Functional nicotinic receptor expression in mesodermal cells transfected with MyoD cDNA

Abstract

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]α-bungarotoxin binding sites; radiolabelled α-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]α-bungarotoxin binding was of high affinity ( K d = 1 nM ), saturable and was inhibited by nicotinic but not muscarinic receptor ligands, with ic 50s of 1–3 nM for α-bungarotoxin, 1–3 μM for d-tubocurarine and 3–10 μM 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 α-bungarotoxin ( ic 50 = 2 to 6 nM ) and d-tubocurarine ( ic 50 = 0.1 to 0.4 μM ); muscarinic receptor ligands had no effect. A time course study showed that α-bungarotoxin binding and carbachol stimulated 22Na uptake developed in parallel, suggesting that the observed functional response was mediated through an interaction at the α-bungarotoxin recognition site. To conclude, the present studies show that transfection with MyoD results in the appearance of a functional cell surface muscle type nicotinic acetylcholine receptor. They further support the contention that MyoD plays a pivotal role in nicotinic acetylcholine receptor regulation in muscle.