Pentameric Ligand-Gated Ion Channel ELIC Research Articles

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.

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.