No abstract

Specific transport proteins package classical neurotransmitters into vesicles so that their release can be regulated by neural activity. Previous studies have suggested that a single activity mediates the vesicular transport of monoamines in the adrenal gland, brain, and other tissues such as mast cells and platelets. However, molecular cloning has recently identified two vesicular transporters for monoamines. Although the predicted proteins are closely related in sequence, they show a range of differences in their physiologic and pharmacologic properties. To clarify further the biological significance of the observed functional differences, we have generated anti-peptide antibodies to the C-termini of the two transporters and used them to determine the distribution and localization of the proteins in the rat. We have detected expression of vesicular monoamine transporter 1 (VMAT1) in adrenal chromaffin cells but not in neural cells. Interestingly, some adrenal chromaffin cells also express VMAT2 but the amount of VMAT2 relative to VMAT1 appears much lower than in the bovine adrenal gland. In contrast, sympathetic ganglion cells express only VMAT2, as do enteric neurons and enterochromaffin-like cells of the stomach. Thus, although adrenal chromaffin cells, sympathetic and enteric neurons derive from the neural crest, they express different vesicular amine transporters. In the CNS, dopamine, norepinephrine, epinephrine, 5-HT, and histamine cell groups all express VMAT2. These findings are consistent with the functional characteristics of VMAT1 and VMAT2 and help to explain several classic pharmacological observations. VMAT2-immunoreactivity is generally stronger in cell bodies, proximal dendrites and axonal processes, indicating the potential for monoamine storage at each of these sites. Surprisingly, dopaminergic interneurons in the olfactory bulb show no detectable immunoreactivity for either VMAT1 or VMAT2.

In central neurons, monoamine neurotransmitters are taken up and stored within two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles (DCVs). Biochemical and pharmacological evidence has shown that this uptake is mediated by specific vesicular monoamine transporters (VMATs). Recent molecular cloning techniques have identified the vesicular monoamine transporter (VMAT2) that is expressed in brain. This transporter determines the sites of intracellular storage of monoamines and has been implicated in both the modulation of normal monoaminergic neurotransmission and the pathogenesis of related neuropsychiatric disease. We used an antiserum against VMAT2 to examine its ultrastructural distribution in rat solitary tract nuclei, a region that contains a dense and heterogeneous population of monoaminergic neurons. We find that both immunoperoxidase and immunogold labeling for VMAT2 localize to DCVs and small synaptic vesicles in axon terminals, the trans-Golgi network of neuronal perikarya, tubulovesicles of smooth endoplasmic reticulum, and potential sites of vesicular membrane recycling. In axon terminals, immunogold labeling for VMAT2 was preferentially associated with DCVs at sites distant from typical synaptic junctions. The results provide direct evidence that a single VMAT is expressed in two morphologically distinct types of regulated secretory vesicles in central monoaminergic neurons.The uptake of monoamines into the secretory vesicles of neurons and adrenal chromaffin cells is mediated by specific vesicular monoamine transporters (VMATs) (1). These monoamine transporters use the proton electrochemical gradient to drive monoamine uptake and are potently inhibited by reserpine and tetrabenazine (1). In the rat, two distinct but closely related proteins with these pharmacological characteristics have been identified: one that is detected in chromaffin cells (VMAT1 or CGAT) and one that is expressed in neurons and other cells (VMAT2 or SVAT) (2-4). Recent evidence has suggested that the VMATs may also play a role in neuroprotection since they have the capacity to sequester neurotoxins into vesicles or other acidic intracellular compartments (3, 5, 6). As a result, the VMATs have been implicated both in the modulation of normal monoaminergic neurotransmission and in the pathogenesis of neuropsychiatric disease (7).To determine the potential sites of intracellular storage of monoamines and sequestration of neurotoxins, we examined the in vivo distribution of the VMAT that is expressed in central neurons. We used an anti-peptide antiserum to characterize the ultrastructural localization of VMAT2 in the nuclei of the solitary tract (NTS), which contain a dense and heterogeneous population of monoaminergic neurons (8). We used two distinct immunocytochemical markers: the avidinbiotin immunoperoxidase complex, a highly sensitive technique that maximizes the detection of low-abundance, membrane-bound antigens (9); and immunogold-silver, a less sensitive but p...

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 aspartate 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 ⌬ H ؉-induced acceleration of reserpine binding. This indicates that substrate recognition can be separated from coupling to the driving force.

The vesicular monoamine transporters (VMATs) package monoamine neurotransmitters into secretory vesicles for regulated exocytotic release. One isoform occurs in the adrenal gland (VMAT1) and another in the brain (VMAT2). To assess their potential for regulation, we have investigated the phosphorylation of the VMATs. Using heterologous expression in Chinese hamster ovary, PC12, and COS cells, we find that rat VMAT2, but not VMAT1, is constitutively phosphorylated. Phosphoamino acid analysis indicates that this phosphorylation occurs on serine residues, and the analysis of VMAT1-VMAT2 chimeras and site-directed mutagenesis localize the phosphorylation sites to serines 512 and 514 at the carboxyl terminus of VMAT2. Since these residues occur in an acidic region, we tested the ability of the acidotropic kinases casein kinase I (CKI) and casein kinase II (CKII) to phosphorylate bacterial fusion proteins containing the carboxyl terminus of VMAT2. Purified CKI and CKII phosphorylate the wild-type carboxyl terminus of VMAT2, but not a double mutant with both serines 512 and 514 replaced by alanine. The protein kinase inhibitor CKI-7 and unlabeled GTP both block in vitro phosphorylation by cell homogenates, indicating a role for CKII and possibly CKI in vivo. Both kinases phosphorylate the VMAT2 fusion protein to a much greater extent than a similar fusion protein containing the carboxyl terminus of VMAT1, consistent with differential phosphorylation of the two transporters observed in intact cells. These results provide the first demonstration of phosphorylation of a vesicular neurotransmitter transporter and a potential mechanism for differential regulation of the two VMATs.