Molecular Forms of the Acetylcholine Receptor from Vertebrate Muscles and Torpedo Electric Organ

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Abstract
Multiple forms of the acetylcholine receptor solubilised from cat denervated muscle were separated by velocity sedimentation centrifugation. The kinetic properties of the two main forms (with sedimentation coefficients of 9 S and 4 S) were investigated using a pure preparation of a suitable probe, [3H]propionyl-alpha-bungaro-toxin. The binding of this toxin to each of these forms of the muscle receptor was consistent with a simple bimolecular reaction with a homogeneous class of binding sites. Negligible dissociation of the receptor-toxin complex was observed. This behaviour was also found for the different forms of the acetylcholine receptor of chick embryo muscle and of Torpedo marmorata electric organ. Association rate constants for binding of the 3H-labelled alpha-toxin to receptor from chick embryo muscle and the 9-S and 4-S forms from cat denervated muscle were 0.54 X 10(5), 1.76 X 10(5) and 2.69 X 10(5) M--1 S--1 respectively, at 25 degrees C. The values obtained for the 9-S and 13-S forms of receptor from T. marmorata were 4.51 X 10(5) and 9.93 X 10(5) M--1 S--1 respectively. The reaction of the 3H-labelled alpha-toxin with the receptor was second-order and linear in the presence of an antagonist, as in its absence, for the 4-S and 9-S forms of the cat denervated muscle receptor. This reaction of the receptor was inhibited by cholinergic ligands, with Ki values for two antagonists tested being greater with the 4-S form than with the 9-S form. Apparent negative interaction is observed for antagonists with this receptor, with Hill coefficients of about 0.78 and 0.64 for the 4-S and 9-S forms respectively. A ligand-induced affinity increase, produced by the agonists but not by the antagonists, was observed in this reaction for both forms of the muscle receptor. Two agonists tested showed no difference between these forms in their high-affinity states in either their binding affinities or Hill coefficients.