TY - JOUR

T1 - Purification and fluorescent labeling of the human serotonin transporter

AU - Rasmussen, Søren G F

AU - Gether, Ulrik

PY - 2005/3/8

Y1 - 2005/3/8

N2 - To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter was solubilized in digitonin followed by nickel affinity and subsequent concanavalin A chromatography. Using this procedure we were able to obtain an overall purification of 700-fold and a yield of approximately 0.1 mg/L of cell culture. The purified transporter displayed pharmacological properties similar to those of hSERT expressed in native tissues and in transfected cell lines. Fluorescent labeling of the purified transporter with the thiol-reactive fluorophore nitrobenxoxadiazol-iodoacetamide (IANBD) and Texas Red bromoacetamide preserved the pharmacological profile of FLAG-hSERT-12H. Collisional quenching experiments revealed that the aqueous quencher iodide was able to cause marked quenching of the fluorescence of the IANBD labeled transporter with a K(SV) of 3.4 +/- 0.10 M(-)(1). In a mutant transporter with five cysteines mutated (5CysKO) we observed a significant reduction in this quenching (K(SV) = 2.1 +/- 0.16 M(-)(1), p <0.01). This reduction was most likely due to labeling of (109)Cys since mutation of this cysteine alone resulted in a reduction in collisional quenching that was similar to that observed with 5CysKO (K(SV) = 2.2 +/- 0.15 M(-)(1)). These data suggest that labeling of (109)Cys contributes substantially to the overall fluorescence of IANBD labeled FLAG-hSERT-12H. On the basis of these data we infer that (109)Cys is embedded in a mixed hydrophobic/hydrophilic environment at the external ends of transmembrane segments 1 and 2. Further use of fluorescent techniques on purified hSERT should prove useful in future studies aimed at understanding the molecular structure and function of Na(+)/Cl(-)-dependent neurotransmitter transporters.

AB - To establish a purification procedure for the human serotonin transporter (hSERT) we expressed in Sf9 insect cells an epitope-tagged version of the transporter containing a FLAG epitope at the N-terminus and a polyhistidine tail at the C-terminus (FLAG-hSERT-12H). For purification, the transporter was solubilized in digitonin followed by nickel affinity and subsequent concanavalin A chromatography. Using this procedure we were able to obtain an overall purification of 700-fold and a yield of approximately 0.1 mg/L of cell culture. The purified transporter displayed pharmacological properties similar to those of hSERT expressed in native tissues and in transfected cell lines. Fluorescent labeling of the purified transporter with the thiol-reactive fluorophore nitrobenxoxadiazol-iodoacetamide (IANBD) and Texas Red bromoacetamide preserved the pharmacological profile of FLAG-hSERT-12H. Collisional quenching experiments revealed that the aqueous quencher iodide was able to cause marked quenching of the fluorescence of the IANBD labeled transporter with a K(SV) of 3.4 +/- 0.10 M(-)(1). In a mutant transporter with five cysteines mutated (5CysKO) we observed a significant reduction in this quenching (K(SV) = 2.1 +/- 0.16 M(-)(1), p <0.01). This reduction was most likely due to labeling of (109)Cys since mutation of this cysteine alone resulted in a reduction in collisional quenching that was similar to that observed with 5CysKO (K(SV) = 2.2 +/- 0.15 M(-)(1)). These data suggest that labeling of (109)Cys contributes substantially to the overall fluorescence of IANBD labeled FLAG-hSERT-12H. On the basis of these data we infer that (109)Cys is embedded in a mixed hydrophobic/hydrophilic environment at the external ends of transmembrane segments 1 and 2. Further use of fluorescent techniques on purified hSERT should prove useful in future studies aimed at understanding the molecular structure and function of Na(+)/Cl(-)-dependent neurotransmitter transporters.

KW - Amino Acid Sequence

KW - Animals

KW - Cloning, Molecular

KW - Cysteine

KW - Fluorescence Polarization

KW - Fluorescent Dyes

KW - Genetic Vectors

KW - Humans

KW - Hydrophobic and Hydrophilic Interactions

KW - Ligands

KW - Membrane Glycoproteins

KW - Membrane Transport Proteins

KW - Molecular Sequence Data

KW - Mutagenesis, Site-Directed

KW - Nerve Tissue Proteins

KW - Oxadiazoles

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Rhodamines

KW - Serotonin Plasma Membrane Transport Proteins

KW - Spectrometry, Fluorescence

KW - Spodoptera

KW - Xanthenes

U2 - 10.1021/bi048022b

DO - 10.1021/bi048022b

M3 - Journal article

C2 - 15736959

VL - 44

SP - 3494

EP - 3505

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 9

ER -