Individual Residues Contribute to Multiple Differences in Ligand Recognition between Vesicular Monoamine Transporters 1 and 2

Abstract

Molecular cloning has identified two vesicular monoamine transporters (VMATs), one expressed in non-neural cells of the periphery (VMAT1) and the other by multiple monoamine cell populations in the brain (VMAT2). Functional analysis has previously shown that VMAT2 has a higher affinity than VMAT1 for monoamine neurotransmitters as well as the inhibitor tetrabenazine. The analysis of chimeric transporters has also identified two major regions required for the high affinity interactions of VMAT2 with these ligands. We have now used site-directed mutagenesis to identify the individual residues responsible for these differences. Focusing on the region that spans transmembrane domains 9 through 12, we have replaced VMAT2 residues with the corresponding residues from VMAT1. Many residues in this region had no effect on the recognition of these ligands, but substitution of Tyr-434 with Phe and Asp-461 with Asn reduced the affinity for tetrabenazine, histamine, and serotonin. Although the ability to affect recognition of multiple ligands suggests a general structural role for these residues, the mutations did not affect dopamine recognition, indicating a more specific role, possibly in recognition of the ring nitrogen that occurs in tetrabenazine, histamine, and serotonin but not dopamine. The mutation K446Q reduced the affinity of VMAT2 for tetrabenazine and serotonin but not histamine, whereas F464Y reduced serotonin affinity and perhaps histamine recognition but not tetrabenazine sensitivity, providing more evidence for specificity. Interestingly, the V max of both VMATs for dopamine exceeded that for serotonin by 3-5-fold, indicating a difference in the speed of packaging of these two neurotransmitters. We also found that VMAT1 has a higher affinity for tryptamine than VMAT2. This mutually exclusive interaction with serotonin and tryptamine also suggests a physiological rationale for the existence of two VMATs. Surprisingly, the residue responsible for this difference, Tyr-434, also accounts for the higher affinity interaction of VMAT2 with tetrabenazine, histamine, and serotonin. Interestingly, replacement of Tyr-434 with alanine increases the affinity of VMAT2 for both serotonin and dopamine and reduces the rate of dopamine transport.

Directed Evolution Reveals Hidden Properties of VMAT, a Neurotransmitter Transporter

“…Cys-431 has been identified in human VMAT2 as a residue that confers sensitivity to thiolreactive reagents, but the reaction is prevented by TBZ (27). Mutations in Tyr-434 reduce the affinity to serotonin and TBZ (28,29). In TM2, Lys-139 has been identified as part of a charge pair with Asp-427 (24).…”

A Leucine-based Motif Mediates the Endocytosis of Vesicular Monoamine and Acetylcholine Transporters

“…cDNAs were cloned into pcDNA1/Amp (Invitrogen, Carlsbad, CA). To facilitate the subcloning of VMAT2 mutants, we used a cDNA with two silent mutations that create a BglII and a SalI site at nucleotides 440 and 1302, respectively (34). A sequence encoding the hemaglutinin epitope, YPYDVPDYA, was inserted after the codon for glycine 96 in the VMAT2 cDNA (35) and after the codon for glycine 105 in the VAChT cDNA (28).…”

Mitochondrial Complex I Inhibitor Rotenone Inhibits and Redistributes Vesicular Monoamine Transporter 2 via Nitration in Human Dopaminergic SH-SY5Y Cells

“…Therefore, using immunoprecipitation as another technique, we established that the nitration of tyrosine residues via VMAT2 was caused by rotenone and the reduced dopamine uptake activity of VMAT2. A previous study demonstrated that tyrosine 434 of VMAT2 plays an important role in dopamine affinity and the activity of dopamine transport (Finn and Edwards, 1997). The replacement of tyrosine 434 with alanine increases the affinity of VMAT2 for dopamine and reduces the rate of dopamine transport.…”