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 ⌬ H ؉, but serotonin failed to inhibit reserpine binding, suggesting that the charge reversal specifically disrupts substrate recognition.Storage of classical neurotransmitters in secretory vesicles provides a mechanism for regulated release by exocytosis. Since neurotransmitters appear in the cytoplasm after either synthesis or reuptake, storage in vesicles depends on transport from the cytoplasm, and several distinct transport activities have been identified (1, 2). Transport into chromaffin granules has served as a model system to investigate the mechanism of vesicular amine transport. This transport activity recognizes multiple monoamine neurotransmitters (dopamine, norepinephrine, and serotonin) with similar affinity and uses a proton electrochemical gradient (⌬ H ϩ, interior positive or acid) across the vesicle membrane to drive uptake, exchanging two protons inside the vesicle for one cytoplasmic amine (3, 4). Despite the suggestion that a single protein is responsible for transport in both the brain and the periphery, two vesicular monoamine transporters (VMATs) 1 have recently been identified by expression cloning (5-7). VMAT1 occurs in the adrenal gland, whereas VMAT2 is expressed in the central nervous system (8, 9).The VMAT cDNAs protect against the parkinsonian toxin MPP ϩ , presumably because transport of the toxin into secretory vesicles sequesters it away from its primary site of action in mitochondria. Supporting a role in detoxification, the Nterminal half of VMATs shows weak sequence similarity to a bacterial multidrug-resistance transpo...

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.

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