Expression of neurotransmitter transporters for structural and biochemical studies

Abstract

Neurotransmitter transporters play essential roles in the process of neurotransmission. Vesicular neurotransmitter transporters mediate storage inside secretory vesicles in a process that involves the exchange of lumenal H(+) for cytoplasmic transmitter. Retrieval of the neurotransmitter from the synaptic cleft catalyzed by sodium-coupled transporters is critical for the termination of the synaptic actions of the released neurotransmitter. Our current understanding of the mechanism of these transporters is based on functional and biochemical characterization but is lacking high-resolution structural information. Very few structures of membrane transport systems from mammalian origin have been solved to atomic resolution, mainly because of the difficulty in obtaining large amounts of purified protein. Development of high yield heterologous expression systems suitable for mammalian neurotransmitter transporters is essential to enable the production of purified protein for structural studies. Such a system makes possible also the production of mutants that can be used in biochemical and biophysical studies. We describe here a screen for the expression of the vesicular monoamine transporter 2 (VMAT2) in cell-free and baculovirus expression systems and discuss the expression of VMAT2 in other systems as well (bacterial, yeast and mammalian cell lines). After screening and optimization, we achieved high yield (2-2.5 mg/l) expression of functional VMAT2 in insect cells. The system was also used for the expression of three additional plasma membrane neurotransmitter transporters. All were functional and expressed to high levels. Our results demonstrate the advantages of the baculovirus expression system for the expression of mammalian neurotransmitter transporters in a functional state.

Fig 1. Cell-free expression of VMAT2

A. Native VMAT2 (lane 1), VMAT2 harboring silent mutations (lanes 2 and 3) and synthetic VMAT2 (lane 4) were synthesized by using the rapid-translation system 100 E. coli HY kit. Reaction was supplemented with ³⁵S-Methionine. Samples were separated on SDS-PAGE and radioactive bands were visualized with a FLA-3000 Phosphor-Imager (Fujifilm, Tokyo). B. His tagged VMAT2 (lane 1 N' tag, lane 2 C' tag) was synthesized using the rapid-translation system 100 Wheat Germ HY kit. Reaction was supplemented with ³⁵S-Methionine. Samples were separated on SDS-PAGE and radioactive bands were visualized with a FLA-3000 Phosphor-Imager. C. EAAC1-His synthesized in the rapid-translation system 100 E. coli HY kit was incubated with Ni-NTA beads. After purification and elution from the beads, sample (equivalent to half of the original reaction) was separated by SDS-PAGE and detected by Coomassie blue staining.

Fig 2. Expression screen in insect cells using three baculovirus vectors

A. Three baculovirus vectors were used for the infection of Sf9 cells: FB-VMAT2 (lane 1), FBG-VMAT2 (lane 2) and FBU-VMAT2 (lane 3). Sf9 cells were harvested 72 hrs post infection, washed in lysis buffer and sonicated. Samples of the lysates (equal to original volume of 10 μl infected cells in suspension) were separated by SDS-PAGE and Western blot analysis with anti-HA antibody was performed. B. Sf9 cells infected using the FB-VMAT2 vector were grown without or with the addition of glucose, Asn, Gln and FBS after the first 24 hours of infection. Cells were harvested 2,3,4 or 5 days (as indicated) post infection and cell lysates were prepared and assayed for [³H]TBZOH binding. The amount of VMAT2 was calculated from the TBZ binding results.

Fig 3. Expression and localization of VMAT2 expressed in insect cells

A. Immunocytochemistry: cells grown on cover slips at 50 % confluency were infected with FBG-VMAT2. Two days post-infection, cells were fixed with methanol at −20°C. Cover slips were incubated with mouse anti His₆ monoclonal antibody, followed by Texas-Red conjugated secondary IgG. Cells were observed with a confocal microscope. A. Differential interference contrast microscopy (DIC) image B. Fluorescence C. Merged image. Cells expressing the wild-type protein without His tag showed no background fluorescence. White bars represent 10 μm.

Fig 4. Expression and purification of VMAT2 expressed in insect cells

A. Sf9 cells were infected using the baculovirus FBG-VMAT2 vector (lanes 2 and 4). Samples were analyzed by SDS-PAGE and transferred onto PVDF membrane. Western blot was performed using anti-His (lanes 1–2) or anti HA (lanes 3–4) antibodies. Lanes 1 and 3 are un-infected control cells. B. Western analysis using anti-His antibody of Sf9 expressed native VMAT2 (lane 1) and GlyQ in which putative glycosylation sites have been mutated, as described under Material and Methods. C. Purification of Sf9 expressed native VMAT2. Sf9 membranes from 800 ml cells infected using FBG-VMAT2 vector were solubilized (lane 1, 0.5 μl from a total of 60 ml solution) and loaded onto NiNTA column (lane 2 unbound material, same amount as lane 1) and eluted with imidazole (lane 3, 5 μl from a total of 6 ml solution). rVMAT2 was further purified on a ConA column as described (lane 4, 2 μl from a total of 1.5 ml solution). Samples were analyzed on SDS-PAGE and stained with coomassie.

Fig 5. VMAT2 expressed in insect cells is functional

A. Binding of the noncompetitive inhibitor of VMAT2, [³H] TBZOH to lysates of Sf9 cells infected by FBG-VMAT2. Constant amount of cell lysate was bound to increasing [³H] TBZOH concentrations for 20 min, followed by fast dilution and filtration on 0.45 μm filters. The calculated Kd is 16.27±1.46 nM, and the Bmax is 1.1±0.03 pmole per 30 μl of original cell volume (R-square = 0.995; the experiment was performed in duplicates and repeated three times). B. Uptake of [³H]-serotonin in VMAT2 reconstituted in proteoliposomes. VMAT2 expressed in Sf9 cells was reconstituted into proteoliposomes as described under “materials and methods”. Proteoliposomes (2 μl samples) were diluted into 200 μl reaction buffer containing 50 nM valinomycin and 100nM [³H]-serotonin. Reaction was stopped at the indicated time points by fast dilution and filtration on 0.22 μm filters. The calculated Km for the transport of serotonin was 228±56 nM.

Fig 6. EAAC1, GLT1 and GAT1 expressed in insect cells are functional

A. Protein from Sf9 cells expressing EAAC1, GAT1 or GLT1 (5 ml each) was used for reconstitution of the transporters into brain lipids. Transport activity was assayed in the presence and absence of sodium as indicated. B. Partial purification of 5 ml cells expressing EAAC1 (lane 1), GAT1 (lane 2) and GLT1 (lane 3). Cell lysates were prepared, and protein was solubilized by addition of 2 % DDM. Solubilizate was bound to 30 μl NiNTA beads for 1 h, then eluted with 450 mM Imidazole, analyzed by SDS-PAGE and visualized with Coomassie stain.