The preparation of HEK293 α 1A or HEK293 α 1B cell membrane stationary phase and the chromatographic affinity study of ligands of α 1 adrenoceptor

Abstract

As a novel bioaffinity chromatographic technique, cell membrane chromatography (CMC) originated in 1996. The cell membrane stationary phase (CMSP) consists of porous silica coated with active cell membranes. By immersing silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes. The present study repeatedly investigated the interaction between ligands and receptors by employing the system of CMC and especially evaluated the accuracy and feasibility of the CMC model in the study of subtype receptors. The cDNA encoding α 1A or α 1B adrenergic receptors (ARs) was transfected into human embryonic kidney 293 (HEK293) cell lines; cell lines stably overexpressing subtype receptors were obtained. HEK293 α 1A or HEK293 α 1B ARs CMSP were prepared by immobilizing relevant cell membranes on silica. In the described chromatography-based CMSP, the retention times of nine α 1 adrenoceptor ligands and calculated capacity factors, as chromatographic parameters, were recorded carefully. These results showed a good correlation with the affinity of the same compounds for the corresponding cloned α 1 adrenoceptor subtype. The rank order of capacity factors was consistent with the affinity rank order obtained from radioligand binding assays. The immobilized subtype-selective CMSP was stable and reproducible. The study demonstrates that the HEK293 α 1A and HEK293 α 1B CMSP can be utilized for initial screening of drug candidates.