Initial synthesis and characterization of an alpha7 nicotinic receptor cellular membrane affinity chromatography column: effect of receptor subtype and cell type.
In this study, cellular membrane fragments from SH-EP1-pCEP4-halpha7 and alpha7 HEK-293 cell lines were used to synthesize cellular membrane affinity chromatography (CMAC) columns containing functional alpha7 nicotinic acetylcholine receptors, CMAC(alpha7 nAChR) columns. 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-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-EP1-pCEP4-halpha4beta2 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 alpha4beta2 HEK-293 cell line, which required no additions to the 2% cholate s/i buffer. The data demonstrate 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. The results are also consistent with the observation that the alpha7 nAChR is localized in lipid rafts in both of these cell lines and that the cholate detergent removed cholesterol from these microdomains.
Gerontology Research Center, National Institutes on Aging, National Institutes of Health, Baltimore, Maryland 21224-6825, USA. moaddelru@grc.nia.nih.gov
Ruin
R
Moaddel
Regina V
RV
Oliveira
Tomoko
T
Kimura
Patrick
P
Hyppolite
Magdalena
M
Juhaszova
Yingxian
Y
Xiao
Kenneth J
KJ
Kellar
Michel
M
Bernier
Irving W
IW
Wainer
D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000737:chemistry; D002038:Bungarotoxins / Q000737:chemistry; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D018914:Cells, Immobilized / Q000737:chemistry; D002846:Chromatography, Affinity / Q000295:instrumentation*; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D018613:Microscopy, Confocal; D009538:Nicotine / Q000737:chemistry; D009928:Organ Specificity; D011725:Pyridines / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000145:classification*; D064569:alpha7 Nicotinic Acetylcholine Receptor
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002038:Bungarotoxins; C579167:Chrna7 protein, human; D011725:Pyridines; D011978:Receptors, Nicotinic; D064569:alpha7 Nicotinic Acetylcholine Receptor; D009538:Nicotine; D016650:Fluorescein-5-isothiocyanate; C082748:epibatidine