Development and characterization of an immobilized human organic cation transporter based liquid chromatographic stationary phase

J Chromatogr B Analyt Technol Biomed Life Sci. 2005 Apr 25;818(2):263-8. doi: 10.1016/j.jchromb.2005.01.015.

Abstract

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.

Publication types

  • Comparative Study

MeSH terms

  • 1-Methyl-4-phenylpyridinium / metabolism
  • Animals
  • Binding Sites
  • Cell Line
  • Cell Membrane / metabolism
  • Chromatography, Liquid / methods*
  • Dogs
  • Humans
  • Organic Cation Transporter 1 / isolation & purification*
  • Organic Cation Transporter 1 / metabolism
  • Protein Binding
  • Stereoisomerism
  • Verapamil / metabolism

Substances

  • Organic Cation Transporter 1
  • Verapamil
  • 1-Methyl-4-phenylpyridinium