Activation and desensitization of Torpedo acetylcholine receptor: evidence for separate binding sites.

Abstract

The acetylcholine [ACh] receptor from T. californica was labeled by reaction with the fluorescent probe 4-[(N-iodoacetoxy)ethyl-N-methyl]amino-7-nitrobenz-2-oxa-1,3-diazole without apparent effect on its in vitro ligand binding and functional properties. Addition of ACh or carbamoylcholine to the labeled-receptor preparations enhanced the fluorescence of the bound probe, and this effect was specific for agonists and inhibited by prior incubation with excess .alpha.-bungarotoxin. Equilibrium fluorescence titrations gave apparent Kd of 0.86 .+-. 0.14 mM for carbamoylcholine and 79 .+-. 11 .mu.M for ACh, in good agreement with the Kd measured for the permeability response of the receptor. Stopped-flow experiments showed that the fluorescence change was a single exponential process whose rate increased with ligand concentration, reaching a saturating value for carbamoylcholine of .apprx. 400 s-1. The equilibrium binding of carbamoylcholine was not significantly affected by prior incubation of the receptor with d-tubocurarine or histrionicotoxin and the Kd was only slightly increased in the presence of lidocaine. These inhibitory ligands do not complete directly with agonists for this low-affinity binding site, suggesting that their mode of action may be indirect.