Chimeric Vesicular Monoamine Transporters Identify Structural Domains That Influence Substrate Affinity and Sensitivity to Tetrabenazine

Abstract

The vesicular monoamine transporters (VMATs) 1 and 2 show close sequence similarity but substantial differences in apparent substrate affinity and drug sensitivity. To identify structural domains that determine these functional characteristics, chimeric transporters were constructed and their properties were analyzed in a heterologous expression system. The results implicate multiple regions in the recognition of serotonin and histamine and the sensitivity to tetrabenazine. Two domains of VMAT2, one extending from transmembrane domain (TMD) 5 to the beginning of TMD8 and the other from the end of TMD9 through TMD12, increase the affinity for serotonin and histamine as well as the sensitivity to tetrabenazine but only in the context of more C-terminal and more N-terminal VMAT2 sequences, respectively. In addition, the extreme N terminus of VMAT2 alone suffices to confer a partial increase in substrate affinity and tetrabenazine sensitivity. Despite these similarities among the interactions with serotonin, histamine, and tetrabenazine, the region of VMAT2 from TMD3 through TMD4 increases serotonin affinity but not histamine affinity or tetrabenazine sensitivity, and whereas the region from TMD5 to TMD8 of VMAT2 increases serotonin affinity in the context of more C-terminal VMAT2 sequences, the region encompassing TMD5 through TMD7 reduces serotonin but not histamine affinity or tetrabenazine sensitivity in the context of more N-terminal VMAT2 sequences. Thus, the chimeric analysis also reveals differences between serotonin recognition and the recognition of both histamine and tetrabenazine that may account for the observed differences in their interaction with the transport protein.

Peptide Mapping of the [125I]Iodoazidoketanserin and [125I]2-N-[(3′-Iodo-4′-azidophenyl)propionyl]tetrabenazine Binding Sites for the Synaptic Vesicle Monoamine Transporter

“…The model is also supported by the work performed using chimeric VMAT1/VMAT2 proteins (44). Replacing TMD1 of VMAT1 with TMD1 of VMAT2 increased the affinity of the transporter for inhibitors and substrates to an intermediate level between VMAT1 and VMAT2.…”

Vesicular monoamine transporter 2: Role as a novel target for drug development

“…129 Experiments employing a chimera of VMAT1 and VMAT2 indicate that 2 domains, TMD5 through TMD8 and TMD9 through TMD12, cooperate to confer the high-affi nity interaction of VMAT2 with TBZ and histamine. 130 In addition, the domain encompassing TMD3 and TMD4 infl uences serotonin affi nity but not histamine affi nity or TBZ sensitivity. 130 The domain encompassing TMD5 through TMD7 of VMAT2 in the context of N-terminal VMAT2 sequences reduces the apparent affi nity for serotonin but not histamine or the sensitivity to TBZ.…”

“…130 In addition, the domain encompassing TMD3 and TMD4 infl uences serotonin affi nity but not histamine affi nity or TBZ sensitivity. 130 The domain encompassing TMD5 through TMD7 of VMAT2 in the context of N-terminal VMAT2 sequences reduces the apparent affi nity for serotonin but not histamine or the sensitivity to TBZ. 130 Tyrosine 434 and aspartate 461 in TMD9 through TMD12 are identifi ed as being responsible for the high-affi nity interaction of TBZ, histamine, and serotonin, but not for DA.…”

“…130 The domain encompassing TMD5 through TMD7 of VMAT2 in the context of N-terminal VMAT2 sequences reduces the apparent affi nity for serotonin but not histamine or the sensitivity to TBZ. 130 Tyrosine 434 and aspartate 461 in TMD9 through TMD12 are identifi ed as being responsible for the high-affi nity interaction of TBZ, histamine, and serotonin, but not for DA. 131 Photoaffi nity labeling of purifi ed rat VMAT2 indicates that TMD1 and TMD10/11 are possibly juxtaposed and may interact in a functionally signifi cant manner.…”

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

“…To identify domains responsible for these differences, we first expressed and analyzed a series of chimeric transport proteins (8). These chimeras retained function, and analysis showed that two major regions from VMAT2, one extending from transmembrane domain (TMD) 5 to the beginning of TMD8 (TMD5-8) and the other from the end of TMD9 through TMD12 (TMD9 -12), are apparently both required for the high affinity interactions characteristic of VMAT2 (8).…”