Grutter, T. et al. Molecular tuning of fast gating in pentameric ligand-gated ion channels. Proc. Natl Acad. Sci. USA 102, 18207-18212

Laboratoire Récepteurs et Cognition, Institut Pasteur, Paris, France.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 01/2006; 102(50):18207-12. DOI: 10.1073/pnas.0509024102
Source: PubMed


Neurotransmitters such as acetylcholine (ACh) and glycine mediate fast synaptic neurotransmission by activating pentameric ligand-gated ion channels (LGICs). These receptors are allosteric transmembrane proteins that rapidly convert chemical messages into electrical signals. Neurotransmitters activate LGICs by interacting with an extracellular agonist-binding domain (ECD), triggering a tertiary/quaternary conformational change in the protein that results in the fast opening of an ion pore domain (IPD). However, the molecular mechanism that determines the fast opening of LGICs remains elusive. Here, we show by combining whole-cell and single-channel recordings of recombinant chimeras between the ECD of alpha7 nicotinic receptor (nAChR) and the IPD of the glycine receptor (GlyR) that only two GlyR amino acid residues of loop 7 (Cys-loop) from the ECD and at most five alpha7 nAChR amino acid residues of the M2-M3 loop (2-3L) from the IPD control the fast activation rates of the alpha7/Gly chimera and WT GlyR. Mutual interactions of these residues at a critical pivot point between the agonist-binding site and the ion channel fine-tune the intrinsic opening and closing rates of the receptor through stabilization of the transition state of activation. These data provide a structural basis for the fast opening of pentameric LGICs.

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    • "Given that certain Cys-loop receptors are gated by glycine, while others are activated by the chemically and structurally very different serotonin, there must naturally be a substantial degree of divergence at critical agonist-binding side chains in the agonist-binding site. Not surprisingly then, most of the ECD side chains that are absolutely conserved lie outside of the agonist-binding loops (Hibbs and Gouaux, 2011), e.g., in the eponymous Cys-loop that is situated between the ECD and the membrane-spanning domain, where it transduces conformational changes from the agonist-binding site to the channel (Kash et al., 2003; Grutter et al., 2005). Only two side chains/motives within agonist-binding loops are conserved across all Cys-loop "
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    ABSTRACT: Cys-loop receptors are ligand-gated ion channels that are activated by a structurally diverse array of neurotransmitters, including acetylcholine, serotonin, glycine, and GABA. After the term "chemoreceptor" emerged over 100 years ago, there was some wait until affinity labeling, molecular cloning, functional studies, and X-ray crystallography experiments identified the extracellular interface of adjacent subunits as the principal site of agonist binding. The question of how subtle differences at and around agonist-binding sites of different Cys-loop receptors can accommodate transmitters as chemically diverse as glycine and serotonin has been subject to intense research over the last three decades. This review outlines the functional diversity and current structural understanding of agonist-binding sites, including those of invertebrate Cys-loop receptors. Together, this provides a framework to understand the atomic determinants involved in how these valuable therapeutic targets recognize and bind their ligands.
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    • "Previous work has shown that loose packing of the β-sandwich hydrophobic core in the extracellular binding domain of eukaryotic pLGICs is a unique structural feature that is absent in the prokaryotic channels, and contributes to their ability to rapidly switch from closed to open channel states when agonists are bound [24]. Additionally, an interaction between the eponymous cys-loop and the M2-M3 loop in eukaryotic pLGICs has been implicated in the control of fast gating [25]. The sequences of these elements are poorly conserved between eukaryotic and prokaryotic channels suggesting that the interaction may not be optimal in prokaryotic channels, leaving the extracellular domain and the ion channel gate poorly coupled. "
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    ABSTRACT: Electrochemical signaling in the brain depends on pentameric ligand-gated ion channels (pLGICs). Recently, crystal structures of prokaryotic pLGIC homologues from Erwinia chrysanthemi (ELIC) and Gloeobacter violaceus (GLIC) in presumed closed and open channel states have been solved, which provide insight into the structural mechanisms underlying channel activation. Although structural studies involving both ELIC and GLIC have become numerous, thorough functional characterizations of these channels are still needed to establish a reliable foundation for comparing kinetic properties. Here, we examined the kinetics of ELIC and GLIC current activation, desensitization, and deactivation and compared them to the GABAA receptor, a prototypic eukaryotic pLGIC. Outside-out patch-clamp recordings were performed with HEK-293T cells expressing ELIC, GLIC, or α1β2γ2L GABAA receptors, and ultra-fast ligand application was used. In response to saturating agonist concentrations, we found both ELIC and GLIC current activation were two to three orders of magnitude slower than GABAA receptor current activation. The prokaryotic channels also had slower current desensitization on a timescale of seconds. ELIC and GLIC current deactivation following 25 s pulses of agonist (cysteamine and pH 4.0 buffer, respectively) were relatively fast with time constants of 24.9±5.1 ms and 1.2±0.2 ms, respectively. Surprisingly, ELIC currents evoked by GABA activated very slowly with a time constant of 1.3±0.3 s and deactivated even slower with a time constant of 4.6±1.2 s. We conclude that the prokaryotic pLGICs undergo similar agonist-mediated gating transitions to open and desensitized states as eukaryotic pLGICs, supporting their use as experimental models. Their uncharacteristic slow activation, slow desensitization and rapid deactivation time courses are likely due to differences in specific structural elements, whose future identification may help uncover mechanisms underlying pLGIC gating transitions.
    Full-text · Article · Nov 2013 · PLoS ONE
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    • "One would expect the switch from a low-affinity tense state to a higher affinity, more relaxed state to underlie the gating process in other members of the Cys-loop receptor superfamily, because of their shared molecular design, implying conservation of function. Functional conservation has been demonstrated convincingly by the creation of chimaeric receptors that combine distinct regions of different superfamily members to build new receptors having the functional properties of both (Eiselé et al. 1993; Grutter et al. 2005). On the other hand, less basic aspects of the mechanism, such as the degree and nature of participation by each subunit, must certainly differ from one superfamily member to the next. "
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