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 determin e whether this subsequently led to the development of functional nicot inic acetylcholine receptors. Transfection of C3H 10T1/2 cells with My oD cDNA resulted in the appearance of [I-125]alpha-bungarotoxin bindin g sites; radiolabelled alpha-toxin binding was not observed in cells t ransfected with a plasmid that lacked MyoD cDNA. Receptor development plateaued over a time course of several days with maximal binding seve n and 11 days after exposure to fusion medium. [I-125]alpha-bungarotox in binding was of high affinity (K(d) = 1 nM), saturable and was inhib ited by nicotinic but not muscarinic receptor ligands, with IC50s of 1 -3 nM for alpha-bungarotoxin, 1-3 muM for d-tubocurarine and 3-10 muM for nicotine. Not only did the cells exhibit a cell surface nicotinic receptor but they also expressed a nicotinic receptor mediated functio nal response. Carbachol resulted in uptake of Na-22 into the cells at concentrations similar to those required for receptor activation at a muscle type nicotinic receptor; furthermore, the functional response w as effectively blocked by nicotinic receptor ligands, including alpha- bungarotoxin (IC50 = 2 to 6 nM) and d-tubocurarine (IC50 = 0.1 to 0.4 muM); muscarinic receptor ligands had no effect. A time course study s howed that a-bungarotoxin binding and carbachol stimulated Na-22 uptak e developed in parallel, suggesting that the observed functional respo nse was mediated through an interaction at the alpha-bungarotoxin reco gnition site. To conclude, the present studies show that transfection with MyoD results in the appearance of a functional cell surface muscl e type nicotinic acetylcholine receptor. They further support the cont ention that MyoD plays a pivotal role in nicotinic acetylcholine recep tor regulation in muscle.