Expression of functional M2 muscarinic acetylcholine receptor in Escherichia coli

The M-2 muscarinic acetylcholine receptor mutant (M-2 mutant), with a lack of glycosylation sites, a deletion in the central part of the third inner loop, and the addition of a six histidine tag at the C-terminus, was fused to maltose binding protein (MBP) at its N-terminus and expressed in Escherichia coli, The expression level was 0.2 nmol receptor per 100 ml culture, as assessed as [H-3]L-quinuclidinyl benzilate ([H-3]QNB) binding activity, when the BL 21 strain was cultured at 37 degrees C to a late growth phase and the expression was induced by isopropyl beta-thiogalactoside at 20 degrees C. No [H-3]QNB binding activity was detected when it was not fused to MBP or when expression was induced at 37 degrees C instead of 20 degrees C. The MBP-M-2 mutant expressed in E. coli showed the same ligand binding activity as the M-2 mutant expressed in the Sporodoptera frugiperda (Sf9)/baculovirus system, as assessed as displacement of [H-3]QNB with carbamylcholine and atropine. The MBPM, mutant was solubilized, purified with Co2+-immobilized Chelating Sepharose gel and SP-Sepharose, and then reconstituted into lipid vesicles with G protein G(o) or G(il) in the presence or absence of cholesterol. The reconstituted vesicles showed GTP-sensitive high affinity binding for carbamylcholine and carbamylcholine-stimulated [S-35]GTP gamma S binding activity in the presence of GDP. The proportion of high affinity sites for carbamylcholine and the extent of carbamylcholine-stimulated [S-35]GTP gamma S binding were the same as those observed for the M-2 mutant expressed in Sf9 cells and were not affected by the presence or absence of cholesterol, These results indicate that the MBP-M-2 mutant expressed in E. coli has the same ability to interact with and activate G proteins as the M-2 mutant expressed in Sf9, and that cholesterol is not essential for the function of the M-2 muscarinic receptor.