Stephan A. Pless

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-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.

Conformational Variability of the Glycine Receptor M2 Domain in Response to Activation by Different Agonists

Models describing the structural changes mediating Cys loop receptor activation generally give little attention to the possibility that different agonists may promote activation via distinct M2 pore-lining domain structural rearrangements. We investigated this question by comparing the effects of different ligands on the conformation of the external portion of the homomeric α1 glycine receptor M2 domain. Conformational flexibility was assessed by tethering a rhodamine fluorophore to cysteines introduced at the 19′ or 22′ positions and monitoring fluorescence and current changes during channel activation. During glycine activation, fluorescence of the label attached to R19′C increased by ∼20%, and the emission peak shifted to lower wavelengths, consistent with a more hydrophobic fluorophore environment. In contrast, ivermectin activated the receptors without producing a fluorescence change. Although taurine and β-alanine were weak partial agonists at the α1R19′C glycine receptor, they induced large fluorescence changes. Propofol, which drastically enhanced these currents, did not induce a glycine-like blue shift in the spectral emission peak. The inhibitors strychnine and picrotoxin elicited fluorescence and current changes as expected for a competitive antagonist and an open channel blocker, respectively. Glycine and taurine (or β-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22′C residue. Thus, results from two separate labeled residues support the conclusion that the glycine receptor M2 domain responds with distinct conformational changes to activation by different agonists.

Ligand-specific Conformational Changes in the α1 Glycine Receptor Ligand-binding Domain

Understanding the activation mechanism of Cys loop ion channel receptors is key to understanding their physiological and pharmacological properties under normal and pathological conditions. The ligand-binding domains of these receptors comprise inner and outer β-sheets and structural studies indicate that channel opening is accompanied by conformational rearrangements in both β-sheets. In an attempt to resolve ligand-dependent movements in the ligand-binding domain, we employed voltage-clamp fluorometry on α1 glycine receptors to compare changes mediated by the agonist, glycine, and by the antagonist, strychnine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. In the inner β-sheet, we labeled residues in loop 2 and in binding domain loops D and E. At each position, strychnine and glycine induced distinct maximal fluorescence responses. The pre-M1 domain responded similarly; at each of four labeled positions glycine produced a strong fluorescence signal, whereas strychnine did not. This suggests that glycine induces conformational changes in the inner β-sheet and pre-M1 domain that may be important for activation, desensitization, or both. In contrast, most labeled residues in loops C and F yielded fluorescence changes identical in magnitude for glycine and strychnine. A notable exception was H201C in loop C. This labeled residue responded differently to glycine and strychnine, thus underlining the importance of loop C in ligand discrimination. These results provide an important step toward mapping the domains crucial for ligand discrimination in the ligand-binding domain of glycine receptors and possibly other Cys loop receptors.

Unnatural Amino Acids as Probes of Ligand-Receptor Interactions and Their Conformational Consequences

G protein-coupled receptors and ion channels couple a wide range of external stimuli to cellular growth and division, metabolism, motility, and a myriad of intra- and intercellular signaling pathways. G protein-coupled receptors initiate complex, interrelated downstream signaling cascades, whereas rapid ionic flux through channels directly supports membrane excitability and mediates cellular functions through second messengers. Because of these characteristics, these ubiquitous transmembrane proteins are valuable therapeutic targets and have provided fertile ground for the development of leading-edge synthetic and chemical biological approaches. Here we summarize recent advances in the use of site-directed incorporation of unnatural amino acids and chemical probes to study ligand-receptor interactions, determine the location of binding sites, and examine the downstream conformational consequences of ligand binding in G protein-coupled receptors and ion channels.

A cation-π interaction at a phenylalanine residue in the glycine receptor binding site is conserved for different agonists.

Cation-π 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-π 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) α1 subunit forms a strong cation-π interaction with the principal agonist, glycine. In the current study, we investigated whether the lower efficacy agonists of the human GlyR β-alanine and taurine also form cation-π interactions with Phe159. By incorporating a series of unnatural amino acids, we found cation-π interactions between Phe159 and the amino groups of β-alanine and taurine. The strengths of these interactions were significantly weaker than for glycine. Modeling studies suggest that β-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 β-alanine and taurine.

Magnitude of a Conformational Change in the Glycine Receptor β1-β2 Loop Is Correlated with Agonist Efficacy

The efficacy of agonists at Cys-loop ion channel receptors is determined by the rate they isomerize receptors to a pre-open flip state. Once the flip state is reached, the shut-open reaction is similar for low and high efficacy agonists. The present study sought to identify a conformational change associated with the closed-flip transition in the α1-glycine receptor. We employed voltage-clamp fluorometry to compare ligand-binding domain conformational changes induced by the following agonists, listed from highest to lowest affinity and efficacy: glycine > β-alanine > taurine. Voltage-clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Agonist affinity and efficacy correlated inversely with maximum fluorescence magnitudes at labeled residues in ligand-binding domain loops D and E, suggesting that large conformational changes in this region preclude efficacious gating. However, agonist affinity and efficacy correlated directly with maximum fluorescence magnitudes from a label attached to A52C in loop 2, near the transmembrane domain interface. Because glycine experiences the largest affinity increase between closed and flip states, we propose that the magnitude of this fluorescence signal is directly proportional to the agonist affinity increase. In contrast, labeled residues in loops C, F, and the pre-M1 domain yielded agonist-independent fluorescence responses. Our results support the conclusion that a closed-flip conformation change, with a magnitude proportional to the agonist affinity increase from closed to flip states, occurs in the microenvironment of Ala-52.

Illuminating the structure and function of Cys-loop receptors

1 Cys‐loop receptors are an important class of ligand‐gated ion channels. They mediate fast synaptic neurotransmission, are implicated in various ‘channelopathies’ and are important pharmacological targets. Recent progress in X‐ray crystallography and electron microscopy has provided a considerable insight into the structure of Cys‐loop receptors. However, data from these experiments only provide ‘snapshots’ of the proteins under investigation. They cannot provide information about the various conformations the protein adopts during transition from the closed to the open and desensitized states. 2 Voltage‐clamp fluorometry helps overcome this problem by simultaneously monitoring movements at the channel gate (through changes in current) and conformational rearrangements in a domain of interest (through changes in fluorescence) in real time. Thus, the technique can provide information on both transitional and steady state conformations and serves as a real time correlate of the channel structure and its function. 3 Voltage‐clamp fluorometry experiments on Cys‐loop receptors have yielded a large body of data concerning the mechanisms by which agonists, antagonists and modulators act on these receptors. They have shed new light on the conformational mobility of both the ligand‐binding and the transmembrane domain of Cys‐loop receptors.

Contributions of conserved residues at the gating interface of glycine receptors

Glycine receptors (GlyRs) are chloride channels that mediate fast inhibitory neurotransmission and are members of the pentameric ligand-gated ion channel (pLGIC) family. The interface between the ligand binding domain and the transmembrane domain of pLGICs has been proposed to be crucial for channel gating and is lined by a number of charged and aromatic side chains that are highly conserved among different pLGICs. However, little is known about specific interactions between these residues that are likely to be important for gating in α1 GlyRs. Here we use the introduction of cysteine pairs and the in vivo nonsense suppression method to incorporate unnatural amino acids to probe the electrostatic and hydrophobic contributions of five highly conserved side chains near the interface, Glu-53, Phe-145, Asp-148, Phe-187, and Arg-218. Our results suggest a salt bridge between Asp-148 in loop 7 and Arg-218 in the pre-M1 domain that is crucial for channel gating. We further propose that Phe-145 and Phe-187 play important roles in stabilizing this interaction by providing a hydrophobic environment. In contrast to the equivalent residues in loop 2 of other pLGICs, the negative charge at Glu-53 α1 GlyRs is not crucial for normal channel function. These findings help decipher the GlyR gating pathway and show that distinct residue interaction patterns exist in different pLGICs. Furthermore, a salt bridge between Asp-148 and Arg-218 would provide a possible mechanistic explanation for the pathophysiologically relevant hyperekplexia, or startle disease, mutant Arg-218 → Gln.

Ligand- and Subunit-specific Conformational Changes in the Ligand-binding Domain and the TM2-TM3 Linker of α1 β2 γ2 GABAA Receptors

Cys-loop receptor ligand binding sites are located at subunit interfaces where they are lined by loops A–C from one subunit and loops D–F from the adjacent subunit. Agonist binding induces large conformational changes in loops C and F. However, it is controversial as to whether these conformational changes are essential for gating. Here we used voltage clamp fluorometry to investigate the roles of loops C and F in gating the α1 β2 γ2 GABAA receptor. Voltage clamp fluorometry involves labeling introduced cysteines with environmentally sensitive fluorophores and inferring structural rearrangements from ligand-induced fluorescence changes. Previous attempts to define the roles of loops C and F using this technique have focused on homomeric Cys-loop receptors. However, the problem with studying homomeric receptors is that it is difficult to eliminate the possibility of bound ligands interacting directly with attached fluorophores at the same site. Here we show that ligands binding to the β2-α1 interface GABA binding site produce conformational changes at the adjacent subunit interface. This is most likely due to agonist-induced loop C closure directly altering loop F conformation at the adjacent α1-β2 subunit interface. However, as antagonists and agonists produce identical α1 subunit loop F conformational changes, these conformational changes appear unimportant for gating. Finally, we demonstrate that TM2-TM3 loops from adjacent β2 subunits in α1 β2 receptors can dimerize via K24′C disulfides in the closed state. This result implies unexpected conformational mobility in this crucial part of the gating machinery. Together, this information provides new insights into the activation mechanisms of Cys-loop receptors.

Distinct conformational changes in activated agonist-bound and agonist-free glycine receptor subunits

Ligand binding to Cys‐loop receptors produces either global conformational changes that lead to activation or local conformational changes that do not. We found that the fluorescence of a fluorophore tethered to R271C in the extracellular M2 region of the α1 glycine receptor increases during glycine activation but not during ivermectin activation. This prompted the hypothesis that this signal reports a glycine‐mediated conformational change not essential for activation. We tested this by investigating whether the fluorescence signal depended on whether the fluorophore was attached to a glycine‐free or a glycine‐bound subunit. Agonist‐free subunits were created by incorporating T204A and R65K mutations, which disrupted glycine binding to both (+) and (−) subunit interfaces. In heteromeric receptors comprising wild‐type and R65K,T204A,R271C triple‐mutant subunits, the fluorescence response exhibited a drastically reduced glycine sensitivity relative to the current response. Two conclusions can be drawn from this. First, because the labeled glycine‐free subunits were activated by glycine binding to neighboring wild‐type subunits, our results provide evidence for a cooperative activation mechanism. However, because the fluorescent label on glycine‐free subunits does not reflect movements at the channel gate, we conclude that glycine binding also produces a local non‐concerted conformational change that is not essential for receptor activation.