Directed evolution reveals hidden properties of VMAT, a neurotransmitter transporter

Abstract

The vesicular neurotransmitter transporter VMAT2 is responsible for the transport of monoamines into synaptic and storage vesicles. VMAT2 is the target of many psychoactive drugs and is essential for proper neurotransmission and survival. Here we describe a new expression system in Saccharomyces cerevisiae that takes advantage of the polyspecificity of VMAT2. Expression of rVMAT2 confers resistance to acriflavine and to the parkinsonian toxin 1-methyl-4-phenylpyridinium (MPP(+)) by their removal into the yeast vacuole. This expression system allowed identification of a new substrate, acriflavine, and isolation of mutants with modified affinity to tetrabenazine (TBZ), a non-competitive inhibitor of VMAT2 that is used in the treatment of various movement disorders including Tourette syndrome and Huntington chorea. Whereas one type of mutant obtained displayed decreased affinity to TBZ, a second type showed only a slight decrease in the affinity to TBZ, displayed a higher K(m) to the neurotransmitter serotonin, but conferred increased resistance to acriflavine and MPP(+). A protein where both types of mutations were combined (with only three amino acid replacements) lost most of the properties of the neurotransmitter transporter (TBZ-insensitive, no transport of neurotransmitter) but displayed enhanced resistance to the above toxicants. The work described here shows that in the case of rVMAT2, loss of traits acquired in evolution of function (such as serotonin transport and TBZ binding) bring about an improvement in older functions such as resistance to toxic compounds. A process that has taken millions of years of evolution can be reversed by three mutations.

FIGURE 1.

rVMAT2 confers resistance against MPP⁺ and acriflavine, which is fully reversed in the presence of TBZ and reserpine. ADU1-7 cells harboring pAES426 with or without rVMAT2 were grown in minimal medium and diluted to comparable densities. After serial dilution, they were plated (5 μl) on rich solid medium (C) supplemented with 1.5 mm MPP⁺ (A), or 40 μm acriflavine (B). Where indicated, the plates contained 2 μm TBZ or 0.5 μm reserpine. Growth was analyzed after two overnights incubation at 30 °C. Growth in liquid media was performed as described under “Experimental Procedures” with increasing concentrations of acriflavine (D) or MPP⁺ (E), IC₅₀ values were calculated from fits of the data obtained by Origin 8 software (the lowest R-square value is 0.98).

FIGURE 2.

rVMAT2-GFP is localized mainly to the vacuolar membrane. ADU1-7 cells expressing rVMAT2-GFP were grown overnight in minimal medium, stained with 10 μm FM4–64, washed, and visualized in a confocal microscope as described under “Experimental Procedures.” Yeast cells in DIC (A), the vacuole can be noticed easily after exposure to hypotonic solution; FM 4-64, red staining, specific to the vacuole membrane (B). rVMAT2-GFP is visible mainly in the vacuole membrane. Some cells also show GFP fluorescence in the endoplasmic reticulum (C). Scale bars are 4 μm.

FIGURE 3.

F136L alters rVMAT2 sensitivity toward TBZ in yeast cells. ADU1-7 cells harboring pAES426 rVMAT2 (A) or F136L (B) were grown in MPP⁺ and analyzed as in Fig. 1. Growth in liquid medium was performed with increasing concentrations of acriflavine. The calculated IC₅₀ values are shown (C). Sensitivity toward TBZ was determined in the presence of 100 μm acriflavine and increasing concentrations of the inhibitor (D). The lowest R-square value is 0.98.

FIGURE 4.

The effect of the mutation F136L on rVMAT2-mediated resistance. ADU1-7 cells harboring empty pAE426 vector (circles), rVMAT2 (squares), or F136L (triangles) were grown overnight in minimal medium and diluted in rich medium containing increasing concentrations of acriflavine (A) or 100 μm acriflavine with increasing concentrations of TBZ (B), ketanserine (C), or reserpine (D). Absorbance was measured at 600 nm after overnight growth at 30 °C. Each curve represents a fit obtained by Origin 8 software (the lowest R-square is 0.96). A representative experiment of at least three replicas is shown.

FIGURE 5.

The F136L mutation modifies the pharmacological profile of rVMAT2. Liposomes (1.5 μl) reconstituted with rVMAT2 (squares) or F136L (triangles) were assayed for serotonin transport as described under “Experimental Procedures.” The reaction was terminated by filtration, and the bound radioactivity was measured by scintillation counting. [³H]Serotonin transport was assayed in the presence of increasing concentrations of TBZ (A), acriflavine (B), MPP⁺ (C), and histamine (D) to determine the concentration required to inhibit transport by 50% (IC₅₀). IC₅₀ were calculated with Origin 8 software. For each compound, a representative experiment of three replicas is shown.

FIGURE 6.

Phenylalanine or tyrosine at position 136 confers high affinity binding of TBZ. ADU1-7 cells harboring pAES426 rVMAT2 with different substitutions at position 136 were grown overnight at 30 °C in minimal medium. 5-μl volumes of serial dilutions were spotted on rich solid medium supplemented with 1.5 mm MPP⁺ (A) and addition of 2 μm TBZ (B). Growth was analyzed after two overnight incubations at 30 °C.

FIGURE 7.

rVMAT2-I425F and V428A confer increased resistance to acriflavine. ADU1-7 cells harboring pAES426 rVMAT2, I425F, or V428A were grown on plates containing YPD (A), YPD with 40 μm acriflavine (B), YPD with 40 μm acriflavine and 2 μm TBZ (C), or YPD with 40 μm acriflavine and 100 nm reserpine (D). Growth in liquid media with different concentrations of acriflavine was analyzed after overnight incubation at 30 °C and is expressed as percent of growth of a culture without added acriflavine (E).

FIGURE 8.

rVMAT2-triple displays the phenotype of a multidrug transporter. Cells harboring empty pAES426 vector, rVMAT2, and rVMAT2-triple were tested for resistance on liquid medium with acriflavine (A), acridine orange (B), or ethidium bromide (C). Growth was analyzed after overnight incubation at 30 °C.

FIGURE 9.

Model of rVMAT2 and the cluster involved in TBZ binding. Charged residues facing the cavity and previously shown to play some role in function are highlighted (A). The helical projection of TM2 (B) and TM11 (C) is shown. Highlighted residues have been shown to modify rVMAT2 function or to provide targets for inhibitors.