Allosteric regulation of the binding of [3H]acetylcholine to m2 muscarinic receptors.
Muscarinic receptors of the m2 subtype expressed in Chinese hamster ovary cells were labeled with [methyl-3H]acetylcholine([3H]ACh), and the rate of dissociation in the presence and absence of several compounds known to exert allosteric effects on labeled antagonist binding was observed. At 25 degrees C, [3H]ACh bound to the receptors with a Kd of 1.2 nM and dissociated with a half-time of 1.6 min. This binding was sensitive to appropriate concentrations of guanine nucleotide and the muscarinic antagonist N-methylscopolamine (NMS). Gallamine, tetrahydroaminoacridine, physostigmine, obidoxime, and 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) all inhibited the binding of [3H]ACh and all slowed the rate of dissociation of [3H]ACh in a concentration-dependent manner. However, the nature of some of the allosteric effects differed from previous studies that used other labeled ligands. In particular, TMB-8, which is very effective in slowing the dissociation of the antagonist [3H]NMS, had much weaker effects on the dissociation of [3H]ACh. Furthermore, TMB-8 was able to partially reverse the stronger effects of gallamine on the dissociation of [3H]ACh, consistent with the possibility that TMB-8 and gallamine share a common site on the receptor. In summary, the binding of ACh to muscarinic receptors is subject to allosteric regulation, and assays using [3H]ACh may be especially useful in the evaluation of potential allosteric regulators of muscarinic systems.
Gnagey
A
A
Department of Psychiatry, University of Vermont, Burlington 05405, USA.
Ellis
J
J
D000109:Acetylcholine / Q000378:metabolism*; D000110:Acetylcholinesterase / Q000378:metabolism; D000494:Allosteric Regulation; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000201:enzymology; D006224:Cricetinae; D005703:Gallamine Triethiodide / Q000494:pharmacology; D005707:Gallic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D019832:N-Methylscopolamine; D011976:Receptors, Muscarinic / Q000378:metabolism*; D012602:Scopolamine Derivatives / Q000378:metabolism
D011976:Receptors, Muscarinic; D012602:Scopolamine Derivatives; C006014:8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate; D005707:Gallic Acid; D000110:Acetylcholinesterase; D000109:Acetylcholine; D005703:Gallamine Triethiodide; D019832:N-Methylscopolamine