Local and Global Ligand-Induced Changes in the Structure of the GABA<sub>A</sub> Receptor

Muroi, Yukiko; Czajkowski, Cynthia; Jackson, Meyer B.

doi:10.1021/bi060222v

Cited by 45 publications

(91 citation statements)

References 45 publications

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“…Perhaps most interestingly, picrotoxin produced a large ⌬F at one of the labeled positions, providing strong evidence that by binding in the pore it produces global conformational change (17). A more recent study on ␣1␤2 and ␣1␤2␥2 ␥-aminobutyric acid type A receptors showed that the ⌬F magnitude monitored at two binding domain residues exhibited subunit dependence (18). The results from these two studies provide strong evidence for ligand-and subunit-specific conformational changes in the ligand binding domain.…”

Section: Tablementioning

confidence: 81%

“…Our aim is to investigate the conformational changes induced by agonists, antagonists, and allosteric modulators by covalently labeling different residues near the extracellular M2 boundary with the sulfhydrylreactive fluorophores, methanethiosulfonate-rhodamine (MTSR) and tetramethylrhodamine-maleimide (TMRM), and simultaneously measuring current changes and fluorescence changes (15,16). This approach has been employed so far in only a few studies on Cys loop receptors (15,17,18). A particular advantage of the GlyR is that it displays little or no desensitization thereby facilitating the quantitation of ligand-induced fluorescence spectral shifts.…”

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confidence: 99%

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

Pless

Dibas

Lester

et al. 2007

Journal of Biological Chemistry

155

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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 ␣1 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 R19C increased by ϳ20%, 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 ␤-alanine were weak partial agonists at the ␣1R19C 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 ␤-alanine) also produced an increase and a decrease, respectively, in the fluorescence of a label attached to the nearby L22C 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. Glycine receptor (GlyR)4 chloride channels mediate inhibitory neurotransmission in the central nervous system (1). They comprise an assembly of five subunits that are each composed of a large N-terminal extracellular ligand-binding domain and four transmembrane ␣-helices (M1-M4). Cryo-electron microscopy images of the homologous Torpedo electropax nicotinic acetylcholine receptor (nAChR) transmembrane region reveals that the pore-lining M2 domains are kinked radially inward to form a central constriction at the membrane midpoint (2). There is currently a great deal of interest in understanding how M2 domains move to open the channel. Although the original model proposed a drastic rotation of M2 domains about their long axes (3), more recent evidence suggests that there is little if any rotation during receptor activation (4 -6). The precise nature of the structural change remains a matter for debate.The central pore kink is likely to introduce a degree of structural discontinuity because the hydrogen bonds responsible for maintaining ␣-helix rigidity are most likely broken. The kink may therefore act as a swivel enabling the outer half of M2 to move asynchronously with the inner half, where the gate is most likely positioned. Indeed, several lines of evidence suggest gating is mediated by a backbone rearrangement at this midpoint (7-10). In addition, a rate equilibrium fr...

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Section: Tablementioning

confidence: 81%

mentioning

confidence: 99%

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

Pless

Dibas

Lester

et al. 2007

Journal of Biological Chemistry

155

View full text Add to dashboard Cite

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“…A direct ligand-fluorophore interaction cannot explain results from labeled residues in loop 2 or the pre-M1 domain as they lie well away from the ligand-binding site. Direct interactions between fluorophore and ligand are difficult to categorically rule out for the remaining labeled residues in binding loops C, D, E, and F. However, many of the sites investigated here have been studied previously in VCF studies on Cys-loop receptors and in each case a variety of criteria has been used to rule out direct ligand-induced quenching or dequenching events (16,19,21,22,27).…”

Section: Discussionmentioning

confidence: 99%

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

Pless¹,

Lynch

2009

Journal of Biological Chemistry

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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 ␣1-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 > ␤-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. Glycine receptors (GlyRs)3 are pentameric chloride-selective ion channels that mediate fast inhibitory neurotransmission (1). They are members of the Cys-loop receptor family that includes the prototypical nicotinic acetylcholine receptor (nAChR), the ␥-aminobutyric acid type-A receptors (GABA A Rs), and serotonin type-3 receptors (5-HT 3 Rs). Recent structural studies have provided a wealth of information on the structure and function of this receptor family (2-6). In Cys-loop receptors, the ligand-binding domain (LBD) preceding the four transmembrane helices consists of two twisted ␤-sheets. The inner (vestibule facing) ␤-sheet comprises seven ␤-strands, while the outer ␤-sheet is formed by three ␤-strands (3). The ligand binding site is located at the interface of adjacent subunits and is lined by six domains: three loops from the principal and the complementary sides, termed A-C and D-F, respectively (3).GlyRs are activated by endogenous amino acid agonists in the following order of efficacy: glycine Ͼ ␤-alanine Ͼ taurine (7, 8). As these amino acids share considerable structural similarity (Fig. 1A), they are likely to compete for the same binding site (9 -11). A recent ground-breaking study on an intermediate pre-open state, the so-called "flip" state (12), has provided new insights into the mechanism of partial agon...

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“…The excised inside-out patch procedure, which has proven to be useful for cyclic nucleotide-gated channels (Zheng and Zagotta, 2003), is less useful for channels gated by extracellular ligands and therefore has not been employed. There are also indications that covalently bound fluorophores can sense structural changes in the GABA A R that presumably originate at binding interfaces, and then propagate to a non-binding subunit that has the fluorescent label (Muroi et al 2006). Concentration-response relationships are possible when the maximal DF/F (e.g.…”

Section: Voltage-clamp Fluorometrymentioning

confidence: 99%

The structural basis of function in Cys-loop receptors

Thompson

Lester

Lummis

2010

Quart. Rev. Biophys.

326

398

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Abstract. Cys-loop receptors are membrane-spanning neurotransmitter-gated ion channels that are responsible for fast excitatory and inhibitory transmission in the peripheral and central nervous systems. The best studied members of the Cys-loop family are nACh, 5-HT 3 , GABA A and glycine receptors. All these receptors share a common structure of five subunits, pseudo-symmetrically arranged to form a rosette with a central ion-conducting pore. Some are cation selective (e.g. nACh and 5-HT 3 ) and some are anion selective (e.g. GABA A and glycine). Each receptor has an extracellular domain (ECD) that contains the ligand-binding sites, a transmembrane domain (TMD) that allows ions to pass across the membrane, and an intracellular domain (ICD) that plays a role in channel conductance and receptor modulation. Cys-loop receptors are the targets for many currently used clinically relevant drugs (e.g. benzodiazepines and anaesthetics). Understanding the molecular mechanisms of these receptors could therefore provide the catalyst for further development in this field, as well as promoting the development of experimental techniques for other areas of neuroscience.In this review, we present our current understanding of Cys-loop receptor structure and function. The ECD has been extensively studied. Research in this area has been stimulated in recent years by the publication of high-resolution structures of nACh receptors and related proteins, which have permitted the creation of many Cys loop receptor homology models of this region. Here, using the 5-HT 3 receptor as a typical member of the family, we describe how homology modelling and ligand docking can provide useful but not definitive information about ligand interactions. We briefly consider some of the many Cys-loop receptors modulators. We discuss the current understanding of the structure of the TMD, and how this links to the ECD to allow channel gating, and consider the roles of the ICD, whose structure is poorly understood. We also describe some of the current methods that are beginning to reveal the differences between different receptor states, and may ultimately show structural details of transitions between them.

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Local and Global Ligand-Induced Changes in the Structure of the GABA_A Receptor

Cited by 45 publications

References 45 publications

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

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

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

The structural basis of function in Cys-loop receptors

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Local and Global Ligand-Induced Changes in the Structure of the GABAA Receptor

Cited by 45 publications

References 45 publications

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

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

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

The structural basis of function in Cys-loop receptors

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Local and Global Ligand-Induced Changes in the Structure of the GABA_A Receptor