The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that inter

The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.

Background and Purpose: Nicotinic acetylcholine receptors (AChRs) are valuable therapeutic targets. To exploit them fully requires rapid assays for the evaluation of potentially therapeutic ligands and improved understanding of the interaction of such ligands with their receptor binding sites. Experimental Approach: A variety of neuromuscular blocking agents (NMBAs) were tested for their ability to inhibit the binding of [125 I]a-bungarotoxin to TE671 cells expressing human muscle AChRs. Association and dissociation rate constants for vecuronium inhibition of functional agonist responses were then estimated by electrophysiological studies on mouse muscle AChRs expressed in Xenopus oocytes containing either wild type or mutant a1 subunits. Key results: The TE671 inhibition binding assay allowed for the rapid detection of competitive nicotinic AChR ligands and the relative IC 50 results obtained for NMBAs agreed well with clinical data. Electrophysiological studies revealed that acetylcholine EC 50 values of muscle AChRs were not substantially altered by non-conservative mutagenesis of phenylalanine at a1:189 and proline at a1:194 to serine. However the a1:Phe189Ser mutation did result in a 3-4 fold increase in the rate of dissociation of vecuronium from mouse muscle AChRs. Conclusions and implications:The TE671 binding assay is a useful tool for the evaluation of potential therapeutic agents. The a1:Phe189Ser substitution, but not a1:Pro194Ser, significantly increases the rate of dissociation of vecuronium from mouse muscle AChRs. In contrast, these non-conservative mutations had little effect on EC 50 values. This suggests that the AChR agonist binding site has a robust functional architecture, possibly as a result of evolutionary 'reinforcement'.

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