Xenopus muscle acetylcholine receptor alpha subunits bind ligands with different affinities.
The acetylcholine receptor (AChR) from vertebrate skeletal muscle is a pentameric complex composed of two identical alpha (the ligand-binding subunit) and one each of beta, gamma, and delta subunits. All muscle-like alpha subunits appear to be encoded by a single gene except for Xenopus where two genes have been identified (X alpha 1a and X alpha 1b). By establishing stable cell lines expressing each Xenopus alpha subunit plus Torpedo beta gamma delta (XaT and XbT), we were able to investigate immunological and ligand-binding properties of these two Xenopus subunits. We determined that each subunit was capable of proper assembly with beta gamma delta subunits, each hybrid AChR cell line responded to bath-applied acetylcholine as determined by 22Na+ influx, but the two hybrid AChRs had distinct ligand-binding properties. The most striking difference between the two hybrids was observed with alpha-bungarotoxin (BuTx) binding. The KD for BuTx binding to XbT hybrids was virtually identical to that of Torpedo and most other muscle AChRs, approximately 10(-10)-10(-11) M. The KD for BuTx binding to XaT hybrids, however, was approximately 1000-fold weaker (6 x 10(-8) M). This weak binding was due primarily to a very rapid BuTx off-rate (4.4 x 10(-2) s-1 for XaT AChRs compared with approximately 1.6 x 10(-5) s-1 for all-Torpedo and XbT AChRs).
Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510.
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D016475:3T3 Cells / Q000378:metabolism; D000109:Acetylcholine / Q000494:pharmacology; D000818:Animals; D002038:Bungarotoxins / Q000378:metabolism; D002217:Carbachol / Q000378:metabolism; D015870:Gene Expression; D015151:Immunoblotting; D007163:Immunosorbent Techniques; D007700:Kinetics; D046911:Macromolecular Substances; D051379:Mice; D009132:Muscles / Q000737:chemistry*; D011950:Receptors, Cholinergic / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects / Q000378:metabolism; D014101:Torpedo; D014162:Transfection; D014982:Xenopus laevis
D002038:Bungarotoxins; D046911:Macromolecular Substances; D011950:Receptors, Cholinergic; D015222:Sodium Channels; D002217:Carbachol; D012964:Sodium; D000109:Acetylcholine