Purohit_2007_Br.J.Pharmacol_150_920

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]alpha-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 alpha1 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 alpha1:189 and proline at alpha1:194 to serine. However the alpha1: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 alpha1:Phe189Ser substitution, but not alpha1: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'.