Molecular Pharmacology of the Monoamine Transporter of the Chromaffin Granule Membrane (original) (raw)
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Biochemistry and molecular biology of the vesicular monoamine transporter from chromaffin granules
The Journal of experimental biology, 1994
Prior to secretion, monoamines (catecholamines, serotonin, histamine) are concentrated from the cytoplasm into vesicles by vesicular monoamine transporters (VMAT). These transporters also carry non-physiological compounds, e.g. the neurotoxin methyl-4-phenylpyridinium. VMAT acts as an electrogenic antiporter (exchanger) of protons and monoamines, using a proton electrochemical gradient. Vesicular transport is inhibited by specific ligands, including tetrabenazine, ketanserin and reserpine. The mechanism of transport and the biochemistry of VMAT have been analyzed with the help of these tools, using mainly the chromaffin granules from bovine adrenal glands as a source of transporter. Although biochemical studies did not suggest a multiplicity of VMATs, two homologous but distinct VMAT genes have recently been cloned from rat, bovine and human adrenal glands. The VMAT proteins are predicted to possess 12 transmembrane segments, with both extremities lying on the cytoplasmic side. They...
The vesiicular monoamine transporter: from chromaffin granule to brain
Neurochemistry International, 1998
All characterized monoaminergic cells utilize the same transport system for the vesicular accumulation of monoamines prior to their release. This system operates in neuronal (catecholaminergic, serotoninergic or histaminergic) as well as in endocrine or neuroendocrine cells. For several decades, chromaffin granules from bovine adrenal medulla have been used as a model system, allowing progress in the understanding of the biophysics, the biochemistry and the pharmacology of the monoamine vesicular transporter. The transporters from rat, bovine and man have been cloned. Surprisingly, two genes encode different isoforms of the protein which are differentially expressed in monoaminergic systems. The conjunction of recombinant DNA techniques and expression m secretory or non-secretory cells with the large body of data obtained on the chromaffin granule transporter has allowed rapid progress in the study of the protein. But interestingly enough, this progress has open new possibilities in the study of biological problems, especially in the brain. The transporter is useful for the determination of the relationship between small and large dense core vesicles, for the understanding of the mechanism of drugs such as 1-methyl-4-phenylpyridinium (Mpp+), tetrabenazine or amphetamines, and as a marker in brain development. The possibility of regulations at the vesicular transporter level and of their effect on the quantum size has to be investigated. The vesicular monoamine transporter is also an important target for brain imaging.
Proceedings of the National Academy of Sciences, 1996
A second isoform of the human vesicular monoamine transporter (hVMAT) has been cloned from a pheochromocytoma cDNA library. The contribution of the two transporter isoforms to monoamine storage in human neuroendocrine tissues was examined with isoform-specific polyclonal antibodies against hVMAT1 and hVMAT2. Central, peripheral, and enteric neurons express only VMAT2. VMAT1 is expressed exclusively in neuroendocrine, including chromaffin and enterochromaffin, cells. VMAT1 and VMAT2 are coexpressed in all chromaffin cells of the adrenal medulla. VMAT2 alone is expressed in histamine-storing enterochromaffin-like cells of the oxyntic mucosa of the stomach. The transport characteristics and pharmacology of each VMAT isoform have been directly compared after expression in digitonin-permeabilized fibroblastic (CV-1) cells, providing information about substrate feature recognition by each transporter and the role of vesicular monoamine storage in the mechanism of action of psychopharmacologic and neurotoxic agents in human. Serotonin has a similar affinity for both transporters. Catecholamines exhibit a 3-fold higher affinity, and histamine exhibits a 30-fold higher affinity, for VMAT2. Reserpine and ketanserin are slightly more potent inhibitors of VMAT2-mediated transport than of VMAT1mediated transport, whereas tetrabenazine binds to and inhibits only VMAT2. N-methyl-4-phenylpyridinium, phenylethylamine, amphetamine, and methylenedioxymethamphetamine are all more potent inhibitors of VMAT2 than of VMAT1, whereas fenfluramine is a more potent inhibitor of VMAT1-mediated monamine transport than of VMAT2mediated monoamine transport. The unique distributions of hVMAT1 and hVMAT2 provide new markers for multiple neuroendocrine lineages, and examination of their transport properties provides mechanistic insights into the pharmacology and physiology of amine storage in cardiovascular, endocrine, and central nervous system function.
Expression of a bovine vesicular monoamine transporter in COS cells
FEBS Letters, 1994
Catecholamines are accumulated in vesicles by a proton gradient-dependent transport, which has mostly been studied in bovine chromaffin granules. The full sequence of a cDNA encoding a vesicular transporter from bovine chromaffi cells, bVMAT*, was recently reported. We now present an analysis of bVMAT,, expressed in transfected COS cells. Comparing the binding of a labelled ligand, ['HITBZOH, and the rate of uptake, we find a much lower molecular turnover number than in chromaffin granules, probably indicating that a majority of expressed transporters are correctly folded and possess the ligand binding site but cannot actively transport monoamines because they are located in compartments which do not possess a proton gradient. The substrate specificity of uptake and its pharmacological sensitivity to various inhibitors closely resemble those previously observed in chromaffin granules. These results suggest that VMAT, is the major transporter in bovine adrenal glands, and raise the question of the significance of the second related transporter, VMAT,, which is also expressed in this tissue.
Proceedings of the National Academy of Sciences, 1983
Catecholamines are transported into chromaffin granules by a Mg2+/ATP-driven process under conditions in which the substrate exists primarily as a positively charged or neutral species. In order to distinguish between these two states, we studied the transport properties of a permanently charged quaternary analogue of epinephrine, (R,S)-dimethylepinephrine. We found that this compound was a classical competitive inhibitor of (R)-[3H]norepinephrine uptake, with a K; of 3.8 mM for the racemic form, or 1.9 mM for the R form. However, the [3H]dimethylepinephrine was not transported at all into granules. Our control for steric hindrance as an explanation for deficient translocation was analysis of the transport properties of isoproterenol, a secondary catecholamine with an isopropyl group around the amine residue. (R)-Isoproterenol was an effective competitive inhibitor of (R)-[3H]norepinephrine transport, with a K; of 91 ,uM. In contrast to dimethylepinephrine, (RS)-[3H]isoproterenol was clearly translocated across the granule membrane, with a Km of 123 aiM, or 61.5 IAM for the R isomer. Thus, the positive charge on dimethylepinephrine and not the size of the amine moiety appeared to be responsible for the lack of translocation. We interpret these data to indicate that, although the positively charged species can interact with the transport site, an uncharged species is the one actually transported. Catecholamines are transported into chromaffin granules by a Mg2+/ATP-driven process, which has become increasingly attractive as a general and simple system to analyze chemiosmotic energy coupling to transport (1-5). Many models have been proposed to explain the process, but distinguishing among them has been made difficult by lack of knowledge of whether the neutral, cationic, or anionic catecholamine species is the true substrate (4, 6-9). Recently, on the basis of observations that both the binding constants for dopamine and serotonin and the calculated cationic fractions were constant between pH 6.8 and 7.6, Knoth et al. (10) concluded that the cationic species was translocated. However, using a similar experimental approach, Scherman and Henry (11) came to the opposite conclusion. They observed that the Km for norepinephrine declined between pH 6.5 and 8.5, and they interpreted this as due to an increase in the concentration of the uncharged species, the proper substrate. Yet, on the basis of entirely different considerations, Johnson and Scarpa (7) initially claimed that the species transported was most likely neutral, and more recently summarized by saying that a conclusion was not yet possible (8). We therefore decided to reinvestigate this question directly
Functional Identification and Molecular Cloning of a Human Brain Vesicle Monoamine Transporter
Journal of Neurochemistry, 1993
A vesicle monoamine transporter was functionally identified, molecularly cloned, and characterized from a human substantia nigra cDNA library. The ATP-dependent transport of 5-[3H)hydroxytryptamine (t3H]5-HT) by digitonin-permeabilized fibroblasts expressing the vesicle monoamine/H+ antiporter in culture exhibited a K, of 0.55 pM. Reserpine and tetrabenazine, inhibitors of two rnonoamine binding sites, effectively blocked r3H]5-HT accumulation with K, values of 34 and 78 nM, respectively. Pretreatment of cells with as little as 10 nM reserpine in the presence of ATP abolished uptake. The rank order for substrate inhibition of 13H]5-HT uptake for both the previously reported rat vMATl and the human transporter clone followed the order 5-HT > dopamine > epinephrine > norepinephrine > 1-methyl-4-phenylpyridinium > 2-phenylethylarnine > histamine. The virtually identical transport characteristics of rvMAT1 and hvMAT1 confirm the relevance of neuropharrnacological studies of rat brain biogenic amine uptake and storage to human brain neurochernistry. Key Words: Human vesicle monoamine transporter type I-Vaccinia virus/SPG bacteriophage hybrid expression-Digitonin permeabilization-Reserpine-Tetrabenazine-Biogenic amines-MPP+ .