Identification of critical residues in loop E in the 5-HT3ASR binding site

Abstract

Background The serotonin type 3 receptor (5-HT 3 R) is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results Thirteen amino acids in the mouse 5-HT 3AS R that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147) to alanine eliminated binding of the 5-HT 3 R antagonist [ 3 H]granisetron. Three tyrosine residues (Y140, Y142 and Y152) also significantly altered the binding of 5-HT 3 R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT 3AS R. Conclusion Our data supports a role for the putative E-loop region of the 5-HT 3 R in the binding of 5-HT, m CPBG, d -tc and lerisetron. 5-HT and m CPBG interact with Y142, d -tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

Cysteine modification reveals which subunits form the ligand binding site in human heteromeric 5‐HT3AB receptors

“…Several of our A subunit cysteine mutants were non-functional (W90, E129, Y143, Y153 and W183), which confirms the critical importance of these residues as reported elsewhere ( Spier, 2000 ; Yan et al 1999 ; Venkataraman et al 2002 ; Beene et al 2004 ; Price & Lummis, 2004 ; Thompson et al 2005 , 2008 ; Sullivan et al 2006 ; Price et al 2008 ). The other A subunit mutants were all modified by MTSEA, which places them on a solvent accessible surface.…”

“…Cysteine substitution of A subunit residues, and covalent modification of these with MTSEA, confirmed the importance and accessibility of residues that have been previously identified in mutagenesis and modelling studies ( Venkataraman et al 2002 ; Reeves et al 2003 ; Thompson et al 2005 , 2008 ; Yan et al 2006 ). MTSEA had significant effects on receptors containing cysteine-substituted residues in both the principal and complementary faces of A subunits.…”

Subunit rotation models activation of serotonin 5-HT3AB receptors by agonists

“…Tyrosines Y141, Y143 and Y153 in loop E are present in both human A and B subunits but they are not conserved in the superfamily. Their mutations affect the binding of antagonists and activation of 5-HT 3A Rs [20,21]. Accordingly, these tyrosines participated in docking of several antagonists [18] and in the network of hydrogen bonding perturbed by rotation/activation.…”

“…A recent review has summarised several residues, which participate in ligand binding and activation of 5-HT 3A Rs [19]. Further residues such as tyrosines Y141, Y143 and Y153 in loop E [20,21], and glutamates E225 and E236 in loop C [22] also contribute to ligand binding. Finally, R246 in the pre-TM 1 region affects channel gating of 5-HT 3A Rs [23].…”

The Cys-loop superfamily of ligand-gated ion channels: the impact of receptor structure on function

“…A lack of identification does not imply an absence of an involvement, only that no data are available, to our knowledge. Information in this Figure was gathered from [3,18,19,[26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41]55,56,[63][64][65][66][67][68][69][70][71][72][73][74][75][76][77].…”