Initial Synthesis and Characterization of an a7 Nicotinic Receptor Cellular Membrane Affinity Chromatography Column:Effect of Receptor Subtype and Cell Type

摘要

In this study,cellular membrane fragments from SH-EP1-pCEP4-h alpha7 and alpha7 HEK-293 cell lines were used to synthesize cellular membrane affinity chromatography (CMAC) columns containing functional alpha7 nicotinic acetylcholine receptors,CMAC(a7 nAChR) columns.The synthesis of stable columns required the addition of cholesterol to the 2% cholate solubilization/immobiliza-tion (s/i) buffer and to the mobile phase.In addition,when membranes from the SH-EP1 cell line were used,L-alpha-phosphatidylserine and L-alpha-phosphatidylethanolamine also had to be added to the s/i buffer.A CMAC(alpha4beta2 nAChR) column was prepared using membrane fragments from a SH-EPl-pCEP4-h alpha4beta2 cell line,and this process required the addition of L-alpha-phosphatidylserine and L-alpha-phosphatidylethanolamine to the s/i buffer,but not cholesterol.The s/i buffers from the three columns were compared with the s/i buffer utilized in the preparation of a CMAC(alpha4beta2 nAChR) column prepared using an alpha4/beta2 HEK-293 cell line,which required no additions to the 2% cholate s/i buffer.The data demonstrate that bom cell type and receptor type affect the protocol required to produce a stable CMAC column and that,at the current time,the development of an optimum immobilization protocol is an empirical process.The results are also consistent wim the observation that the a7 nAChR is localized in lipid rafts in both of these cell lines and that the cholate detergent removed cholesterol from these microdomains.