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Characterization of a Mouse Serotonin 5‐HT 3 Receptor Purified from Mammalian Cells
Author(s) -
Hovius Ruud,
Tairi AnaPaula,
Blasey Horst,
Bernard Alain,
Lundström Kenneth,
Vogel Horst
Publication year - 1998
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1998.70020824.x
Subject(s) - receptor , 5 ht3 receptor , ligand (biochemistry) , molecular mass , chemistry , agonist , 5 ht receptor , biochemistry , binding site , affinity chromatography , size exclusion chromatography , gel electrophoresis , competitive antagonist , serotonin , enzyme
A serotonin 5‐HT 3 receptor was functionally expressed to high levels and on a large scale in mammalian cells with the Semliki Forest virus system. Conditions were optimized to maximize detergent solubilization of the receptor, while preserving ligand binding activity. An efficient one‐step purification yielding ∼50% of the histidine‐tagged 5‐HT 3 receptor was achieved with immobilized metal ion chromatography. The expressed receptor, in both membranes and purified preparations, exhibited wild‐type ligand binding properties, characterized by one class of binding sites. The purity of the receptor was shown by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, yielding a single band at 65 kDa, and was confirmed by the specific ligand binding activity of ∼5 nmol/mg of protein. Deglycosylation of the receptor reduced the estimated relative molecular mass to 49 kDa. The apparent molecular mass of the functional receptor complex was determined by size exclusion chromatography to be 280 kDa, suggesting that the 5‐HT 3 receptor is a pentameric homooligomer. The secondary structure of the 5‐HT 3 receptor as determined by circular dichroism appeared to consist of mainly α‐helices (50%) and β‐strands (24%), with minor contributions from nonregular structure (9%). The binding of either agonist or antagonist did not alter the secondary structure of the receptor.