Interaction of a Semirigid Agonist with Torpedo Acetylcholine Receptor
- 16 March 2000
- journal article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 39 (14), 3867-3876
- https://doi.org/10.1021/bi992151o
Abstract
The binding of the semirigid agonist [3H]arecolone methiodide to the Torpedo nicotinic acetylcholine receptor has been correlated with its functional properties measured both in flux studies with Torpedo membrane vesicles and by single-channel analysis after reconstitution in giant liposomes. Under both equilibrium and preequilibrium conditions, the binding of arecolone methiodide is similar to that of other agonists such as acetylcholine. At equilibrium, it binds to two sites per receptor with high affinity (Kd = 99 ± 12 nM), and studies of its dissociation kinetics suggest that each of these sites is made up of two subsites that are mutually exclusive at equilibrium. The kinetics of arecolone methiodide binding were monitored by the changes in the receptor intrinsic fluorescence, and the data are consistent with a model in which the initial binding event is followed by sequential conformational transitions of the receptor−ligand complex. In flux studies, arecolone methiodide was approximately 3-fold more potent (EC50 = 31 ± 5 μM) than acetylcholine but its maximum flux rate was 4−10-fold lower. This phenomenon has been studied further by single-channel analysis of Torpedo receptors reconstituted in giant liposomes. Whereas the flexible agonist carbamylcholine (5 μM) was shown to induce channels with conductances of 56 and 34 pS with approximately equal frequency, arecolone methiodide (2 μM) preferentially induced the channel of lower conductance. These results are interpreted in terms of a simple model in which the rigidity of arecolone methiodide restrains the conformation that the receptor−ligand complex can adopt, thus favoring the lower conductance state.Keywords
This publication has 15 references indexed in Scilit:
- A membrane fusion strategy for single‐channel recordings of membranes usually non‐accessible to patch‐clamp pipette electrodesFEBS Letters, 1987
- Structural and electronic requirements for potent agonists at a nicotinic receptorEuropean Journal of Pharmacology, 1986
- Single-channel currents from acetylcholine receptors in embryonic chick muscle. Kinetic and conductance properties of gaps within burstsBiophysical Journal, 1984
- Channel properties of the purified acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayer membranesBiophysical Journal, 1984
- Flickering of a nicotinic ion channel to a subconductance stateBiophysical Journal, 1983
- Two‐component desensitization at the neuromuscular junction of the frog.The Journal of Physiology, 1983
- Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patchesPflügers Archiv - European Journal of Physiology, 1981
- Purification of Torpedo californica post-synaptic membranes and fractionation of their constituent proteinsBiochemical Journal, 1980
- Agonist-mediated changes of the acetylcholine receptor in its membrane environmentJournal of Molecular Biology, 1978
- Determination of dose‐response curves by quantitative ionophoresis at the frog neuromuscular junction.The Journal of Physiology, 1978