title: The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors.
abstract: The competitive antagonist d-tubocurarine (curare) has greater potency at mouse than at human 5-hydroxytryptamine 3A (5-HT3A) receptors, despite 84% amino acid sequence identity between the receptors. Within the ligand binding domain of this receptor are six loops (A-F). A previous report demonstrated that loop C of the 5-HT3A receptor contributed to differential potency between the receptors [Hope, A. G. et al. (1999) Mol. Pharmacol. 55, 1037-1043]. The present study tested the hypothesis that loop F plays a significant role in conferring interspecies curare potency differences. Wild-type, chimeric, and point mutant 5-HT3A receptors were expressed in Xenopus oocytes, and two-electrode voltage clamp electrophysiological recordings were performed. Our data suggest that loops C and F contribute to curare potency, given that the curare IC50's (concentration of drug that produces 50% inhibition of the response) for chimeric human receptors with substitutions of mouse residues in loop C (40.07 +/- 2.52 nM) or loop F (131.8 +/- 5.95 nM) were intermediate between those for the mouse (12.99 +/- 0.77 nM) and human (1817 +/- 92.36 nM) wild-type receptors. Two human point mutant receptors containing mouse receptor substitutions in loop F (H-K195E or H-V202I) had significantly lower curare IC50's than that of the human receptor. The human double mutant receptor, H-K195E,V202I, had the same curare IC50 (133.8 +/- 6.38 nM) as that of the human receptor containing all six loop F mouse substitutions. These results demonstrate that two loop F residues make a significant contribution in determining curare potency at the 5-HT3A receptor.
authors.affiliation: Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, 3601 Fourth Street, Lubbock, Texas 79430, USA.
authors.forename: Ran
authors.initials: R
authors.lastname: Zhang
authors.forename: Xiaofei
authors.initials: X
authors.lastname: Wen
authors.forename: Julius
authors.initials: J
authors.lastname: Militante
authors.forename: Brent
authors.initials: B
authors.lastname: Hester
authors.forename: Heather E
authors.initials: HE
authors.lastname: Rhubottom
authors.forename: Hongwei
authors.initials: H
authors.lastname: Sun
authors.forename: Nancy J
authors.initials: NJ
authors.lastname: Leidenheimer
authors.forename: Dong
authors.initials: D
authors.lastname: Yan
authors.forename: Michael M
authors.initials: MM
authors.lastname: White
authors.forename: Tina K
authors.initials: TK
authors.lastname: Machu
mesh_terms: D000596:Amino Acids; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D004594:Electrophysiology; D006801:Humans; D020128:Inhibitory Concentration 50; D051379:Mice; D003473:Neuromuscular Nondepolarizing Agents / Q000494:pharmacology; D009865:Oocytes; D017354:Point Mutation; D011985:Receptors, Serotonin / Q000187:drug effects* / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3; D013045:Species Specificity; D014161:Transduction, Genetic; D014403:Tubocurarine / Q000494:pharmacology*; D014981:Xenopus
chemical_list: D000596:Amino Acids; C509409:HTR3A protein, human; C511230:Htr3a protein, mouse; D003473:Neuromuscular Nondepolarizing Agents; D011985:Receptors, Serotonin; D044406:Receptors, Serotonin, 5-HT3; D014403:Tubocurarine