Multiple Tyrosine Residues Contribute to GABA Binding in the GABA(C) Receptor Binding Pocket

Abstract

The ligand binding site of Cys-loop receptors is dominated by aromatic amino acids. In GABA(C) receptors, these are predominantly tyrosine residues, with a number of other aromatic residues located in or close to the binding pocket. Here we examine the roles of these residues using substitution with both natural and unnatural amino acids followed by functional characterization. Tyr198 (loop B) has previously been shown to form a cation-π interaction with GABA; the current data indicate that none of the other aromatic residues form such an interaction, although the data indicate that both Tyr102 and Phe138 may contribute to stabilization of the positively charged amine of GABA. Tyr247 (loop C) was very sensitive to substitution and, combined with data from a model of the receptor, suggest a π-π interaction with Tyr241 (loop C); here again functional data show aromaticity is important. In addition the hydroxyl group of Tyr241 is important, supporting the presence of a hydrogen bond with Arg104 suggested by the model. At position Tyr102 (loop D) size and aromaticity are important; this residue may play a role in receptor gating and/or ligand binding. The data also suggest that Tyr167, Tyr200, and Tyr208 have a structural role while Tyr106, Trp246, and Tyr251 are not critical. Comparison of the agonist binding site "aromatic box" across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences.

Figure 1

Sequence alignments for the ligand binding domain of the GABA_C and other Cys loop receptor subunits. Residues with similar chemical properties are shaded. The approximate locations of the loops that constitute the binding pocket are indicated with black bars. Those residues examined in this study are stared.

Figure 2

(A) Model of the GABA_C receptor binding site showing GABA docked into the binding pocket. For ease of viewing, only two of the five subunits are shown. Inset: Enlargement of the binding pocket showing GABA surrounded by aromatic binding site residues. (B) The location of GABA docked into the GABA_C receptor binding pocket is consistent with a cation-π interaction at Y198 as previously shown. The model reveals there is also the potential for aromatic stabilization of GABA by Tyr102 and Phe138, as suggested by the data. The location of Arg104, which has previously been shown to be critical for GABA binding, is also consistent with the presence of a hydrogen bond with Tyr247 as suggested by the data.

Figure 3

(A) Typical responses to GABA application to mutant GABA_C receptors expressed in oocytes. (B) Typical concentration response data from which the parameters in Tables 2 and 3 are determined.

Figure 4

Location of aromatic residues tested in this study that do not directly contribute to the binding pocket on the principle (LHS) and complementary (RHS) faces. The data show that Tyr167, Tyr200, and Tyr208 are important for correct receptor function suggesting a role in the structure and/or gating of the receptor.