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

J Patrick Finn,Robert H Edwards

doi:10.1074/jbc.273.7.3943

Abstract

The two closely related vesicular monoamine transporters (VMATs) 1 and 2 differ substantially in ligand recognition. The neuronal VMAT2 exhibits a higher affinity for monoamine substrates and in particular for histamine as well as a greater sensitivity to the inhibitor tetrabenazine than the nonneuronal VMAT1. The analysis of chimeric transport proteins has previously shown that two major domains, one spanning transmembrane domains (TMDs) 5-8 (TMD5-8) and the other, TMDs 9-12 (TMD9-12), are required for the high affinity interactions characteristic of VMAT2. Using site-directed mutagenesis to replace residues in TMD5-8 of VMAT2 with the equivalent residues from VMAT1, we now show that the sensitivity of VMAT2 to tetrabenazine requires Ala-315, and this interaction occurs independently of the interaction with residues in TMD9-12. The ability to recognize histamine as a substrate depends on Pro-237, and the contribution of TMD9-12 to histamine recognition appears to involve a common mechanism. In contrast, the replacement of many residues in TMD5-8 of VMAT2 with equivalent residues from VMAT1 improves the recognition of both serotonin and tryptamine, and these mutations show a dominant effect on the recognition of both tryptamine and serotonin over mutations in TMD9-12. The results indicate that different ligands interact through distinct mechanisms with the VMATs and that the recognition of each ligand involves multiple, independent interactions with the transport protein.

Highlights

Neurotransmitters are packaged into secretory vesicles so that their release can be regulated by exocytosis
We have extended the analysis to TMD5– 8 in VMAT2
Tetrabenazine Sensitivity—The majority of VMAT1 substitutions introduced into TMD5– 8 of VMAT2 do not influence the sensitivity to tetrabenazine as measured by the IC50 for inhibition of [3H]serotonin transport (Table I)

Summary

EXPERIMENTAL PROCEDURES

Mutagenesis—Mutations were introduced into wild-type VMAT2 using single-stranded, uracil-containing DNA as described previously [9, 10]. The fragments were subcloned back into appropriate wild-type cDNAs. Transient Expression and Membrane Preparation—Plasmid DNA was prepared, COS1 cells were transfected, and membranes were isolated for analysis as described previously [9]. A frozen aliquot of membranes was thawed on ice, and 10 ␮l (50 –100 ␮g of protein) was added to 200 ␮l of SH buffer at 29 °C containing 4 mM KCl, 2 mM MgSO4, 2.5 mM ATP, and either 20 nM [1,2-3H]serotonin (NEN Life Science Products), 20 nM [2,5,6-3H]dopamine (NEN Life Science Products ), or 29 nM [2,5-3H]histamine (Amersham Life Science, Inc.). To determine the Km for dopamine and serotonin, a range of nonradioactive substrate concentrations was added to the reaction solution, and transport was measured after incubation for 1 min at 29 °C. The protein concentration of each membrane sample was measured using a Bradford assay (Bio-Rad), and the unpaired two-tailed t test was used for all statistical comparisons

RESULTS AND DISCUSSION

62 Ϯ 12 193 Ϯ 48a 159 Ϯ 9a 158 Ϯ 39a 119 Ϯ 29a

33 Ϯ 14 159 Ϯ 9a 119 Ϯ 29a