Article thumbnail
Location of Repository

Functional, biochemical and structural characterisation of pentameric ligand-gated ion channels

By Eveline Wijckmans


Pentameric ligand-gated ion channels (pLGICs) are of high clinical relevance due to their central role in fast synaptic neurotransmission in both the central and peripheral nervous system. They are involved in the disease mechanism of numerous disorders including Alzheimer’s disease, myasthenia gravis, epilepsy and startle disease. Unraveling the 3-dimensional structures of pLGICs can provide insights in ligand recognition, channel gating, ion selection and ionpermeation. Consequently, the structural insights can improve our understanding of the pathophysiology and can aid in improving the treatment strategies for pLGIC related disorders. This research project aimed at extending the current knowledge by focusing on a group of novel pLGIC homologues derived from the extremophile Alvinella pompejana and by characterizing a novel allosteric binding site in ELIC. We applied the Basic Local Alignment Search Tool algorithm with human pLGIC sequences as search models to identify seven homologues, named Alpo1-7, in the proteome of the extremophile Alvinella pompejana. Exploring the proteome of Alvinella pompejana was a strategic choice since there are numerous reports that proteins derived from extremophile species tend to be more stable and thus more amenable for challenging structural studies. Based on the sequence analysis, Alpo1-4 were assumed to be cation-selective with close similarity to nicotinic acetylcholine receptors and to lesser extend to 5-HT3A receptors, while Alpo5-7 appeared to be more closely related to anion-selective pLGICs among which glycine receptors and GABAA receptors. To confirm those functional hypotheses, we performed a ligand screen with two-electrode voltage clamp electrophysiology and found that Alpo5 and Alpo6 can be activated by GABA, glycine and taurine and thus, as expected based on sequence analysis, that they are closely related to human glycine receptors. Moreover, we could confirm the activation of Alpo7 by elevated proton concentrations, as described earlier by Juneja et al. Unfortunately, were not able to confirm the function as pLGICs of the presumably cation-selective channels Alpo1-4 with the high-throughput screening technique. Therefore, we did initial patch clamp electrophysiology experiments with purified, detergent-solubilized Alpo4. Those experiments provided a first glimpse of the functional activation of Alpo4 by the typical nicotinic acetylcholine receptor agonists acetylcholine, nicotine and choline. Moreover, the findings were supported with additional calcium imaging data. Structural studies with pLGICs, or with any membrane protein in general, are particularly challenging due to difficulties in recombinant expression, membrane extraction, stable purification and eventual structure determination. All seven identified homologues, Alpo1-7, could be readily expressed in Spodoptera frugiperda 9 insect cells. Five of them, Alpo1, Alpo4, Alpo6 and Alpo7, could be extracted out of the membrane in a stable oligomeric state by a wide variety of detergents. With Alpo1, Alpo4 and Alpo6, we were able to tackle the next bottleneck, being the stable purification consisting of immobilized metal affinity chromatography and subsequent size exclusion chromatography. Those results indicate that these proteins, derived from an extremophile species, display favorable biochemical characteristics and thus, that they have great potential to succeed in the atomic resolution structural studies. For structural studies within this PhD project, we focused mainly on Alpo4. Despite excellent biochemical behavior, the protein appears to be resistant to crystallization, a phenomenon that confronts many membrane protein crystallographers. Luckily, recent advances in cryo-electron microscopy (cryo-EM) offer a valuable alternative for high-resolution structure determination. Ongoing cryo-EM studies with Alpo4 indicate that the protein sample, resulting in high-contrast monographs displaying monodisperse protein with an equal representation of side and top views, is particularly promising for high-resolution structure determination with cryo-EM. Insights in allosteric modulation of pLGICs are notably interesting as allosteric modulators tweak receptor function while preserving the normal fluctuations in neurotransmitter signaling at the synapse, which holds promising therapeutic potential. In the second part of the research, we focused on the investigation of molecular determinants of chlorpromazine modulation in ELIC. ELIC is a prokaryotic pLGIC, which has been optimized for crystallization and therefore, often functions as a model system for studying structural determinants of function in pLGICs. Chlorpromazine is a frequently used drug to treat psychotic disorders and was previously used as a tool to probe the ion channel pore of nicotinic acetylcholine receptors. In this study, we unveiled that chlorpromazine binds to ELIC at a novel allosteric site, behind the functional and structural important β8-β9 loop. Insights originate from a combination of X-ray crystallographic studies with ELIC in complex with chlorpromazine or its brominated derivate, bromopromazine, electrophysiological experiments based on the substituted-cysteine accessibility method and molecular dynamics simulations. The novel allosteric binding site is part of the almost continuous path of allosteric sites in pLGICs stretching from one extreme end at the top of the N-terminal α-helix to the bottom of the intracellular entrance of the channel pore.AKNOWLEDGEMENTS I Table of Contents I List of Abbreviations III Samenvatting VII Summary IX 1 General introduction 1 1.1 Synaptic neurotransmission 1 1.2 Pentameric ligand-gated ion channels 2 1.2.1 pLGICs are present among prokaryotes and eukaryotes, sharing a conserved architectural fold 4 1.2.2 pLGICs can adopt 3 different functional conformations 6 1.2.3 The structural basis of pLGIC function 6 1.2.4 Diseases associated with human pLGICs 25 2 Objectives 31 3 Functional, Biochemical and Structural characterization of Alvinella pompejana Cys-loop receptor homologues 33 3.1 Abstract 33 3.2 Introduction 34 3.3 Materials & Methods 35 3.3.1 Bio-informatics 35 3.3.2 Construct design 36 3.3.3 Expression 37 3.3.4 Two-electrode voltage clamp 38 3.3.5 Functional characterization of Alpo4 39 3.3.6 Detergent screen 40 3.3.7 Large-scale purification 41 3.3.8 FSEC-based thermostability assay 41 3.3.9 Biochemical optimization for crystallization 42 3.3.10 Crystallization 42 3.3.11 Negative stain electron microscopy 42 3.3.12 Single particle cryo electron microscopy 43 3.4 Results 44 3.4.1 Seven CLR homologues are identified in the proteome of Alvinella pompejana 44 3.4.2 Alpo5-wt and Alpo6-wt can be activated by glycine, GABA and taurine 45 3.4.3 Alpo4 can be activated by acetylcholine, nicotine and choline 47 3.4.4 Several Alvinella pompejana CLR homologues can be purified in a monodisperse, oligomeric state 48 3.4.5 Purified Alvinella pompejana CLR homologues are thermostable 54 3.4.6 The flexibility of Alpo4 can be drastically decreased with limited proteolysis 55 3.4.7 Crystallization 56 3.4.8 Initial structural models of Alpo4, determined with electron microscopy 56 3.5 Discussion and future perspectives 57 3.6 Supporting information 62 4 An allosteric binding site in a Cys-loop receptor ligand-binding domain unveiled in the crystal structure of ELIC in complex with chlorpromazine 63 4.1 Abstract 63 4.2 Introduction 63 4.3 Materials & Methods 65 4.3.1 Chlorpromazine and analogues 65 4.3.2 Protein expression and crystallization 65 4.3.3 X-ray crystal structure determination 66 4.3.4 Mutagenesis and two-electrode voltage clamp recordings 66 4.3.5 Modification of engineered Cys residues in ELIC with MTS-PZ 67 4.3.6 Molecular Dynamics Simulations of ELIC 68 4.4 Results 68 4.4.1 X-ray crystal structures of ELIC in complex with chlorpromazine or bromopromazine 68 4.4.2 Conformational change of the β8-β9 loop 71 4.4.3 Cysteine-scanning mutagenesis of the β8-β9 loop binding site 72 4.4.4 Binding stability of chlorpromazine 74 4.5 Discussion 76 4.6 Supporting information 81 5 Concluding discussion 85 6 References 89 Author contributions a Acknowledgements a Conflict of interest a Professional career cnrpages: 100status: publishe

Topics: pentameric ligand-gated ion channels, cys-loop receptors
Year: 2017
OAI identifier:
Provided by: Lirias
Download PDF:
Sorry, we are unable to provide the full text but you may find it at the following location(s):
  • (external link)
  • (external link)
  • Suggested articles

    To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.