The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors

Ran Zhang, Xiaofei Wen, Julius Militante, Brent Hester, Heather E. Rhubottom, Hongwei Sun, Nancy J. Leidenheimer, Dong Yan, Michael M. White, Tina K. Machu

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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.

Original languageEnglish
Pages (from-to)1194-1204
Number of pages11
JournalBiochemistry
Volume46
Issue number5
DOIs
StatePublished - 6 Feb 2007

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Zhang, R., Wen, X., Militante, J., Hester, B., Rhubottom, H. E., Sun, H., Leidenheimer, N. J., Yan, D., White, M. M., & Machu, T. K. (2007). The role of loop F residues in determining differential d-tubocurarine potencies in mouse and human 5-hydroxytryptamine 3A receptors. Biochemistry, 46(5), 1194-1204. https://doi.org/10.1021/bi0616100