Ligand-Gated Ion Channel ELIC Research Articles

ALLOSTERIC MODULATION OF THE PENTAMERIC LIGAND‐GATED ION CHANNEL ELIC BY FUNCTIONALLY ACTIVE NANOBODIES

Pentameric ligand‐gated ion channels (pLGICs) or Cys‐loop receptors are a class of ion channels involved in fast synaptic transmission in the central and peripheral nervous systems. Members of this family include the nicotinic acetylcholine receptors, serotonin type‐3 receptors, GABAA/C (gamma‐aminobutyric acid) receptors and glycine receptors. These receptors are the target for a wide variety of therapeutics, including benzodiazepines such as diazepam (Valium), which act as positive allosteric modulators (PAMs) of the GABAA receptor and are prescribed as a sedative, anxiolytic or anti‐epileptic. In this study, we employed the prokaryote ligand‐gated ion channel ELIC as a model to understand the structural mechanism of allosteric modulation. We raised nanobodies, which are antibody fragments derived from camelids and have the potential to trap conformationally transient states, and used them as crystallization chaperones for ELIC. Functional characterization of ELIC nanobodies using electrophysiological techniques demonstrated that these nanobodies are active as channel modulators. We demonstrate that nanobodies can either inhibit or potentiate agonist‐evoked channel responses, suggesting that nanobodies can act as negative (NAMs) or positive allosteric modulators (PAMs), respectively. The X‐ray crystal structure of ELIC in complex with a nanobody reveals that a nanobody binds at an allosteric binding site and near to a binding site previously known to mediate potentiation of ELIC by the benzodiazepine flurazepam. Given the structural conservation of allosteric binding sites between ELIC and human receptors our study reveals common mechanisms for allosteric modulation in this family of channels. Our expanded knowledge of the structural determinants of allosteric modulation opens opportunities for the development of new therapeutics targeting pLGICs in ion channel‐related disorders.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Allosteric Modulation of the Pentameric Ligand-Gated Ion Channel ELIC by Barbiturates

Allosteric Modulation of the Pentameric Ligand-Gated Ion Channel ELIC by Barbiturates

Allosteric Modulation of the Pentameric Ligand-Gated Ion Channel ELIC by Functionally Active Nanobodies

Allosteric Modulation of the Pentameric Ligand-Gated Ion Channel ELIC by Functionally Active Nanobodies

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC

Structural Basis of Alcohol Inhibition of the Pentameric Ligand-Gated Ion Channel ELIC

The structural basis for alcohol modulation of neuronal pentameric ligand-gated ion channels (pLGICs) remains elusive. We determined an inhibitory mechanism of alcohol on the pLGIC Erwinia chrysanthemi (ELIC) through direct binding to the pore. X-ray structures of ELIC co-crystallized with 2-bromoethanol, in both the absence and presence of agonist, reveal 2-bromoethanol binding in the pore near T237(6') and the extracellular domain (ECD) of each subunit at three different locations. Binding to the ECD does not appear to contribute to the inhibitory action of 2-bromoethanol and ethanol as indicated by the same functional responses of wild-type ELIC and mutants. In contrast, the ELIC-α1β3GABAAR chimera, replacing the ELIC transmembrane domain (TMD) with the TMD of α1β3GABAAR, ispotentiated by 2-bromoethanol and ethanol. Theresults suggest a dominant role of the TMD in modulating alcohol effects. The X-ray structures andfunctional measurements support a pore-blocking mechanism for inhibitory action of short-chain alcohols.

Direct Pore Binding as a Mechanism for Isoflurane Inhibition of the Pentameric Ligand-gated Ion Channel ELIC.

Pentameric ligand-gated ion channels (pLGICs) are targets of general anesthetics, but molecular mechanisms underlying anesthetic action remain debatable. We found that ELIC, a pLGIC from Erwinia chrysanthemi, can be functionally inhibited by isoflurane and other anesthetics. Structures of ELIC co-crystallized with isoflurane in the absence or presence of an agonist revealed double isoflurane occupancies inside the pore near T237(6′) and A244(13′). A pore-radius contraction near the extracellular entrance was observed upon isoflurane binding. Electrophysiology measurements with a single-point mutation at position 6′ or 13′ support the notion that binding at these sites renders isoflurane inhibition. Molecular dynamics simulations suggested that isoflurane binding was more stable in the resting than in a desensitized pore conformation. This study presents compelling evidence for a direct pore-binding mechanism of isoflurane inhibition, which has a general implication for inhibitory action of general anesthetics on pLGICs.

Conformational Changes Underlying Desensitization of the Pentameric Ligand-Gated Ion Channel ELIC

Structural rearrangements underlying functional transitions of pentameric ligand-gated ion channels (pLGICs) are not fully understood. Using (19)F nuclear magnetic resonance and electron spin resonance spectroscopy, we found that ELIC, a pLGIC from Erwinia chrysanthemi, expanded the extracellular end and contracted the intracellular end of its pore during transition from the resting to an apparent desensitized state. Importantly, the contraction at the intracellular end of the pore likely forms a gate to restrict ion transport in the desensitized state. This gate differs from the hydrophobic gate present in the resting state. Conformational changes of the TM2-TM3 loop were limited to the N-terminal end. The TM4 helices and the TM3-TM4 loop appeared relatively insensitive to agonist-mediated structural rearrangement. These results indicate that conformational changes accompanying functional transitions are not uniform among different ELIC regions. This work also revealed the co-existence of multiple conformations for a given state and suggested asymmetric conformational arrangements in a homomeric pLGIC.

The Prokaryote Ligand-Gated Ion Channel ELIC Captured in a Pore Blocker-Bound Conformation by the Alzheimer’s Disease Drug Memantine

Pentameric ligand-gated ion channels (pLGIC) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules, including the Alzheimer's drug memantine, block ion conduction at vertebrate pLGICs by plugging the channel pore. We show that memantine has similar potency in ELIC, a prokaryotic pLGIC, when it contains an F16'S pore mutation. X-ray crystal structures, using both memantine and its derivative, Br-memantine, reveal that the ligand is localized at the extracellular entryway of the channel pore, and the pore is in a more closed conformation than wild-type ELIC in both the presence and absence of memantine. However, using voltage clamp fluorometry we observe fluorescence changes in opposite directions during channel activation and pore block, revealing an additional conformational transition not apparent from the crystal structures. These results have important implications for drugs such as memantine, which block channel pores.

The Prokaryote Ligand-Gated Ion Channel Elic Captured in a Pore Blocker-Bound Conformation by the Alzheimer's Disease Drug Memantine

Pentameric ligand-gated ion channels (pLGICs) catalyze the selective transfer of ions across the cell membrane in response to a specific neurotransmitter. A variety of chemically diverse molecules block ion conduction by plugging the channel pore. Understanding the structural basis of pore block is important, as some of these molecules, including the antipsychotic chlorpromazine, the local anaesthetic lidocaine and the Alzheimer's disease drug memantine exert their therapeutic effects by specifically blocking these and other receptors. In this study, we report X-ray crystal structures of a prokaryote model ion channel ELIC containing a pore mutation F16'S, which is inhibited by pore blockers with affinities similar to human pLGICs. Structures in complex with memantine and its brominated derivative, Br-memantine, reveal that these pore blockers bind at the extracellular entryway of the channel pore, and cause a structural change, stabilizing the pore-lining helices in a conformation that is more closed than in the unbound structure. In addition, we observe that memantine binds at the agonist binding site, where it can act as a competitive antagonist. We further investigated the conformational dynamics of the pore domain by using voltage clamp fluorometry, which demonstrated that the pore helix orientation changes in opposite directions during pore block and channel activation, consistent with the crystal structures. These data are the first to reveal a conformational change in the pore initiated by a channel blocker, and have important implications for the action of drugs such as memantine which bind to channel pores.

A Unified Model of the GABAA Receptor Comprising Agonist and Benzodiazepine Binding Sites

We present a full-length α1β2γ2 GABA receptor model optimized for agonists and benzodiazepine (BZD) allosteric modulators. We propose binding hypotheses for the agonists GABA, muscimol and THIP and for the allosteric modulator diazepam (DZP). The receptor model is primarily based on the glutamate-gated chloride channel (GluCl) from C. elegans and includes additional structural information from the prokaryotic ligand-gated ion channel ELIC in a few regions. Available mutational data of the binding sites are well explained by the model and the proposed ligand binding poses. We suggest a GABA binding mode similar to the binding mode of glutamate in the GluCl X-ray structure. Key interactions are predicted with residues α1R66, β2T202, α1T129, β2E155, β2Y205 and the backbone of β2S156. Muscimol is predicted to bind similarly, however, with minor differences rationalized with quantum mechanical energy calculations. Muscimol key interactions are predicted to be α1R66, β2T202, α1T129, β2E155, β2Y205 and β2F200. Furthermore, we argue that a water molecule could mediate further interactions between muscimol and the backbone of β2S156 and β2Y157. DZP is predicted to bind with interactions comparable to those of the agonists in the orthosteric site. The carbonyl group of DZP is predicted to interact with two threonines α1T206 and γ2T142, similar to the acidic moiety of GABA. The chlorine atom of DZP is placed near the important α1H101 and the N-methyl group near α1Y159, α1T206, and α1Y209. We present a binding mode of DZP in which the pending phenyl moiety of DZP is buried in the binding pocket and thus shielded from solvent exposure. Our full length GABAA receptor is made available as Model S1.

Inhibition of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC by Divalent Cations

Author SummaryPentameric ligand-gated ion channels (pLGICs) are ionotropic neurotransmitter receptors that mediate electrical signaling at chemical synapses. The pLGIC family includes receptors for acetylcholine, serotonin, GABA and glycine, which share a similar structural organization and activation mechanism: the channels are closed in the absence of ligands and open when neurotransmitters bind to a conserved site in the extracellular domain. In many family members, activation by the neurotransmitter can be affected by modulators (including several drugs in therapeutic use), which bind to different sites on the channel. Channel function can be modulated also by divalent cations, which either potentiate or inhibit pLGICs at physiological concentrations. Here, we analyze this mechanism in the pLGIC ELIC, a prokaryotic family member of known structure. We show that divalent cations such as calcium or zinc inhibit ELIC by occupying an extracellular site remote from the ligand-binding region thereby interfering with gating. Although the site of interaction is not conserved between different family members, we present evidence that regulation of other pLGICs involves the same region. Our study has thus provided insights into a regulatory process that appears to be general for the pLGIC family in both eukaryotes and prokaryotes.

Pentameric ligand-gated ion channel ELIC is activated by GABA and modulated by benzodiazepines.

GABA(A) receptors are pentameric ligand-gated ion channels involved in fast inhibitory neurotransmission and are allosterically modulated by the anxiolytic, anticonvulsant, and sedative-hypnotic benzodiazepines. Here we show that the prokaryotic homolog ELIC also is activated by GABA and is modulated by benzodiazepines with effects comparable to those at GABA(A) receptors. Crystal structures reveal important features of GABA recognition and indicate that benzodiazepines, depending on their concentration, occupy two possible sites in ELIC. An intrasubunit site is adjacent to the GABA-recognition site but faces the channel vestibule. A second intersubunit site partially overlaps with the GABA site and likely corresponds to a low-affinity benzodiazepine-binding site in GABA(A) receptors that mediates inhibitory effects of the benzodiazepine flurazepam. Our study offers a structural view how GABA and benzodiazepines are recognized at a GABA-activated ion channel.

Structure of the pentameric ligand-gated ion channel ELIC cocrystallized with its competitive antagonist acetylcholine

ELIC, the pentameric ligand-gated ion channel from Erwinia chrysanthemi, is a prototype for Cys-loop receptors. Here we show that acetylcholine is a competitive antagonist for ELIC. We determine the acetylcholine–ELIC cocrystal structure to a 2.9-Å resolution and find that acetylcholine binding to an aromatic cage at the subunit interface induces a significant contraction of loop C and other structural rearrangements in the extracellular domain. The side chain of the pore-lining residue F247 reorients and the pore size consequently enlarges, but the channel remains closed. We attribute the inability of acetylcholine to activate ELIC primarily to weak cation-π and electrostatic interactions in the pocket, because an acetylcholine derivative with a simple quaternary-to-tertiary ammonium substitution activates the channel. This study presents a compelling case for understanding the structural underpinning of the functional relationship between agonism and competitive antagonism in the Cys-loop receptors, providing a new framework for developing novel therapeutic drugs.

Ligand Activation of the Prokaryotic Pentameric Ligand-Gated Ion Channel ELIC

While the pentameric ligand-gated ion channel ELIC has recently provided first insight into the architecture of the family at high resolution, its detailed investigation was so far prevented by the fact that activating ligands were unknown. Here we describe a study on the functional characterization of ELIC by electrophysiology and X-ray crystallography. ELIC is activated by a class of primary amines that include the neurotransmitter GABA at high micro- to millimolar concentrations. The ligands bind to a conserved site and evoke currents that slowly desensitize over time. The protein forms cation selective channels with properties that resemble the nicotinic acetylcholine receptor. The high single channel conductance and the comparably simple functional behavior make ELIC an attractive model system to study general mechanisms of ion conduction and gating in this important family of neurotransmitter receptors.