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

Abstract

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.

Synaptic Vesicle Protein 2 Binds Adenine Nucleotides

“…Expression of SV2 in HEK293 fibroblasts did not increase ATP uptake into mitochondria-free membrane preparations, indicating that SV2 neither is an ATP transporter nor affects ATP transport. Our results can be contrasted to the effect of exogenous expression of the vesicular monoamine transporter (26,27) or of the vesicular acetylcholine transporter (28), both of which increase uptake of their substrates into microsomes. We also examined ATP uptake into synaptic vesicle preparations.…”

Emulating proton-induced conformational changes in the vesicular monoamine transporter VMAT2 by mutagenesis

“…Thus, we tested the possibility that protonation of one of the essential membrane-embedded carboxyl residues in rVMAT2 allows the conformational change necessary for reserpine binding. D33 (TM1) and E313 (TM7) are both highly conserved within the DHA12 subfamily (34), and have both been found to be essential for transport activity (29,35). Moreover, we recently demonstrated the importance of the equivalent residues in a bacterial homolog of rVMAT2 (34), and highlighted them both as putative proton-binding sites.…”

Functionally Important Carboxyls in a Bacterial Homologue of the Vesicular Monoamine Transporter (VMAT)

“…The corresponding positions in rVMAT2 (Asp-33 and Glu-313) are also important for transport activity, although their precise role is unclear. Specifically, when Asp-33 is mutated to Asn, practically no transport activity could be detected, but ⌬ Hϩ -dependent reserpine binding was unaffected (58). This indicates that the mutant protein is still coupled to at least one proton (59).…”

“…This indicates that the mutant protein is still coupled to at least one proton (59). Moreover, serotonin failed to inhibit reserpine binding to the mutated transporter, suggesting a role for Asp-33 in substrate binding as well (58).…”