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Development of an immobilized mutant organic cation transporter column for on‐line screening using chromatographic techniques
Author(s) -
Moaddel Ruin,
Bighi Federica,
Yamaguchi Rika,
Patel Sharvil,
Wainer Irving W
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.a1142-c
Subject(s) - organic cation transport proteins , chemistry , chromatography , elution , membrane , verapamil , mutant , yield (engineering) , transfection , transporter , stationary phase , high performance liquid chromatography , ligand (biochemistry) , receptor , biochemistry , materials science , gene , organic chemistry , metallurgy , calcium
Purpose To develop and validate and immobilize in a liquid chromatographic column format two point mutations of the human Organic Cation Transporters (R488M and G465R). Methods The membranes used in the study were obtained from a transfected MDCK cell line that expressed hOCT1‐R488M or G465R and hOCT1‐transfected MDCK cells [hOCT(+)]. The cells were homogenized, solubilized, and subsequently mixed with 160 mg of immobilized artificial membrane (IAM) stationary phase and the resulting mixture was dialyzed for 2 days. The resulting stationary phase mixture was packed into a HR 5/2 glass column to yield a 150 mm x 5 mm (ID) chromatographic bed. The column was placed in a HPLC system containing an on‐line scintillation detector. A series of nine displacer ligands with [ 3 H]‐MPP as the marker ligand were run through the R488M and the G465R column to obtain elution profiles showing a front and plateau regions. Results The R488M point mutation resulted in a change of the binding properties of the catechols and failed to exhibit the enantioselectivity observed for verapamil on the hOCT1 transporter, while the G465R exhibited similar binding properties as the hOCT1 however resulted in a non‐functional transporter. Conclusions The results suggest that the R488M mutation is not located at the transport site but in the vicinity of this site, while the G465R is believed to be in the transport site.