Involvement of tryptophan residue(s) in the specific binding of agonists/antagonists to 5-HT 3 receptors in NG108-15 clonal cells

Abstract

Chemical modification of the 5-HT 3 receptors in membranes from NG108-15 hybridoma cells was achieved using protein modifying reagents specific for various amino acid residues: N-bromosuccinimide for tryptophan, dithiothreitol for cystine, sodium tetrathionate for cysteine, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide and N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline for aspartic and glutamic acids, diethylpyrocarbonate for histidine, tetranitromethane for tyrosine and 2,3-butanedione for arginine. Among all the reagents tested, N-bromosuccinimide produced the largest alteration in the specific binding of [ 3H]zacopride onto 5-HT 3 receptors. A significant reduction in B max, (~50%) with no change in K d were noted on [ 3H]zacopride specific binding to membranes which were incubated with 40 μM N-bromosuccinimide for 60 min at 25°. The occupancy of 5-HT 3 receptor binding sites by various 5-HT 3 agonists and antagonists (phenylbiguanide, ondansetron, granisetron, MDL 72222) prevented, at least partially, any subsequent reduction in [ 3H]zacopride specific binding by N-bromosuccinimide treatment. However, neither m-chloro-phenylbiguanide, among the agonists, nor zacopride, among the antagonists, were able to prevent the effect of N-bromosuccinimide, suggesting that variations might exist in the molecular mechanisms implicated in the binding of 5-HT 3 ligands to the recognition site on 5-HT 3 receptors. Nevertheless, these data support the suggestion that tryptophan residue(s) are probably involved in the binding of agonists and antagonists onto 5-HT 3 receptors in NG108-15 cell membranes.