Characterization of a Multiple Ligand-Gated Ion Channel Cellular Membrane Affinity Chromatography Column and Identification of Endogenously Expressed Receptors in Astrocytoma Cell Lines

• Published in: Analytical chemistry (Washington) Vol. 80; no. 22; pp. 8673 - 8680
• Main Authors: Kitabatake, T, Moaddel, R, Cole, R, Gandhari, M, Frazier, C, Hartenstein, J, Rosenberg, A, Bernier, M, Wainer, I. W
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 15.11.2008
• Subjects: Analytical chemistry; Astrocytoma - genetics; Astrocytoma - pathology; Cell Line, Tumor; Cell Membrane - metabolism; Cells; Chemistry; Chromatographic methods and physical methods associated with chromatography; Chromatography; Chromatography, Affinity - methods; Exact sciences and technology; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Ion Channel Gating; Ion Channels - metabolism; Ions; Ligands; Membranes; Microscopy, Confocal; Nicotine; Other chromatographic methods; Protein Binding; Proteins; Receptors, GABA-A - analysis; Receptors, GABA-A - metabolism; Receptors, N-Methyl-D-Aspartate - analysis; Receptors, N-Methyl-D-Aspartate - metabolism; Receptors, Nicotinic - analysis; Receptors, Nicotinic - metabolism; Frontal chromatography; Immobilization; Confocal microscopy; Ionic channel; Marker; Cell line; Affinity chromatography; Membrane; Aspartic acid; Acetylcholine; Inhibitor; Column chromatography; Astrocytoma; Diazepam; Nicotine
• ISSN: 0003-2700; 1520-6882
• DOI: 10.1021/ac8016407

Cellular membranes obtained from the 1321N1 and A172 astrocytoma cell lines were immobilized on a chromatographic phase to create cellular membrane affinity chromatography (CMAC) columns, CMAC(1321N1) and CMAC(A172). The columns were characterized using frontal affinity chromatography with [3H]-epibatidine as the marker ligand and epibatidine, nicotine, and methyllycaconitine as the displacers. The results indicated that the columns contained homomeric α7 nicotinic acetylcholine receptors (α7 nAChR) and heteromeric nicotinic acetylcholine receptors (α x β y nAChRs), which was confirmed by the addition of subtype-specific inhibitors, α-bungarotoxin (α7 nAChR) and κ-bungarotoxin (α x β y nAChR) to the mobile phase. The presence of two additional ligand-gated ion channels (LGICs), γ-aminobutyric acid (GABAA) and N-methyl-d-aspartic acid (NMDA), was established using frontal affinity chromatography with flunitrazepam and diazepam (GABAA receptor) and MK-801 and NMDA (NMDA receptor). The presence of the four LGICs was confirmed using confocal microscopy and flow cytometry. The results indicate that the CMAC(1321N1) and CMAC(A172) columns contain four independently functioning LGICs, that the columns can be used to characterize binding affinities of small molecules to each of the receptors, and that the CMAC approach can be used to probe the expression of endogenous membrane receptors.