus DNA for expression in Spodoptera frugiperda (Sf9) insect cells. Using this recombinant baculovirus and cultured Sf9 cells, rVMAT2 has been expressed at levels of 7.8x10(6) transporters per cell, as assessed by [3H]dihydrotetrabenazine binding. A 1l culture of infected cells produced approx. 15 nmol (900 microg) of transporter. rVMAT2 expressed in the Sf9 cells bound [3H]dihydrotetrabenazine with a KD of 31.2 nM and a Bmax of 19.9 pmol/mg. Two polypeptides of 55 and 63 kDa were identified using the photolabel, 7-azido-8-[125I]iodoketanserin ([125I]AZIK). Photoaffinity labelling of rVMAT2 by 1 nM [125I]AZIK was protectable by 10 microM tetrabenazine and 10 microM 7-aminoketanserin. Digitonin-solubilized VMAT2 was purified to greater than 95% homogeneity using immobilized Ni2+-affinity chromatography, followed by lectin (Concanavalin A) chromatography. The purified transporter migrates as a single broad band with a molecular mass of approx. 63kDa, as analyzed by SDS/PAGE. The purified transporter retained the ability to bind ligands ([125I]AZIK and [3H]dihydrotetrabenazine). The purified VMAT2 bound [3H]dihydrotetrabenazine with a KD of 86.2 nM. As is the case with the monoamine transporter from bovine chromaffin granule membranes, purified VMAT2 is covalently modified by dicyclohexylcarbodi-imide (DCCD) and is specifically labelled by [14C]DCCD. This labelling is inhibited by tetrabenazine and ketanserin. These data indicate that VMAT2 can be overexpressed using the baculovirus expression system and purified.

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 microM tetrabenazine (TBZ), 10 microM 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.

Characterization and crystallization of a minimal catalytic core domain from mammalian type II adenylyl cyclase.

Adenylyl cyclases play a pivotal role in signal transduction by carrying out the regulated synthesis of cyclic AMP. The nine cloned mammalian adenylyl cyclases all share two conserved regions of sequence, C1 and C2, which are homologous to each other and are together responsible for catalytic activity. Recombinant C1 and C2 domains catalyze the synthesis of cyclic AMP when they are mixed and activated by forskolin, and C2 domains alone also manifest reduced levels of forskolin-stimulated enzyme activity. Using limited proteolysis and mass spectrometry, we have mapped the boundaries of a minimal stable and active C2 catalytic domain to residues 871-1090 of type II adenylyl cyclase. We report the properties and crystallization of this trimmed domain, termed IIC2-delta 4. Crystals belong to space group P4n2(1)2, where n = 1 or 3; a = b = 81.3, and c = 180.5 A; and there are two molecules per asymmetric unit related by an approximate body centering operation. Flash-frozen crystals diffract anisotropically to 2.2 A along the c* direction and to 2.8 A along the a* and b* directions using synchrotron radiation.

Structure of the adenylyl cyclase catalytic core.

Mammalian adenylyl cyclases contain two conserved regions, C1 and C2, which are responsible for forskolin- and G-protein-stimulated catalysis. The structure of the C2 catalytic region of type II rat adenylyl cyclase has an alpha/beta class fold in a wreath-like dimer, which has a central cleft. Two forskolin molecules bind in hydrophobic pockets at the ends of cleft. The central part of the cleft is lined by charged residues implicated in ATP binding. Forskolin appears to activate adenylyl cyclase by promoting the assembly of the active dimer and by direct interaction within the catalytic cleft. Other adenylyl cyclase regulators act at the dimer interface or on a flexible C-terminal region.

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.

Catalytic mechanism of the adenylyl and guanylyl cyclases: modeling and mutational analysis.

The adenylyl and guanylyl cyclases catalyze the formation of 3', 5'-cyclic adenosine or guanosine monophosphate from the corresponding nucleoside 5'-triphosphate. The guanylyl cyclases, the mammalian adenylyl cyclases, and their microbial homologues function as pairs of homologous catalytic domains. The crystal structure of the rat type II adenylyl cyclase C2 catalytic domain was used to model by homology a mammalian adenylyl cyclase C1-C2 domain pair, a homodimeric adenylyl cyclase of Dictyostelium discoideum, a heterodimeric soluble guanylyl cyclase, and a homodimeric membrane guanylyl cyclase. Mg2+ATP or Mg2+GTP were docked into the active sites based on known stereochemical constraints on their conformation. The models are consistent with the activities of seven active-site mutants. Asp-310 and Glu-432 of type I adenylyl cyclase coordinate a Mg2+ ion. The D310S and D310A mutants have 10-fold reduced Vmax and altered [Mg2+] dependence. The NTP purine moieties bind in mostly hydrophobic pockets. Specificity is conferred by a Lys and an Asp in adenylyl cyclase, and a Glu, an Arg, and a Cys in guanylyl cyclase. The models predict that an Asp from one domain is a general base in the reaction, and that the transition state is stabilized by a conserved Asn-Arg pair on the other domain.