FUNCTIONAL-PROPERTIES OF MUSCARINIC RECEPTOR SUBTYPES HM1, HM3 AND HM5 EXPRESSED IN SF9 CELLS USING THE BACULOVIRUS EXPRESSION SYSTEM

The human muscarinic ACh receptor subtypes m1, m3 and m5 have been exp ressed in Sf9 cells using the baculovirus expression system. Stimulati on of all three subtypes with CCh caused an increase in inositol-1,4,5 -trisphosphate and intracellular Ca++. The increase in cytosolic free Ca++ was to a large extent due to influx. The levels of receptors (< 0 .1-1 pmol/mg protein) increased with infection time in a narrow time s pan (24-36 h). The changes in the receptor densities did not significa ntly affect the EC(50) values of CCh-mediated Ca++ mobilization with t he m3 or the m5 subtype. The EC(50) value was higher with the m1 recep tor at low expression levels (approximate to 100 fmol/mg protein), and it decreased with an increase in receptor density. The receptor subty pes displayed no gross differences in their response to oxotremorine-M , which behaved as a full agonist, or to oxotremorine and pilocarpine, which were less active. With the m3 subtype, there was an increase in the maximal response to oxotremorine with longer infection times. The results demonstrate that the recombinant muscarinic receptors, expres sed in Sf9 cells, show many of the characteristics of endogenously exp ressed receptors when studied at low expression levels and that the re ceptor density may significantly affect the receptor pharmacology.