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

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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 [3H]-methyl phenyl pyridinium ([3H]-MPP+) as the marker ligand and MPP+, verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The Kd values calculated from the chromatographic studies correlated with previously reported Ki values (r2 = 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.

Introduction

Transport proteins are found in the liver, kidney and intestines and play an essential role in the metabolism and excretion of endogenous and exogenous compounds [1], [2], [3], [4]. The solute carrier (SLC) transporters have 255 members in humans, the majority are highly specific transporters, however, some of the superfamilies are polyspecific.

Two polyspecific transporters superfamilies that are of particular interest in the drug development process are the SLC21 and SLC22 superfamilies. The SLC21 superfamily (organic anion transporter) is composed of nine members, which are involved in the transport of large anionic, amphipathic compounds. The SLC22 superfamily (major facilitator superfamily) [2] has 12 members in humans and rats including the organic cation transporters OCT1, OCT2 and OCT3, the carnitine transporter, and several organic anion transporters. The rOCT1 has been isolated from rats and shown to consist of 556 amino acid residues with 12 transmembrane domains and three glycosylation sites on the extracellular loop between the first and second transmembrane domain [3], [4].

OCTs are believed to mediate the bidirectional transport of small organic cations (50–350 amu) such as tetraethyl ammonium (TEA) and 1-methyl-4-phenyl pyridinium (MPP+) [1], [3]. Other compounds have been shown to bind to the human OCT1 (hOCT1) without being transported, and have been shown to act as transport inhibitors. These compounds include verapamil, quinidine, quinine, disopyramide and dopamine [1].

A key element in drug development programs is the measurement of the binding affinities of lead drug candidates for the hOCT1 and the determination whether these compounds are hOCT1 substrates or inhibitors. Currently, cellular uptake studies are used to determine IC50 values, which can then be converted to Kis based on the Cheng–Prusoff equation [5]. Although this method provides reliable results, they are time consuming and laborious.

This laboratory has previously developed an alternative method for the study of binding interactions between compounds and receptors or drug transporters [6]. This approach is based upon liquid chromatography utilizing stationary phases containing immobilized membranes from cells expressing the target protein. This program has included the study of the drug exporter P-glycoprotein (Pgp), which is a member of the ABC transporter superfamily [4].

In these studies, membranes from a cell line expressing Pgp and from a cell line that does not express Pgp were immobilized on an immobilized artificial membrane (IAM) liquid chromatographic stationary phase [7], [8], [9] or on the surface of a glass capillary to create open tubular columns: Pgp(+)-OT and Pgp(−)-OT [10]. The Pgp-IAM stationary phases were used in frontal affinity chromatography studies to determine the binding affinities (Kd values) of Pgp substrates and inhibitors and were able to identify competitive, allosteric and enantioselective interactions between ligands and the Pgp transporter. The open tubular columns, Pgp(+)-OT and Pgp(−)-OT, were used to differentiate between specific and non-specific interactions between compounds and the immobilized membranes.

In the current study, this experimental approach has been extended to the development of a hOCT1(+)-IAM stationary phase. The column was prepared from a previously described stably transfected MDCK cell line, which expresses hOCT1 [11]. In addition, cellular membranes from the wild-type MDCK cell line were also immobilized onto the IAM stationary phase to produce a hOCT1(−)-IAM stationary phase.

Columns were prepared from both stationary phases and tested to determine the binding activity and specificity of the immobilized hOCT1. The columns were characterized using frontal displacement chromatography with [3H]-MPP+ as the marker ligand and MPP+, verapamil, quinidine, quinine, nicotine, dopamine and vinblastin as the displacers. The results demonstrate that the hOCT1(+) membranes were successfully immobilized on the IAM stationary phase with retention of the ability to specifically bind known hOCT1 ligands and to determine Kd values. The hOCT1(+)-IAM column was also able to determine an enantioselective binding interaction involving (R)-verapamil and to identify an additional high affinity MPP+ binding site.

Section snippets

Materials

(R, S)-Verapamil, (R)-verapamil, (S)-verapamil, N-methyl-4-phenyl pyridinium iodide (MPP+), tetraethyl ammonium chloride (TEA), quinine, quinidine, nicotine tartrate, dopamine, vinblastin sulphate, benzamidine, salts, cholate, leupeptin, phenyl methyl sulfonyl fluoride (PMSF), EDTA, Trizma, CHAPS, bovine serum albumin (BSA), glycerol, pepstatin, sodium chloride and dithiothreitol were purchased from Sigma (St. Louis, MO, USA). HPLC grade methanol, ammonium acetate and 0.1 M ammonium hydroxide

Results

When [3H]-MPP+ was chromatographed on the hOCT1(−)-IAM and hOCT1(+)-IAM columns, elution profiles containing front and plateau regions were observed for both columns (Fig. 1). The midpoint of the breakthrough curve occurred at 10 min on the hOCT1(−)-IAM column and 17 min on the hOCT1(+)-IAM column, representing breakthrough volumes of 2.0 and 3.4 ml, respectively. Since the void volume of the chromatographic system, column and detector, was 0.7 min, the results indicate that [3H]-MPP+ was retained

Conclusions

The data from this study indicate that membranes from the hOCT1-MDCK and MDCK cell lines have been successfully immobilized onto the IAM stationary phase, creating hOCT1(+)-IAM and hOCT1(−)-IAM stationary phases. The data also demonstrate that columns containing these stationary phases can be used to determine binding affinities to the immobilized hOCT1 and that the calculated Kd values correlate with Ki values obtained using cellular uptake or membrane binding techniques. In addition, the

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These authors contributed equally to the work.

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