Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase.
Membranes from a stably transfected cell line that expresses the human organic cation 1 transporter (hOCT1) have been immobilized on the immobilized artificial membrane (IAM) liquid chromatographic stationary phase to form the hOCT1(+)-IAM stationary phase. Membranes from the parent cell line that does not express the hOCT1 were also immobilized to create the hOCT1(-)-IAM stationary phase. Columns were created using both stationary phases, and frontal displacement chromatography experiments were conducted using [(3)H]-methyl phenyl pyridinium ([(3)H]-MPP(+)) as the marker ligand and MPP(+), verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The K(d) values calculated from the chromatographic studies correlated with previously reported K(i) values (r(2)=0.9987; p<0.001). The data indicate that the hOCT1(+)-IAM column can be used for the on-line determination of binding affinities to the hOCT1 and that these affinities are comparable to those obtained using cellular uptake studies. In addition, the chromatographic method was able to identify a previously undetected high affinity binding site for MPP(+) and to determine that hOCT1 bound (R)-verapamil to a greater extent than (S)-verapamil.
Moaddel
R
R
Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224, USA. moaddelru@grc.nia.nih.gov
Yamaguchi
R
R
Ho
P C
PC
Patel
S
S
Hsu
C-P
CP
Subrahmanyam
V
V
Wainer
I W
IW
D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D004285:Dogs; D006801:Humans; D027702:Organic Cation Transporter 1 / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding; D013237:Stereoisomerism; D014700:Verapamil / Q000378:metabolism
D027702:Organic Cation Transporter 1; D014700:Verapamil; D015655:1-Methyl-4-phenylpyridinium