Involvement of tryptophan residue(s) in the specific binding of agonists/antagonists to 5-HT3 receptors in NG108-15 clonal cells.
Chemical modification of the 5-HT3 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-HT3 receptors. A significant reduction in Bmax (approximately 50%) with no change in Kd were noted on [3H]zacopride specific binding to membranes which were incubated with 40 microM N-bromosuccinimide for 60 min at 25 degrees. The occupancy of 5-HT3 receptor binding sites by various 5-HT3 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-HT3 ligands to the recognition site on 5-HT3 receptors. Nevertheless, these data support the suggestion that tryptophan residue(s) are probably involved in the binding of agonists and antagonists onto 5-HT3 receptors in NG108-15 cell membranes.
INSERM U288, Faculté de Médecine Pitié-Salpêtrière, Paris, France.
M C
MC
Miquel
M B
MB
Emerit
H
H
Gozlan
M
M
Hamon
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001549:Benzamides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D001665:Binding Sites / Q000187:drug effects; D001643:Bridged Bicyclo Compounds / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019086:Bridged Bicyclo Compounds, Heterocyclic; D001974:Bromosuccinimide / Q000494:pharmacology; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006822:Hybrid Cells / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D051381:Rats; D011985:Receptors, Serotonin / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism; D012702:Serotonin Antagonists / Q000378:metabolism / Q000494:pharmacology*; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D001549:Benzamides; D001643:Bridged Bicyclo Compounds; D019086:Bridged Bicyclo Compounds, Heterocyclic; D008562:Membrane Glycoproteins; D011985:Receptors, Serotonin; D012702:Serotonin Antagonists; D014364:Tryptophan; C055971:zacopride; D001974:Bromosuccinimide