LIGAND-BINDING PROPERTIES OF MUSCARINIC ACETYLCHOLINE-RECEPTOR SUBTYPES (M1-M5) EXPRESSED IN BACULOVIRUS-INFECTED INSECT CELLS

Five subtypes of muscarinic acetylcholine receptors (m1-m5) have been expressed in insect cells (Spodoptera frugiperda, Sf9) using the bacul ovirus system. Up to 6 nmol of muscarinic acetylcholine receptors were produced by 1 liter culture; 0.3 to 0.6 (human mi), 3 to 6 (human m2) , 2 to 4 (rat m3), 1 to 2 (rat m4) and 0.5 to 1 (human m5) nmol. Piren zepine, AF-DX116 and hexahidrosiladifenidol showed the highest affinit y for the mi, m2 and m3 subtype, respectively, indicating that these r eceptors expressed in Sf9 cells retain the same substrate specificity as those in mammalian tissues or cultured cells. Among 32 kinds of mus carinic ligands examined in the present studies, prifinium was found t o have the highest affinity for the m4 subtype, and pilocarpine, oxotr emorine, McN-A343 and promethazine the highest affinity for the m5 sub type, although the differences in the affinities among the five subtyp es were less than 10-fold. Alcuronium increased the binding of [H-3]N- methylscopalamine to the m2 subtype, but not the mi, m4 and m5 subtype s and only slightly to the m3 subtype. Similar but smaller effects of fangchinoline and tetrandrine were found for [H-3]N-methylscopalamine binding to only the m3 subtype. These effects may also be useful for t he discrimination of individual subtypes.