Initial Synthesis and Characterization of an 7 Nicotinic Receptor Cellular Membrane Affinity Chromatography Column: Effect of Receptor Subtype and Cell Type

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• 作者：Ruin Moaddel ; Regina V. Oliveira ; Tomoko Kimura ; Patrick Hyppolite ; Magdalena Juhaszova ; Yingxian Xiao ; Kenneth J. Kellar ; Michel Bernier ; Irving W. Wainer
• 刊名：Analytical Chemistry
• 出版年：2008
• 出版时间：January 1, 2008
• 年：2008
• 卷：80
• 期：1
• 页码：48 - 54
• 全文大小：187K
• 年卷期：v.80,no.1(January 1, 2008)
• ISSN：1520-6882

文摘

In this study, cellular membrane fragments from SH-EP1-pCEP4-h7 and 7 HEK-293 cell lines were used tosynthesize cellular membrane affinity chromatography(CMAC) columns containing functional 7 nicotinic acetylcholine receptors, CMAC(7 nAChR) columns. Thesynthesis of stable columns required the addition ofcholesterol to the 2% cholate solubilization/immobilization (s/i) buffer and to the mobile phase. In addition,when membranes from the SH-EP1 cell line were used,L--phosphatidylserine and L--phosphatidylethanolaminealso had to be added to the s/i buffer. A CMAC(42nAChR) column was prepared using membrane fragmentsfrom a SH-EP1-pCEP4-h42 cell line, and this processrequired the addition of L--phosphatidylserine and L--phosphatidylethanolamine to the s/i buffer, but not cholesterol. The s/i buffers from the three columns werecompared with the s/i buffer utilized in the preparationof a CMAC(42 nAChR) column prepared using an 42HEK-293 cell line, which required no additions to the 2%cholate s/i buffer. The data demonstrate that both cell typeand receptor type affect the protocol required to producea stable CMAC column and that, at the current time, thedevelopment of an optimum immobilization protocol is anempirical process. The results are also consistent with theobservation that the 7 nAChR is localized in lipid raftsin both of these cell lines and that the cholate detergentremoved cholesterol from these microdomains.