Identification of conformationally sensitive residues essential for inhibition of vesicular monoamine transport by the noncompetitive inhibitor tetrabenazine

Abstract

Vesicular monoamine transporter 2 (VMAT2) transports monoamines into storage vesicles in a process that involves exchange of the charged monoamine with two protons. VMAT2 is a member of the DHA12 family of multidrug transporters that belongs to the major facilitator superfamily of secondary transporters. Tetrabenazine (TBZ) is a non-competitive inhibitor of VMAT2 that is used in the treatment of hyperkinetic disorders associated with Huntington disease and Tourette syndrome. Previous biochemical studies suggested that the recognition site for TBZ and monoamines is different. However, the precise mechanism of TBZ interaction with VMAT2 remains unknown. Here we used a random mutagenesis approach and selected TBZ-resistant mutants. The mutations clustered around the lumenal opening of the transporter and mapped to either conserved proline or glycine, or to residues immediately adjacent to conserved proline and glycine. Directed mutagenesis provides further support for the essential role of the latter residues. Our data strongly suggest that the conserved α-helix breaking residues identified in this work play an important role in conformational rearrangements required for TBZ binding and substrate transport. Our results provide a novel insight into the mechanism of transport and TBZ binding by VMAT2.

Keywords: Directed Evolution; Ion-coupled Transporters; MFS Superfamily; Membrane Proteins; Multidrug Transporters; Neurotransmitter Transport; Neurotransmitters; PXXP Motifs.

FIGURE 1.

Gly³⁰⁸ functions as a pivotal molecular hinge required for TBZ binding. A, replacement of Gly³⁰⁸ by Ala (G308A) confers resistance to TBZ in yeast cells, whereas a Pro replacement rescues TBZ-resistant phenotype. ADU1–7 cells transformed with pAES426 (empty vector) or pAES426 harboring either rVMAT2 or Gly³⁰⁸ mutants were grown in minimal medium and diluted to comparable densities. 5 μl of serial dilutions of the culture were spotted on rich solid medium (YPD) supplemented with 1.5 mm MPP⁺ (upper panel), or 40 μm acriflavine (lower panel). Where indicated, the plates contained 2 μm TBZ. Growth was analyzed after 48 h at 30 °C. The plates are representative of at least three independent experiments. B, time course of [³H]serotonin ([³H]5HT) transport by Gly³⁰⁸ mutants reconstituted in proteoliposomes. The uptake assay was performed as described under “Experimental Procedures.” Ammonium-loaded proteoliposomes (1 μl) were diluted into 200 μl of reaction buffer to generate the pH gradient that drives serotonin transport and 50 nm valinomycin was added to prevent the generation of a membrane potential by the electrogenic exchange of 2H⁺ with one serotonin molecule. All data are mean ± S.E. of 2–3 experiments. C, multiple sequence alignment of members of the SLC18 family in the region of residues 304–312 (TM7). Multiple alignment was performed using Clustal Omega (38). A conservation logo was created using WebLogo 3.3 (39, 40). Only a fraction of the alignment corresponding to residues 304–312 is shown. D, kinetic properties of rVMAT2 and Gly³⁰⁸ mutants. Proteoliposomes were prepared from HEK293 cells expressing either rVMAT2 or Gly³⁰⁸ mutants. Protein determination and serotonin uptake in proteoliposomes and [³H]TBZOH binding were performed as described under ”Experimental Procedures.“ The “specificity constant” V_max/K_m was obtained by simply dividing the corresponding values given in the table and the units are those shown. TBZ sensitivity was assessed by calculating the amount of ligand required to inhibit serotonin transport by 50% (IC₅₀). The uptake assay was performed in the presence of increasing concentrations of TBZ (0–200 nm) for 20 min. Reserpine sensitivity was assessed by calculating the concentration required to inhibit serotonin transport by 50% (IC₅₀) (0–100 nm).

FIGURE 2.

Determination of protein amounts in proteoliposomes. A, typical result of the dot blot experiment. rVMAT2-WT overexpressed in SF9 cell and purified to homogeneity served as a standard and positive control. The dot blot was performed as described under “Experimental Procedures.” B, quantification of protein amounts in rVMAT2 mutants. The amount of protein is expressed as the percent of wild type. The results shown are representative and for specific batches used for determination of K_m and V_max throughout the paper.

FIGURE 3.

Chemical structure of selected substrates and inhibitors of wild type VMAT2. A, serotonin; B, dopamine; C, MPP⁺; D, acriflavine; E, tetrabenazine; F, reserpine.

FIGURE 4.

The highly conserved Pro³¹⁴ is crucial for TBZ binding and plays a role in the transport function of rVMAT2. A, replacement of Pro³¹⁴ by Leu or Thr confers resistance to TBZ in yeast cells. ADU1–7 cells were transformed with pAES426 (empty vector) or pAES426 harboring rVMAT2 and Pro³¹⁴ mutants. Growth was assayed and analyzed as described in the legend to Fig. 1A. B, time course of [³H]serotonin transport by Pro³¹⁴ mutants reconstituted in proteoliposomes. The uptake assay was performed as described in the legend to Fig. 1B. All data are mean ± S.E. of 2–3 experiments. C, conservation of Pro³¹⁴. The sequence of rVMAT2 was used to query the National Center for Biotechnology Information (NCBI) using the psi-BLAST tool as provided by the NCBI server with default parameters. Multiple sequence alignment and the conservation logo were created as described in the legend to Fig. 1C. Only a fraction of the alignment corresponding to residues 311–320 is shown. D, kinetic properties of Pro³¹⁴ mutants. Proteoliposomes were prepared from HEK293 cells expressing rVMAT2 or Pro³¹⁴ mutants. K_D for [³H]TBZOH binding, K_m, V_max for [³H]serotonin uptake, and IC₅₀ for reserpine and TBZ, were determined as described in the legend to Fig. 1D.

FIGURE 5.

Multiple sequence alignment of the members of SLC18 family. A, alignment in the region of residues 304–320 (TM7). B, alignment in the region of residues 27–47 (TM1). Multiple sequence alignment was performed as described in the legend to Fig. 1C. Pro³¹⁴ and Val⁴¹ are highlighted in black. PXXP domains are highlighted in gray.

FIGURE 6.

Substitution of the conserved amino acid Val⁴¹ by Phe impairs the pharmacological properties of rVMAT2. A, the V41F mutation confers resistance to TBZ and eliminates the ability to support growth on acriflavine. ADU1–7 cells were transformed with pAES426 (empty vector) or pAES426 harboring rVMAT2 or Val⁴¹ mutants. Where indicated, the plates contained 2 μm TBZ. Growth was assayed and analyzed as described in the legend to Fig. 1A. B, time course of [³H]serotonin transport by Val⁴¹ mutants reconstituted in proteoliposomes. The uptake assay was performed as described in the legend to Fig. 1B. C, conservation of Val⁴¹. A conservation logo was created as described in the legend to Fig. 4C. Only a fraction of the alignment corresponding to residues 37–45 is shown. D, kinetic properties of Val⁴¹ mutants. Proteoliposomes were prepared from HEK293 cells expressing rVMAT2 and Val⁴¹ mutants. K_D for [³H]TBZOH binding, K_m, V_max for [³H]serotonin uptake, and IC₅₀ for reserpine, were determined as described in the legend to Fig. 1D.

FIGURE 7.

Val¹³² is important for the binding and transport activity of rVMAT2. A, mutations of Val¹³² cause TBZ and reserpine-resistant phenotypes in yeast. ADU1–7 cells were transformed with pAES426 (empty vector) or pAES426 harboring rVMAT2 or Val¹³² mutants. Where indicated, the plates contained 2 μm TBZ or 0.1 μm reserpine. Growth was assayed and analyzed as described in the legend to Fig. 1A. B, time course of [³H]serotonin transport by Val¹³² mutants reconstituted in proteoliposomes. The uptake assay was performed as described in the legend to Fig. 1B, except that nonspecific accumulation of [³H]serotonin was measured in the presence of 15 μm nigericin, which disrupts the proton gradient, and was subtracted from the total transport. C, conservation of Val¹³². A conservation logo was created as described in the legend to Fig. 4C. Only a fraction of the alignment corresponding to residues 131–137 is shown. D, kinetic properties of Val¹³² mutants. Proteoliposomes were prepared from HEK293 cells expressing rVMAT2 and Val¹³² mutants. K_D for [³H]TBZOH binding, K_m, V_max for [³H]serotonin uptake, and IC₅₀ for reserpine were determined as described in the legend to Fig. 1D.

FIGURE 8.

[³H]Serotonin transport and [³H]TBZOH binding by Pro⁴² and Gly¹³³ mutants reconstituted in proteoliposomes. The uptake assay was performed as described in the legend to Fig. 1B, except that nonspecific accumulation of [³H]serotonin was measured in the presence of 15 μm nigericin, which disrupts the proton gradient, and was subtracted from the total transport. A and B, time course for the Pro⁴² and Gly¹³³ replacements, respectively; C, binding of [³H]TBZOH; D, kinetic constants of P42G and G133A.

FIGURE 9.

Clustering of the mutations in the lumenal (A) and cytoplasmic (B) facing model of rVMAT2. Helices are shown as schematics and viewed along the plane of the membrane with the cytoplasm to the bottom. The key helices, TM1, -2, and -7, are opaque and all the others are transparent.