A microtechnique for quantification of detergent-solubilized muscarinic and nicotinic acetylcholine receptors using a semi-automated cell harvestor.
A specific method for the rapid assay of muscarinic acetylcholine receptors (mAChR), either detergent-solubilized or in neuroblastoma cells, is described. This method is also applicable to the assay of nicotinic acetylcholine receptors. The procedure employs a cell harvestor and microtiter plates, and has the advantage of requiring small quantities of radioligand, microgram quantities of detergent-solubilized cholinergic receptor or less cells. The binding parameters such as the equilibrium dissociation constants (Kd) of mAChR and nicotinic acetylcholine receptor (nAChR) and inhibition constants (Ki) for antagonists determined by the present method are in excellent agreement with values determined by other methods. This assay procedure for mAChR and nAChR should facilitate the rapid screening of cholinergic agonists/antagonists and also the further purification and characterization of mAChR.
D000818:Animals; D001285:Atropine / Q000494:pharmacology; D002038:Bungarotoxins / Q000378:metabolism; D004072:Digitonin; D066298:In Vitro Techniques; D008832:Microchemistry; D019832:N-Methylscopolamine; D009447:Neuroblastoma / Q000378:metabolism; D011092:Polyethylene Glycols; D011813:Quinuclidinyl Benzilate / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism; D012602:Scopolamine Derivatives / Q000378:metabolism; D012995:Solubility
D002038:Bungarotoxins; D011976:Receptors, Muscarinic; D011978:Receptors, Nicotinic; D012602:Scopolamine Derivatives; D011092:Polyethylene Glycols; D011813:Quinuclidinyl Benzilate; D001285:Atropine; D004072:Digitonin; D019832:N-Methylscopolamine