15 research outputs found
Tyrosine Residues That Control Binding and Gating in the 5-Hydroxytryptamineâ Receptor Revealed by Unnatural Amino Acid Mutagenesis
The mechanism by which agonist binding triggers pore opening in ligand-gated ion channels is poorly understood. Here, we used unnatural amino acid mutagenesis to introduce subtle changes to the side chains of tyrosine residues (Tyr141, Tyr143, Tyr153, and Tyr234), which dominate the 5-HTâ receptor binding site. Heterologous expression in oocytes, combined with radioligand binding data and a model of 5-HT (serotonin) computationally docked into the binding site, has allowed us to determine which of these residues are responsible for binding and/or gating. We have shown that Tyr 143 forms a hydrogen bond that is essential for receptor gating but does not affect binding, whereas a hydrogen bond formed by Tyr153 is involved in both binding and gating of the receptor. The aromatic group of Tyr234 is essential for binding and gating, whereas its hydroxyl does not affect binding but plays a steric role in receptor gating. Tyr141 is not involved in agonist binding or receptor gating but is important for antagonist interactions. These data, combined with a new model of the nonliganded 5-HTâ receptor, lead to a mechanistic explanation of the interactions that initiate the conformational change leading to channel opening. Thus, we suggest that agonist entry into the binding pocket may displace Tyr143 and Tyr153 and results in their forming new hydrogen bonds. These bonds may form part of the network of bond rearrangements that trigger the conformational change leading to channel opening. Similar rearrangements may initiate gating in all Cys-loop receptors
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 cationâÏ 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 ÏâÏ 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 âaromatic boxâ across the superfamily of Cys-loop receptors reveals some interesting parallels and divergences
Cisâtrans isomerization at a proline opens the pore of a neurotransmitter-gated ion channel
5-Hydroxytryptamine type 3 (5-HT_3) receptors are members of the Cys-loop receptor superfamily. Neurotransmitter binding in these proteins triggers the opening (gating) of an ion channel by means of an as-yet-uncharacterized conformational change. Here we show that a specific proline (Pro 8*), located at the apex of the loop between the second and third transmembrane helices (M2âM3), can link binding to gating through a cisâtrans isomerization of the protein backbone. Using unnatural amino acid mutagenesis, a series of proline analogues with varying preference for the cis conformer was incorporated at the 8* position. Proline analogues that strongly favour the trans conformer produced non-functional channels. Among the functional mutants there was a strong correlation between the intrinsic cisâtrans energy gap of the proline analogue and the activation of the channel, suggesting that cisâtrans isomerization of this single proline provides the switch that interconverts the open and closed states of the channel. Consistent with this proposal, nuclear magnetic resonance studies on an M2âM3 loop peptide reveal two distinct, structured forms. Our results thus confirm the structure of the M2âM3 loop and the critical role of Pro 8* in the 5-HT_3 receptor. In addition, they suggest that a molecular rearrangement at Pro 8* is the structural mechanism that opens the receptor pore
Unnatural amino acid mutagenesis in mapping ion channel function
Unnatural amino acid mutagenesis makes possible the site-specific incorporation of synthetic amino acids, enabling detailed structureâfunction studies as well as the incorporation of biophysical probes. This method has been adapted for use with heterologous expression in Xenopus oocytes, allowing experiments on ion channels
A Cation-Ï Binding Interaction with a Tyrosine in the Binding Site of the GABA_C Receptor
GABA_C (Ï) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HTâ, and glycine receptors. As in other members of this family, the agonist binding site of GABA_C receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-Ï interaction to a tryptophan, the GABA_C binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-Ï binding ability of the side chain and ECâ
â for receptor activation, thus demonstrating a cation-Ï interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-Ï binding energies that reflect the nature of the cationic center of the agonist
A Cation-Ï Binding Interaction with a Tyrosine in the Binding Site of the GABA_C Receptor
GABA_C (Ï) receptors are members of the Cys-loop superfamily of neurotransmitter receptors, which includes nicotinic acetylcholine (nACh), 5-HTâ, and glycine receptors. As in other members of this family, the agonist binding site of GABA_C receptors is rich in aromatic amino acids, but while other receptors bind agonist through a cation-Ï interaction to a tryptophan, the GABA_C binding site has tyrosine at the aligning positions. Incorporating a series of tyrosine derivatives at position 198 using unnatural amino acid mutagenesis reveals a clear correlation between the cation-Ï binding ability of the side chain and ECâ
â for receptor activation, thus demonstrating a cation-Ï interaction between a tyrosine side chain and a neurotransmitter. Comparisons among four homologous receptors show variations in cation-Ï binding energies that reflect the nature of the cationic center of the agonist
CationâÏ Interactions in Ligand Recognition by Serotonergic (5-HT_(3A)) and Nicotinic Acetylcholine Receptors: The Anomalous Binding Properties of Nicotine
A series of tryptophan analogues has been introduced into the binding site regions of two ion channels, the ligand-gated nicotinic acetylcholine and serotonin 5-HT_(3A) receptors, using unnatural amino acid mutagenesis and heterologous expression in Xenopus oocytes. A cationâÏ interaction between serotonin and Trp183 of the serotonin channel 5-HT3AR is identified for the first time, precisely locating the ligand-binding site of this receptor. The energetic contribution of the observed cationâÏ interaction between a tryptophan and the primary ammonium ion of serotonin is estimated to be approximately 4 kcal/mol, while the comparable interaction with the quaternary ammonium of acetylcholine is approximately 2 kcal/mol. The binding mode of nicotine to the nicotinic receptor of mouse muscle is examined by the same technique and found to differ significantly from that of the natural agonist, acetylcholine