Reserpine Binding Research Articles

Evaluating the insecticidal potential of alkaloids for the management of Thrips palmi: in vivo and in silico perspectives

Insecticidal potential of seven commonly available alkaloids against melon thrips (Thrips palmi Karny) was investigated through in vivo experiments and the bioactivity was explained via in silico approaches. In vivo screening showed highest mortality of T. plami larvae for reserpine (43%), closely followed by tropinone (41%) after 24 h of incubation. After 48 h, tropinone surpassed reserpine with 83% mortality, indicating its prolonged insecticidal activity. A detailed bioassay of tropinone revealed its LC50 values as 1187.9 and 686.9 µg mL−1 after 24 and 48 h, respectively. While studying the molecular interactions between the alkaloids and four physiologically important target proteins of T. palmi, tropinone demonstrated the highest ligand efficiency and lowest predicted inhibitory constant, particularly when forming complexes with CathB protein. However, binding energy calculations of the docked complexes showed most favorable binding of reserpine with CathB. To clear the ambiguity, considering both binding energy and ligand efficiency as the evaluation parameters, a molecular dynamics study was carried out, which predicted higher stability of CathB-tropinone complex than CathB-reserpine complex in terms of the total energy of the system. These in silico findings aligned well with the in vivo results, confirming tropinone as a promising candidate for effective thrips management programs.

Structural insights into vesicular monoamine storage and drug interactions.

Biogenic monoamines-vital transmitters orchestrating neurological, endocrinal and immunological functions1-5-are stored in secretory vesicles by vesicular monoamine transporters (VMATs) for controlled quantal release6,7. Harnessing proton antiport, VMATs enrich monoamines around 10,000-fold and sequester neurotoxicants to protect neurons8-10. VMATs are targeted by an arsenal of therapeutic drugs and imaging agents to treat and monitor neurodegenerative disorders, hypertension and drug addiction1,8,11-16. However, the structural mechanisms underlying these actions remain unclear. Here we report eight cryo-electron microscopy structures of human VMAT1 in unbound form and in complex with four monoamines (dopamine, noradrenaline, serotonin and histamine), the Parkinsonism-inducing MPP+, the psychostimulant amphetamine and the antihypertensive drug reserpine. Reserpine binding captures a cytoplasmic-open conformation, whereas the other structures show a lumenal-open conformation stabilized by extensive gating interactions. The favoured transition to this lumenal-open state contributes to monoamine accumulation, while protonation facilitates the cytoplasmic-open transition and concurrently prevents monoamine binding to avoid unintended depletion. Monoamines and neurotoxicants share a binding pocket that possesses polar sites for specificity and a wrist-and-fist shape for versatility. Variations in this pocket explain substrate preferences across the SLC18 family. Overall, these structural insights and supporting functional studies elucidate the mechanism of vesicular monoamine transport and provide the basis to develop therapeutics for neurodegenerative diseases and substance abuse.

Synthesis and Discovery of Arylpiperidinylquinazolines: New Inhibitors of the Vesicular Monoamine Transporter.

Methamphetamine, a human vesicular monoamine transporter 2 (VMAT2) substrate, releases dopamine, serotonin, and norepinephrine from vesicles into the cytosol of presynaptic neurons and induces reverse transport by the monoamine transporters to increase extracellular neurotransmitters. Currently available radioligands for VMAT2 have considerable liabilities: The binding of [3H]dihydrotetrabenazine ([3H]DHTB) to a site on VMAT2 is not dependent on ATP, and [3H]reserpine binds almost irreversibly to VMAT2. Herein we demonstrate that several arylpiperidinylquinazolines (APQs) are potent inhibitors of [3H]reserpine binding at recombinant human VMAT2 expressed in HEK-293 cells. These compounds are biodiastereoselective and bioenantioselective. The lead radiolabeled APQ is unique because it binds reversibly to VMAT2 but does not bind the [3H]DHTB binding site. Furthermore, experimentation shows that several novel APQ ligands have high potency for inhibition of uptake by both HEK-VMAT2 cells and mouse striatal vesicles and may be useful tools for characterizing drug-induced effects on human VMAT2 expression and function.

5] Noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives: Purification of photolabeled protein

The vesicular monoamine transporter (VMAT) accumulates monoamines inside secretory granules or synaptic vesicles by catalyzing an H+/ monoamine antiport. Monoamine uptake is coupled to the generation of a proton electrochemical gradient by an ATP-dependent H + pump of the vacuolar type. The uptake is extremely efficient and in the adrenal medulla chromaffin cells, the monoamine gradient between the cytosol and the secretory granules has been estimated to five orders of magnitude. This chapter discusses noncovalent and covalent labeling of vesicular monoamine transporter with tetrabenazine and ketanserin derivatives protein. The vesicular monoamine transporter from various origins is cloned and expressed in various cell types. Two isoforms, VMAT1 and VMAT2, are identified; they have different localizations and slightly different properties. Two different binding sites are also identified: the first one binds reserpine, whereas the second one binds tetrabenazine (TBZ) and ketanserin (KET). The H+ electrochemical gradient generated in the presence of ATP greatly accelerates reserpine binding, but has no effect on TBZ and KET binding.

Charged Residues in Transmembrane Domains II and XI of a Vesicular Monoamine Transporter Form a Charge Pair That Promotes High Affinity Substrate Recognition

Vesicular monoamine transporters package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. Both vesicular monoamine transporter 1 and 2 contain several charged residues predicted to reside within transmembrane domains (TMDs), and conservation of these residues in multiple species and in other members of the gene family suggest important roles in transporter structure and function. To determine the role of these residues, we have used site-directed mutagenesis. Replacement of Asp-263 in TMD6 with Asn (D263N) had no effect on transport activity. However, replacement of Lys-139 in TMD2 with Ala (K139A), Asp-400 in TMD10 with Asn (D400N), or Asp-427 in TMD11 with Asn (D427N) eliminated transport activity despite normal levels of protein expression. Remarkably, the double mutant K139A/D427N showed substantial transport activity, suggesting that Lys-139 and Asp-427 interact to form an ion pair in the native protein and hence that TMD2 occurs next to TMD11. Nonetheless, the double mutant showed reduced apparent affinity for serotonin and reduced ability of serotonin to inhibit reserpine binding, suggesting that although not required for activity, the ion pair promotes high affinity interaction with the substrate. In addition, a double mutant in which the polarity of the charged residues was reversed (K139D/D427K) showed no active transport. Remarkably, however, this mutant displayed normal reserpine binding that remained coupled to DeltaH+, but serotonin failed to inhibit reserpine binding, suggesting that the charge reversal specifically disrupts substrate recognition.

Molecular and Biochemical Studies of Rat Vesicular Monoamine Transporter

Transport of the monoamines serotonin, doparnine, norepinephrine, epinephrine, and histamine into storage organelles in a variety of cells is catalyzed by vesicular monoamine transporters (VMATs). Accumulation of the neurotransmitter depends on the proton electrochemical gradient generated by the vesicular H + -adenosine triphosphatase and involves the VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. A model of the mechanism of action of VMAT has been proposed based on a large body of biochemical data. A clue for the molecular basis of some of these processes has been obtained using diethyl pyrocarbonate (DEPC), a reagent relatively specific for His residues. The inhibition by DEPC is specific for His groups because transport could be restored by hydroxylamine. Cloning of VMAT has made it feasible to try to identify the residue(s) modified by DEPC. DEPC inhibited transport but had no effect on binding of reserpine, indicating that the inhibition of transport was not because of a direct interaction with either of the known binding sites. The result suggests that either proton transport or a conformational change induced by proton transport is inhibited by DEPC. Practically identical results have been obtained with phenylglyoxal, a reagent specific for Arg residues.

Modification of the pH Profile and Tetrabenazine Sensitivity of Rat VMAT1 by Replacement of Aspartate 404 with Glutamate

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine, and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient (Delta micro H+) across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. Mutagenic analysis of the role of two conserved Asp residues located in transmembrane segments X and XI of rat VMAT type I reveals an important role of these two residues in catalysis. Replacement of Asp 431 with either Glu or Ser inhibits VMAT-mediated [3H]serotonin transport. The mutated proteins are unimpaired in ligand recognition as measured with the high affinity ligand [3H]reserpine or coupling to the proton electrochemical gradient as judged by its ability to accelerate [3H]reserpine binding. Therefore, the Asp residue is needed as such in this position and even a conservative replacement with Glu generates a protein that can catalyze only partial reactions but cannot complete the transport cycle. Replacement of Asp 404 with either Ser or Cys inhibits all VMAT-mediated reactions measured. However, replacement with Glu generated a protein that catalyzed [3H]serotonin transport with modified properties. Whereas the mutated protein binds [3H]reserpine to normal levels and the pH optimum of this reaction is only slightly affected, the optimum pH for transport activity shifted to the acid side and became very sharp; in addition the sensitivity to the inhibitor tetrabenazine increased significantly in this mutated protein. The results point to the need of a carboxyl moiety in position 404. A slight change in its relative location or in the environment around it has a significant effect on the pK of group(s) involved in steps after ligand recognition and coupling to the first H+.

The pharmacological profile of the vesicular monoamine transporter resembles that of multidrug transporters

Vesicular neurotransmitter transporters function in synaptic vesicles and other subcellular organelles and they were thought to be involved only in neurotransmitter storage. Several findings have led us to test novel aspects of their function. Cells expressing a c-DNA coding for one of the rat monoamine transporters (VMAT1) become resistant to the neurotoxin N-methyl-4-phenylpyridinium (MPP +) [Liu et al. (1992) Cell, 70, 539–551]. The basis of the resistance is the VMAT1-mediated transport and sequestration of the toxin into subcellular compartments. In addition, the deduced sequence of VMAT1 predicts a protein that shows a distinct homology to a class of bacterial drug resistance transporters (TEXANs) that share some substrates with mammalian multidrug resistance transporters (MDR) such as the P-glycoprotein. These findings induced us to test whether compounds that are typically transported by MDR interact also with vesicular transporters. The use of [ 3H]reserpine binding to determine drug interactions with VMAT allowed assessment of the ability of various drugs to bind to the substrate site of the transporter. Cytotoxic compounds such as ethidium, isometamidium, tetraphenylphosphonium, rhodamine, tacrine and doxorubicin, interact specifically with vesicular monoamine transporters. Verapamil, a calcium channel blocker, is also a competitive inhibitor of transport. In the case of rhodamine, fluorescence measurements in digitonin-permeabilized cells demonstrated ATP-dependent VMAT-mediated transport. The results imply that even though the bacterial and vesicular transporters are structurally different from the P-glycoprotein, they share a similar substrate range. These findings suggest a novel possible way of protection from the effects of toxic compounds by removal to subcellular compartments.

Identification of Residues Involved in Substrate Recognition by a Vesicular Monoamine Transporter

To identify the residues involved in substrate recognition by recently cloned vesicular monoamine transporters (VMAT1 and VMAT2), we have mutagenized the conserved residues in a cytoplasmic loop between transmembrane domains two and three of VMAT2. Although studies of related bacterial antibiotic resistance proteins indicate an important functional role for this region, we found no effect of these mutations on VMAT2 activity. However, replacement of aspartate 33 in the first predicted transmembrane domain with an asparagine (D33N) eliminates transport. D33N shows normal levels of expression and normal binding at equilibrium to the potent inhibitor reserpine. However, in contrast to wild-type VMAT2, serotonin inhibits reserpine binding to D33N very poorly, indicating a specific defect in substrate recognition. Replacement of three serine residues in transmembrane domain three with alanine (Stmd3A) shows a similarly selective but even more profound defect in substrate recognition. The results suggest that by analogy to receptors and plasma membrane transporters for monoamines, the cationic amino group of the ligand interacts with an asparte in the first transmembrane domain of VMAT2 and hydroxyl groups on the catechol or indole ring interact with a group of serines in the third transmembrane domain. Importantly, D33N and Stmd3A retain coupling to the proton electrochemical gradient as measured by the delta microH(+)-induced acceleration of reserpine binding. This indicates that substrate recognition can be separated from coupling to the driving force.

Time-course of modifications elicited by reserpine on the density and mRNA synthesis of the vesicular monoamine transporter, and on the density of the membrane dopamine uptake complex.

The long-term effects of a unique injection of reserpine (5 mg/kg s.c.) on the vesicular monoamine transporter and dopamine uptake complex have been investigated, in parallel with behavioral and neurochemical effects. Early after treatment, a dramatic decrease in locomotor activity, as well as a marked depletion in striatal dopamine (DA), associated with a prominent enhancement in dopaminergic turnover were observed in reserpine-treated rats. From 2 to 60 days after reserpine injection, a recovery in locomotor activity occurred, in parallel with an increased DA content in the striatum, reaching about 50% of controls at day 60. At this time, the dopamine turnover was quite normal. The density of the dopamine uptake sites in the striatum, studied with 3H GBR12783, was unchanged after reserpine treatment at any time studied up to 60 days. By contrast, the density of binding sites for 3H dihydrotetrabenazine (3H TBZOH), a marker for the vesicular monoamine transporter, remained dramatically decreased in the striatum all over the time of the study (> -90% of controls at day 2 and -80% at day 30 and 60). A lesser decrease (-60%) was observed in the substantia nigra pars compacta (SNc), 2 and 30 days after reserpine treatment. This suggests that at least 60% of the vesicular monoamine transporter is sensitive to reserpine in this cell bodies region, indicating that this proportion of the transporter is integrated in functional vesicles, a prerequisite for reserpine binding.(ABSTRACT TRUNCATED AT 250 WORDS)

Histidine-419 plays a role in energy coupling in the vesicular monoamine transporter from rat

Vesicular monoamine transporters (VMAT) catalyze transport of serotonin, dopamine, epinephrine and norepinephrine into subcellular storage organelles in a variety of cells. Accumulation of the neurotransmitter depends on the proton electrochemical gradient across the organelle membrane and involves VMAT-mediated exchange of two lumenal protons with one cytoplasmic amine. It has been suggested in the past that His residues play a role in H + movement or in its coupling to active transport in H +-symporters and antiporters. Indeed VMAT-mediated transport is inhibited by reagents specific for His residues. We have identified one His residue in VMAT1 from rat which is conserved in other vesicular neurotransmitter transporters. Mutagenesis of this His (H419) to either Arg or Cys completely inhibits [ 3H]serotonin and [ 3H]dopamine accumulation. Mutagenesis also inhibits other H +-dependent partial reactions of VMAT such as the acceleration of binding of the high affinity ligand reserpine, but does not inhibit the [ 3H]reserpine binding which is not dependent on H + translocation. It is concluded that His-419 plays a role in energy coupling in r-VMAT1.

Axonal transport of dopamine-containing vesicles labelled in vivo with [3H]reserpine.

Axonal transport of the vesicular monoamine transporter was assayed in the rat brain by in vivo binding of the specific ligand [3H]reserpine. Because of the marked localization of reserpine binding sites in dopaminergic cell bodies and nerve terminals, the dopaminergic nigrostriatal pathway was chosen for the study of the axonal transport of the monoamine carrier present in the membrane of synaptic vesicles. When labelled reserpine was injected into the substantia nigra, a delayed accumulation of radioactivity in the ipsilateral striatum was observed approximately 4 h after the injection. Similarly, injection into the right striatum was followed by a substantial accumulation of radioactivity in the ipsilateral substantia nigra, which was prevented by peripheral injection of unlabelled reserpine or tetrabenazine. This process was rapid and dependent on microtubules. In senescent rats, the amount of retrogradely transported [3H]reserpine was significantly reduced. These results demonstrate that labelled reserpine may be used to monitor in vivo fast axonal transport in central neurons.

Mutants of the Bacillus subtilis multidrug transporter Bmr with altered sensitivity to the antihypertensive alkaloid reserpine

The Bacillus subtilis multidrug transporter Bmr effluxes structurally diverse toxic compounds out of bacterial cells. Antihypertensive alkaloid reserpine reverses Bmr-mediated multidrug resistance by inhibiting drug transport. We have obtained a mutant of the bmr gene that provides a normal level of multidrug resistance, which can, however, only be reversed by very high concentrations of reserpine. Reduction of Bmr sensitivity to reserpine has been caused by the substitution of Leu for Val286 in the Bmr molecule. This mutation also led to a dramatic decrease of [3H]reserpine binding to membrane vesicles prepared from the Bmr-overexpressing bacteria. Leucine is larger than valine by one methylene group. Substitution of Val286 with a smaller residue, glycine, had an opposite effect. It led to increased sensitivity of Bmr to reserpine and increased affinity of reserpine binding to the membranes prepared from Bmr-overexpressing bacteria. Neither of the mutations significantly changed the sensitivity of Bmr to rescinnamine, a structural analog of reserpine. The results suggest that Val286 is involved in the formation of the reserpine-binding site of the Bmr molecule.

Reserpine binding to a vesicular amine transporter expressed in Chinese hamster ovary fibroblasts.

The potent antihypertensive drug reserpine inhibits the transport of biogenic amines into adrenal chromaffin granules and synaptic vesicles. Reserpine acts by binding almost irreversibly to the vesicular amine transporter, and this interaction has been used both to study the mechanism of transport and to purify the protein responsible. Recent isolation of a cDNA for the rat chromaffin granule amine transporter (CGAT) by selection in the neurotoxin 1-methyl-4-phenylpyridinium now permits an analysis of the interaction with reserpine at a molecular level. Using membranes from stable transformants expressing the transporter, we show that reserpine binds specifically and quantitatively to CGAT. As with the native protein in bovine chromaffin granules, a pH gradient accelerates reserpine binding, and amine substrates compete for binding with reserpine. However, 1-methyl-4-phenylpyridinium and tetrabenazine, the other principal inhibitor of vesicular amine transport, compete very poorly with reserpine for binding, suggesting that they interact with CGAT at distinct sites.

Modification of arginyl or histidyl groups affects the energy coupling of the amine transporter.

We have characterized the effects of phenylglyoxal and diethyl pyrocarbonate (DEPC) on the catalytic cycle of the amine transporter in chromaffin granule membrane vesicles. Both reagents inhibited transport in a dose-dependent reaction (with IC50 values of 8 and 1 mM, respectively). The inhibition by DEPC was specific for histidyl groups since transport could be restored by treatment with hydroxylamine. Neither phenylglyoxal nor DEPC inhibited binding of either R1- or R2-type ligands, indicating that the inhibition of transport is not due to a direct interaction with either of the known binding sites. Interestingly, however, the acceleration of reserpine binding (an R1 ligand) by a transmembrane H+ gradient is inhibited by both reagents at concentrations identical to those which inhibit transprot. As previously demonstrated, transport of one proton across the transporter is required for this acceleration to take place [Rudnick, G., Steiner-Mordoch, S., Fishkes, H., Stern-Bach, Y., & Schuldiner, S. (1990) Biochemistry 29, 603-608]. Therefore, we suggest that either proton transport or a conformational change induced by proton transport is inhibited by both types of reagents.

Analysis of the interaction of reserpine with actin by the photoaffinity labelling method

The interaction of reserpine with one of the cytoskeletal proteins, actin, was analyzed by the photoaffinity labelling method using [ 3H]reserpine. Reserpine bound sufficiently to G- or oligomeric actin, but hardly to F-actin under the same experimental conditions. This result could be explained if reserpine binds to a specific region of the G-actin molecule that is responsible for actin-actin interactions. It is concordant with this idea that [ 3H]reserpine bound only to specific proteolytic fragments of actin. When reserpine was mixed with crude extracts of two kinds of tissues, chicken gizzard smooth muscle and bovine adrenal medulla, it bound to the 42 kDa protein of sodium dodecyl sulfate-polyacrylamide gel electrophoresis in both cases. Its molecular size and abundance suggest strongly that this 42 kDa protein is actin. The binding of reserpine to actin was inhibited by dopamine in a dose-dependent manner. These results suggest that actin could be one of the target molecules of reserpine.

Persistent occultation of the vesamicol receptor

By binding to a specific receptor, the drug vesamicol [(-)-trans-2-(4-phenylpiperidino)cyclohexanol] noncompetitively inhibits acetylcholine active transport into synaptic vesicles. An analog [(+/-)-trans-5-amino-2-hydroxy-3-(4-phenylpiperidino) tetralin] of vesamicol has been discovered that causes time- (t1/2 = 2.6 min) and temperature-dependent loss of vesamicol binding that is only slowly reversible (t1/2 for recovery = 5.4 h). Assuming a simple two step process of ligand binding followed by a conformational change, an apparent dissociation constant of 4 x 10(-11) M can be calculated. Other analogs of the vesamicol family of drugs also display similar high-affinity binding to the receptor in a manner which resembles the time dependent effects of reserpine binding to chromaffin granules.

Energetics of reserpine binding and occlusion by the chromaffin granule biogenic amine transporter

The energetics of reserpine binding to the bovine adrenal biogenic amine transporter suggest that H+ ion translocation converts the transporter to a form which binds reserpine essentially irreversibly. Reserpine binding to bovine adrenal chromaffin granule membrane vesicles is accelerated by generation of a transmembrane pH difference (delta pH) (interior acid) or electrical potential (delta psi) (interior positive). Both components of the electrochemical H+ potential (delta mu H+) must be dissipated to block reserpine binding, and generation of either one stimulates the binding rate. Reserpine binding is less dependent than amine transport on the delta pH, suggesting that translocation of fewer H+ ions is required to expose the high-affinity site than are required for net transport. Bound reserpine dissociates very slowly, if at all, from the transporter. Binding is stable to 1% cholate, 1.5% Triton X-100, 1 M SCN-, and 8 M urea, but sodium dodecyl sulfate (0.035%) and high temperatures (100 degrees C) released bound reserpine, indicating that binding is noncovalent. The results raise the possibility that the transporter, by translocating one H+ ion outward down its concentration gradient, is converted to a form that can either transport a neutral substrate molecule inward or occlude reserpine in a dead-end complex.

Reserpine binding to chromaffin granules suggests the existence of two conformations of the monoamine transporter

The binding of [3H]reserpine ([3H]RES) to purified bovine chromaffin granule membranes has been studied at low membrane concentration. Saturation isotherms indicated a dissociation equilibrium constant KD of 30 pM and a density of binding sites of 8 pmol/mg of protein at 30 degrees C. The association rate constant was 4.0 X 10(5) M-1 s-1, and the calculated dissociation rate constant was 1.2 X 10(-5) s-1, corresponding to a half-lifetime of about 16 h. Although this dissociation was too low to be measured directly, [3H]RES binding was indeed reversible since it was lost after addition of the detergent Triton X-100. Dihydrotetrabenazine (TBZOH) inhibited [3H]RES binding in a time-dependent manner, EC50 varying from 37 nM after a 1-h incubation to 600 nM after 16 h. On the contrary, [3H]RES binding inhibition by the substrate noradrenaline was time independent. It is proposed that the transporter exists in two different conformations which bind exclusively either tetrabenazine (TBZ) or RES and which are in equilibrium. The effects of detergents were consistent with this two-conformation model. The transporter solubilized by cholate bound [3H]TBZOH, but not [3H]RES. On the other hand, addition of cholate to membrane-bound [3H]RES solubilized the membrane without releasing the ligand from its binding site. It is proposed that the TBZ-binding conformation is obtained by solubilization with cholate and that RES stabilizes the RES-binding conformation, allowing its solubilization by this detergent.

Effects of the sulfhydryl reagent N-ethylmaleimide on reserpine binding to the catecholamine transporter in chromaffin granule membranes.

1. Catecholamines are transported into chromaffin granules via a carrier-mediated, active-transport process which is inhibited by micromolar concentrations of the sulfhydryl reagent, N-ethylmaleimide (NEM). Reserpine is a very potent, competitive inhibitor of the catecholamine transporter and can be used to investigate the characteristics of the catecholamine transporter. 2. The purpose of this study was to determine whether [3H]reserpine binding to the catecholamine transporter present in chromaffin granule membranes isolated from bovine adrenal glands was also inhibited by NEM and, if so, whether this was a direct or an indirect effect of NEM on the catecholamine transporter. 3. Both [3H]norepinephrine transport into and [3H]reserpine binding to the chromaffin granule ghosts isolated from bovine adrenal glands are inhibited by NEM, with IC50 values of 0.63 +/- 0.02 and 2.8 +/- 0.66 microM, respectively. 4. Mg and ATP protected both the [3H]norepinephrine transport into the ghosts and the [3H]reserpine binding to the transporter from inhibition by NEM, shifting the IC50 values to 260 +/- 43 and 120 +/- 29 microM, respectively. 5. NEM inhibition of the catecholamine transport and reserpine binding appears to be due to an action on the proton translocator associated with the Mg ATPase enzyme rather than a direct action on the catecholamine transporter since (a) the concentration of NEM required to inhibit formation of a membrane potential is similar to that required to inhibit [3H]norepinephrine transport into and [3H]reserpine binding to the ghosts and (b) Mg and ATP protected the proton translocation and [3H]norepinephrine transport into the ghosts, and [3H]reserpine binding to the ghosts, from inhibition by NEM.(ABSTRACT TRUNCATED AT 250 WORDS)