Functional nicotinic receptor expression in mesodermal cells transfected with MyoD cDNA.
Previous studies had shown that MyoD promoted nicotinic acetylcholine subunit gene expression; the present experiments were done to determine whether this subsequently led to the development of functional nicotinic acetylcholine receptors. Transfection of C3H 10T1/2 cells with MyoD cDNA resulted in the appearance of [125I]alpha-bungarotoxin binding sites; radiolabelled alpha-toxin binding was not observed in cells transfected with a plasmid that lacked MyoD cDNA. Receptor development plateaued over a time course of several days with maximal binding seven and 11 days after exposure to fusion medium. [125I]alpha-bungarotoxin binding was of high affinity (Kd = 1 nM), saturable and was inhibited by nicotinic but not muscarinic receptor ligands, with IC50s of 1-3 nM for alpha-bungarotoxin, 1-3 microM for d-tubocurarine and 3-10 microM for nicotine. Not only did the cells exhibit a cell surface nicotinic receptor but they also expressed a nicotinic receptor mediated functional response. Carbachol resulted in uptake of 22Na into the cells at concentrations similar to those required for receptor activation at a muscle type nicotinic receptor; furthermore, the functional response was effectively blocked by nicotinic receptor ligands, including alpha-bungarotoxin (IC50 = 2 to 6 nM) and d-tubocurarine (IC50 = 0.1 to 0.4 microM); muscarinic receptor ligands had no effect. A time course study showed that alpha-bungarotoxin binding and carbachol stimulated 22Na uptake developed in parallel, suggesting that the observed functional response was mediated through an interaction at the alpha-bungarotoxin recognition site.(ABSTRACT TRUNCATED AT 250 WORDS)
Quik
M
M
Department of Pharmacology, McGill University, Montreal, Quebec, Canada.
Odeh
R
R
Philie
J
J
Szyf
M
M
D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D018076:DNA, Complementary; D008648:Mesoderm / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008809:Mice, Inbred C3H; D017570:MyoD Protein / Q000235:genetics*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology; D012964:Sodium / Q000493:pharmacokinetics; D014162:Transfection
D002038:Bungarotoxins; D018076:DNA, Complementary; D017570:MyoD Protein; D011978:Receptors, Nicotinic; D002217:Carbachol; D012964:Sodium