Initial synthesis and characterization of an alpha7 nicotinic receptor cellular membrane affinity chromatography column: Effect of receptor subtype and cell type

Abstract

Objective:In this study, cellular membrane fragments from SH‐EP1‐pCEP4‐hα7 and α7 HEK‐293 cell lines were used to synthesize cellular membrane affinity chromatography (CMAC) columns containing functional α7 nicotinic acetylcholine receptors, CMAC(α7 nAChR) columns.Methods:The synthesis of stable columns required the addition of cholesterol 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‐α‐phosphatidyl serine and L‐α‐phosphatidyl ethanolamine also had to be added to the s/i buffer.Results:A CMAC(α4β2 nAChR) column was prepared using membrane fragments from a SH‐EP1‐pCEP4‐hα4β2 cell line and this process required the addition of L‐α‐phosphatidyl serine and L‐α‐phosphatidyl ethanolamine to the s/i buffer, but not cholesterol. The s/i buffers from the 3 columns were compared with the s/i buffer utilized in the preparation of a CMAC (α4β2 nAChR) column prepared using an α4β2 HEK‐293 cell line, which required no additions to the 2% cholate s/i buffer.Conclusion:The data demonstrates that both 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.