A Cation-π Interaction in the Binding Site of the Glycine Receptor Is Mediated by a Phenylalanine Residue

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-{pi} interaction with the agonist, whereas in GABAA receptors, a Tyr performs this role. The glycine receptor binding site, however, contains predominantly Phe residues. Homology models suggest that two of these Phe side chains, Phe159 and Phe207, and possibly a third, Phe63, are positioned such that they could contribute to a cation-{pi} interaction with the primary amine of glycine. Here, we test this hypothesis by incorporation of a series of fluorinated Phe derivatives using unnatural amino acid mutagenesis. The data reveal a clear correlation between the glycine EC50 value and the cation-{pi} binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-{pi} interaction between glycine and Phe159. The data thus provide an anchor point for locating glycine in its binding site, and demonstrate for the first time a cation-{pi} interaction between Phe and a neurotransmitter

Exploring a potential palonosetron allosteric binding site in the 5-HT 3 receptor

Palonosetron (Aloxi) is a potent second generation 5-HT3 receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT3 receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr73, Phe130, Ser 163, and Asp165) and in the 5-HT3B receptor subunit (His73, Phe130, Glu170, and Tyr143) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT3A) and heteromeric (5-HT3AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT3A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking

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-HT_3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC_(50) (from ∼10-fold more potent to ∼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 [^3H]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-HT_3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor

Multiple Tyrosine Residues Contribute to GABA Binding in the GABA_C Receptor Binding Pocket

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

Trans−cis Switching Mechanisms in Proline Analogues and Their Relevance for the Gating of the 5-HT3 Receptor

Trans-cis isomerization of a proline peptide bond is a potential mechanism to open the channel of the 5-HT3 receptor. Here, we have used the metadynamics method to theoretically explore such a mechanism. We have determined the free energy surfaces in aqueous solution of a series of dipeptides of proline analogues and evaluated the free energy difference between the cis and trans isomers. These theoretical results were then compared with data from mutagenesis experiments, in which the response of the 5-HT3 receptor was measured when the proline at the apex of the M2-M3 transmembrane domain loop was mutated. The strong correlation between the experimental and the theoretical data supports the existence of a trans-cis proline switch for opening the 5-HT3 receptor ion channel

A Hydrogen Bond in Loop A Is Critical for the Binding and Function of the 5-HT_3 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-HT_3 receptor: Asn128 and Glu129. The data show that substitution of Asn128, with a range of alternative natural and unnatural amino acids, changed the EC_(50) (from ∼10-fold more potent to ∼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 [^3H]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-HT_3 receptor binding site and show that loop A plays a critical role in both the ligand binding and function of this receptor

Photo-antagonism of the GABAA receptor

Neurotransmitter receptor trafficking is fundamentally important for synaptic transmission and neural network activity. GABAA receptors and inhibitory synapses are vital components of brain function, yet much of our knowledge regarding receptor mobility and function at inhibitory synapses is derived indirectly from using recombinant receptors, antibody-tagged native receptors and pharmacological treatments. Here we describe the use of a set of research tools that can irreversibly bind to and affect the function of recombinant and neuronal GABAA receptors following ultraviolet photoactivation. These compounds are based on the competitive antagonist gabazine and incorporate a variety of photoactive groups. By using site-directed mutagenesis and ligand-docking studies, they reveal new areas of the GABA binding site at the interface between receptor β and α subunits. These compounds enable the selected inactivation of native GABAA receptor populations providing new insight into the function of inhibitory synapses and extrasynaptic receptors in controlling neuronal excitation