A hydrogen bond in loop A is critical for the binding and function of the 5-HT3 receptor.
The binding sites of Cys-loop receptors are formed from at least six loops (A-F). Here we have used mutagenesis, radioligand binding, voltage clamp electrophysiology, and homology modeling to probe the role of two residues in loop A of the 5-HT3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC50 (from approximately 10-fold more potent to approximately 10-fold less potent than that of the wild type), increased the maximal peak current for mCPBG compared to 5-HT (R max) 2-19-fold, and decreased n H, indicating this residue is involved in receptor gating; we propose Asn128 faces away from the binding pocket and plays a role in facilitating transitions between conformational states. Substitutions of Glu129 resulted in functional receptors only when the residue could accept a hydrogen bond, but with both these and other substitutions, no [(3)H]granisetron binding could be detected, indicating a role in ligand binding. We propose that Glu129 faces into the binding pocket, where, through its ability to hydrogen bond, it plays a critical role in ligand binding. Thus, the data support a modified model of the 5-HT3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor.
Price
Kerry L
KL
Department of Biochemistry, University of Cambridge, UK.
Bower
Kiowa S
KS
Thompson
Andrew J
AJ
Lester
Henry A
HA
Dougherty
Dennis A
DA
Lummis
Sarah C R
SC
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001216:Asparagine / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D018698:Glutamic Acid / Q000235:genetics; D006860:Hydrogen Bonding; D008024:Ligands; D051379:Mice; D008954:Models, Biological; D008956:Models, Chemical; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
D008024:Ligands; D044406:Receptors, Serotonin, 5-HT3; D018698:Glutamic Acid; D001216:Asparagine