Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor.
The ligand binding pocket of Cys-loop receptors consists of a number of binding loops termed A-F. Here we examine the 5-HT3 receptor loop A residues Asn-128, Glu-129 and Phe-130 using modelling, mutagenesis, radioligand binding and functional studies on HEK 293 cells. Replacement of Asn-128 results in receptors that have wild type [3H]granisetron binding characteristics but large changes (ranging from a five-fold decrease to a 1500-fold increase) in the 5-HT EC50 when compared to wild type receptors. Phe-130 mutant receptors show both increases and decreases in Kd and EC50 values, depending on the amino acid substituted. The most critical of these residues appears to be Glu-129; its replacement with a range of other amino acids results in non-binding and non-functional receptors. Lack of binding and function in some, but not all, of these receptors is due to poor membrane expression. These data suggest that Glu-129 is important primarily for receptor expression, although it may also play a role in ligand binding; Phe-130 is important for both ligand binding and receptor function, and Asn-128 plays a larger role in receptor function than ligand binding. In light of these results, we have created two new homology models of the 5-HT3 receptor, with alternative positions of loop A. In our preferred model Glu-129 and Phe-130 contribute to the binding site, while the location of Asn-128 immediately behind the binding pocket could contribute to the conformation changes that result in receptor gating. This study provides a new model of the 5-HT3 receptor binding pocket, and also highlights the importance of experimental data to support modelling studies.
Sullivan
Nora L
NL
Department of Biochemistry, University of Cambridge, Cambridge, UK.
Thompson
Andrew J
AJ
Price
Kerry L
KL
Lummis
Sarah C R
SC
D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry*; D001665:Binding Sites; D002478:Cells, Cultured; D005455:Fluorescent Antibody Technique; D018698:Glutamic Acid / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry*; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000378:metabolism
D044406:Receptors, Serotonin, 5-HT3; D012701:Serotonin; D018698:Glutamic Acid; D010649:Phenylalanine; D001216:Asparagine