1 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet2 Eyepath Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet3 Neuropharm and Genetics Lab, Department of Neuroscience and Pharmacology, Faculty of Health and Medical Sciences, Københavns Universitet
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.
Biochemistry, 2005, Vol 44, Issue 9, p. 3494-505
Amino Acid Sequence; Animals; Cloning, Molecular; Cysteine; Fluorescence Polarization; Fluorescent Dyes; Genetic Vectors; Humans; Hydrophobic and Hydrophilic Interactions; Ligands; Membrane Glycoproteins; Membrane Transport Proteins; Molecular Sequence Data; Mutagenesis, Site-Directed; Nerve Tissue Proteins; Oxadiazoles; Protein Binding; Protein Structure, Tertiary; Rhodamines; Serotonin Plasma Membrane Transport Proteins; Spectrometry, Fluorescence; Spodoptera; Xanthenes