Rat alpha3/beta4 subtype of neuronal nicotinic acetylcholine receptor stably expressed in a transfected cell line: pharmacology of ligand binding and function.
We stably transfected human kidney embryonic 293 cells with the rat neuronal nicotinic acetylcholine receptor (nAChR) alpha3 and beta4 subunit genes. This new cell line, KXalpha3 beta4R2, expresses a high level of the alpha3/beta4 receptor subtype, which binds (+/-)- [3H]epibatidine with a Kd value of 304+/-16 pM and a Bmax value of 8942 +/- 115 fmol/mg protein. Comparison of nicotinic drugs in competing for alpha3/beta4 receptor binding sites in this cell line and the binding sites in rat forebrain (predominantly alpha4/beta2 receptors) revealed marked differences in their Ki values, but similar rank orders of potency for agonists were observed, with the exception of anatoxin-A. The affinity of the competitive antagonist dihydro-beta-erythroidine is >7000 times higher at alpha4/beta2 receptors in rat forebrain than at the alpha3/beta4 receptors in these cells. The alpha3/beta4 nAChRs expressed in this cell line are functional, and in response to nicotinic agonists, 86Rb+ efflux was increased to levels 8-10 times the basal levels. Acetylcholine, (-)-nicotine, cytisine, carbachol, and (+/-)-epibatidine all stimulated 86Rb+ efflux, which was blocked by mecamylamine. The EC50 values for acetylcholine and (-)-nicotine to stimulate 86Rb+ effluxes were 114 +/- 24 and 28 +/- 4 microM, respectively. The rank order of potency of nicotinic antagonists in blocking the function of this alpha3/beta4 receptor was mecamylamine > d-tubocurarine > dihydro-beta-erythroidine > hexamethonium. Mecamylamine, d-tubocurarine, and hexamethonium blocked the function by a noncompetitive mechanism, whereas dihydro-beta-erythroidine blocked the function competitively. The KXalpha3 beta4R2 cell line should prove to be a very useful model for studying this subtype of nAChRs.
Department of Pharmacology, Georgetown University School of Medicine, Washington, DC 20007, USA.
Y
Y
Xiao
E L
EL
Meyer
J M
JM
Thompson
A
A
Surin
J
J
Wroblewski
K J
KJ
Kellar
D000818:Animals; D001667:Binding, Competitive; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008024:Ligands; D009538:Nicotine / Q000494:pharmacology; D018722:Nicotinic Agonists / Q000494:pharmacology*; D011725:Pyridines / Q000494:pharmacology*; D051381:Rats; D011978:Receptors, Nicotinic / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D014162:Transfection
D019086:Bridged Bicyclo Compounds, Heterocyclic; D002118:Calcium; D008024:Ligands; D009538:Nicotine; D018722:Nicotinic Agonists; D011725:Pyridines; D011978:Receptors, Nicotinic; D012413:Rubidium; D012964:Sodium; C082748:epibatidine