In the Cys loop superfamily of ligand-gated ion channels, a global conformational change, initiated by agonist binding, results in channel opening and the passage of ions across the cell membrane. The detailed mechanism of channel gating is a subject that has lent itself to both structural and electrophysiological studies. Here we defined a gating interface that incorporates elements from the ligand binding domain and transmembrane domain previously reported as integral to proper channel gating. An overall analysis of charged residues within the gating interface across the entire superfamily showed a conserved charging pattern, although no specific interacting ion pairs were conserved. We utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study extensively the gating interface of the mouse muscle nicotinic acetylcholine receptor. We found that charge reversal, charge neutralization, and charge introduction at the gating interface are often well tolerated. Furthermore, based on our data and a reexamination of previously reported data on ␥-aminobutyric acid, type A, and glycine receptors, we concluded that the overall charging pattern of the gating interface, and not any specific pairwise electrostatic interactions, controls the gating process in the Cys loop superfamily.The Cys loop superfamily of neurotransmitter-gated ion channels plays a prominent role in mediating fast synaptic transmission. Receptors for acetylcholine (nicotinic ACh receptor, nAChR), 2 serotonin (5-HT 3 receptor), ␥-aminobutyric acid (GABA, types A and C receptors), and glycine are known, and the receptors are classified as excitatory (cation-conducting; nAChR and 5-HT 3 ) or inhibitory (anion-conducting; GABA and glycine). Malfunctions in these receptors are responsible for a number of "channelopathies," and the receptors are targets of pharmaceutical efforts toward treatments for a wide range of neurological disorders, including Alzheimer disease, Parkinson disease, addiction, schizophrenia, and depression (1, 2). The receptors share a common architecture, are significantly homologous, and are known to have evolved from a single ancestral gene that coded for an ACh receptor.The gating mechanism for the Cys loop superfamily is one of the most challenging questions in molecular neuroscience. At issue is how the binding of a small molecule neurotransmitter can induce a structural change in a large, multisubunit, integral membrane protein sufficient to open (gate) a previously closed ion channel contained within the receptor (3, 4). All evidence indicates that the neurotransmitter-binding site is quite remote (50 -60 Å) from the channel gate, the region that blocks the channel when the neurotransmitter is absent and that must move to open the channel.The quest for a gating mechanism has been greatly aided by several recent structural advances. First, crystal structures of the soluble acetylcholine-binding protein (AChBP) (5-7), which is homologous to the extracellular ...

The binding pockets of Cys-loop receptors are dominated by aromatic amino acids. In the GABA A receptor ␣ 1 Phe65, ␤ 2 Tyr97, ␤ 2 Tyr157, and ␤ 2 Tyr205 are present at the ␤ 2 /␣ 1 interface and have been implicated in forming an important part of the GABA binding site. Here, we have probed interactions of these residues using subtle chemical changes: unnatural amino acid mutagenesis was used to introduce a range of Phe analogs, and mutant receptors expressed in oocytes were studied using voltage-clamp electrophysiology. Serial mutations at ␤ 2 97 revealed a ϳ20-fold increase in EC 50

Cys-loop receptor binding sites characteristically contain many aromatic amino acids. In nicotinic ACh and 5-HT3 receptors, a Trp residue forms a cation-π 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-π 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-π binding ability of the fluorinated Phe derivatives at position 159, but not at positions 207 or 63, indicating a single cation-π 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-π interaction between Phe and a neurotransmitter.

Cation-interactions have been demonstrated to play a major role in agonist-binding in Cys-loop receptors. However, neither the aromatic amino acid contributing to this interaction nor its location is conserved among Cys-loop receptors. Likewise, it is not clear how many different agonists of a given receptor form a cation-interaction or, if they do, whether it is with the same aromatic amino acid as the major physiological agonist. We demonstrated previously that Phe159 in the glycine receptor (GlyR) ␣1 subunit forms a strong cation-interaction with the principal agonist, glycine. In the current study, we investigated whether the lower efficacy agonists of the human GlyR ␤-alanine and taurine also form cation-interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-interactions between Phe159 and the amino groups of ␤-alanine and taurine. The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that ␤-alanine and taurine are orientated subtly differently in the binding pocket, with their amino groups further from Phe159 than that of glycine. These data therefore show that similar agonists can have similar but not identical orientations and interactions in the binding pocket and provide a possible explanation for the lower potencies of ␤-alanine and taurine.

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the alphaVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the alphaVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism.