SOLUBILIZATION OF MUSCARINIC RECEPTOR SUBTYPES FROM BACULOVIRUS-INFECTED SF9 INSECT CELLS

Five different subtypes (human m1, m2, m5 and rat m3, m4) of muscarini c acetylcholine receptors (mAChR) were produced in insect Sf9 cells by infection with recombinant baculoviruses. N[H-3]methylscopolamine ([H -3]NMS) has a similar affinity to each of these mAChR subtypes in cell membranes, while pirenzepine, -dihydro-6H-pyrido-(2,3-b)(1,4)benzo-di azepin-6-on (AF-DX 116) and (+/-)-p-fluoro-hexahydrosiladifenidol (p-F -HHSiD) have a higher affinity for m1, m2 and m3, respectively, than f or the other subtypes, indicating the maintenance of subtype specifici ty of mAChR in this system. Digitonin (1%, w/w) with sodium cholate (0 .1%, w/w) solubilized 51% of m1, 36% of m2, 3% of m3, 28% of m4 and 17 % of m5 mAChR from these cell membranes with retention of the [H-3]NMS binding activity. Optimization of cholate concentrations resulted in solubilization of up to 50-60% for m1, m2 and m4, but up to 25% for m5 and 7% for m3. Optimal concentrations of cholate differed from one su btype to another. Sucrose monolaurate solubilized 21-43% of m1, m2 and m4, but only up to 12% for m5 and 2% for m3. cholamidopropyl)dimethyl ammonio-1-propanesulfonate (CHAPS) was practically ineffective in mACh R solubilization from Sf9 cell membranes for all subtypes investigated . Solubilization with digitonin and cholate had little influence on [H -3]NMS affinity for m2 and m4, but decreased mi and m5 affinity by 10- fold and that of m3 by more than 50-fold. These results indicate that the solubility and stability of mAChR in detergents differ among the s ubtypes, in spite of their structural similarities. These differences should be taken into account when comparing the five subtypes, particu larly when determining the proportion of each subtype in a given tissu e by precipitating the solubilized mAChR with subtype-specific antibod ies.