Jp. Kukkonen et al., FUNCTIONAL-PROPERTIES OF MUSCARINIC RECEPTOR SUBTYPES HM1, HM3 AND HM5 EXPRESSED IN SF9 CELLS USING THE BACULOVIRUS EXPRESSION SYSTEM, The Journal of pharmacology and experimental therapeutics, 279(2), 1996, pp. 593-601
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.