Defining the roles of Asn-128, Glu-129 and Phe-130 in loop A of the 5-HT3 receptor

Mol Membr Biol. Sep-Oct 2006;23(5):442-51. doi: 10.1080/09687860600831539.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Asparagine / chemistry*
  • Binding Sites
  • Cells, Cultured
  • Fluorescent Antibody Technique
  • Glutamic Acid / chemistry*
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutation
  • Phenylalanine / chemistry*
  • Protein Conformation
  • Protein Structure, Tertiary
  • Receptors, Serotonin, 5-HT3 / chemistry*
  • Receptors, Serotonin, 5-HT3 / genetics
  • Receptors, Serotonin, 5-HT3 / metabolism*
  • Serotonin / metabolism

Substances

  • Receptors, Serotonin, 5-HT3
  • Serotonin
  • Glutamic Acid
  • Phenylalanine
  • Asparagine