The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors.
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.
Zhang
Ran
R
Department of Pharmacology and Neuroscience, Texas Tech University Health Sciences Center, 3601 Fourth Street, Lubbock, Texas 79430, USA.
Wen
Xiaofei
X
Militante
Julius
J
Hester
Brent
B
Rhubottom
Heather E
HE
Sun
Hongwei
H
Leidenheimer
Nancy J
NJ
Yan
Dong
D
White
Michael M
MM
Machu
Tina K
TK
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
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