Electrostatic interactions regulate desensitization of the nicotinic acetylcholine receptor.
To determine the importance of electrostatic interactions for agonist binding to the nicotinic acetylcholine receptor (AChR), we examined the affinity of the fluorescent agonist dansyl-C6-choline for the AChR. Increasing ionic strength decreased the binding affinity in a noncompetitive manner and increased the Hill coefficient of binding. Small cations did not compete directly for dansyl-C6-choline binding. The sensitivity to ionic strength was reduced in the presence of proadifen, a noncompetitive antagonist that desensitizes the receptor. Moreover, at low ionic strength, the dansyl-C6-choline affinities were similar in the absence or presence of proadifen, a result consistent with the receptor being desensitized at low ionic strength. Similar ionic strength effects were observed for the binding of the noncompetitive antagonist [(3)H]ethidium when examined in the presence and absence of agonist to desensitize the AChR. Therefore, ionic strength modulates binding affinity through at least two mechanisms: by influencing the conformation of the AChR and by electrostatic effects at the binding sites. The results show that charge-charge interactions regulate the desensitization of the receptor. Analysis of dansyl-C6-choline binding to the desensitized conformation using the Debye-Hückel equation was consistent with the presence of five to nine negative charges within 20 A of the acetylcholine binding sites.
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Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
Pedersen
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D000818:Animals; D001665:Binding Sites; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D003619:Dansyl Compounds / Q000493:pharmacokinetics; D004557:Electric Organ / Q000502:physiology; D004996:Ethidium / Q000493:pharmacokinetics; D005456:Fluorescent Dyes; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D018722:Nicotinic Agonists / Q000493:pharmacokinetics; D018733:Nicotinic Antagonists / Q000493:pharmacokinetics; D009994:Osmolar Concentration; D011487:Protein Conformation; D000644:Quaternary Ammonium Compounds / Q000493:pharmacokinetics; D011869:Radioligand Assay; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000378:metabolism*; D055672:Static Electricity; D014101:Torpedo
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D018722:Nicotinic Agonists; D018733:Nicotinic Antagonists; D000644:Quaternary Ammonium Compounds; D011978:Receptors, Nicotinic; C027933:(1-(5-dimethylaminonaphthalene)sulfonamido)-n-hexanoic acid-beta-N-trimethylammonium ethyl ester; D002217:Carbachol; D004996:Ethidium