Erwinia Ligand-gated Ion Channel Research Articles

Lipid nanodisc scaffold and size alter the structure of a pentameric ligand-gated ion channel

Lipid nanodiscs have become a standard tool for studying membrane proteins, including using single particle cryo-electron microscopy (cryo-EM). We find that reconstituting the pentameric ligand-gated ion channel (pLGIC), Erwinia ligand-gated ion channel (ELIC), in different nanodiscs produces distinct structures by cryo-EM. The effect of the nanodisc on ELIC structure extends to the extracellular domain and agonist binding site. Additionally, molecular dynamic simulations indicate that nanodiscs of different size impact ELIC structure and that the nanodisc scaffold directly interacts with ELIC. These findings suggest that the nanodisc plays a crucial role in determining the structure of pLGICs, and that reconstitution of ion channels in larger nanodiscs may better approximate a lipid membrane environment.

Membrane thickness affects ELIC activity & M4 helix structure.

Pentameric ligand gated ion channels (pLGICs) are a superfamily of ion channels involved in fast neural transmission. Some examples include the GABA(A) receptor and nicotinic acetylcholine receptor (nAchR). In the nAchR, it has been shown that thick membranes promote coupling of agonist binding to channel activation. However, the effect of membrane thickness on the ion transport activity of any pLGIC is not known. To investigate this, we reconstituted the model pLGIC, Erwinia ligand gated ion channel (ELIC), in liposomes consisting of di-monounsaturated phosphatidylcholine (PC) of varying acyl chain length (16-22), and assayed ion flux using a sequential mixing fluorescence quenching assay. ELIC exhibited maximal agonist responses in di-20:1PC liposomes with a dramatic decrease in thinner or thicker membranes. To verify that differences in agonist responses are not a function of changes in channel orientation in the liposomes, we further assayed an ELIC construct with a quenchable fluorescent label in the N-terminal ECD. Minimal differences in the amount of outwardly facing channels were observed in different membrane conditions confirming that changes in agonist responses are primarily a function of channel activity. To investigate the structural basis of ELIC sensitivity to membrane thickness, we obtained apo and agonist-bound structures of ELIC in a circularized nanodisc with di16:1PC, di20:1PC or di22:1PC using single particle cryo-EM. In the agonist-bound structures, the membrane facing helix, M4, is poorly resolved in thin (di16:1PC) and thick (di22:1PC) membranes but clearly resolved in di20:1PC, indicating a correlation between the structure and dynamics of M4 and the optimal membrane thickness that supports channel function. These findings indicate that ELIC function is promoted by thicker membranes and suggests that sensing of membrane thickness is mediated by M4, as has been proposed for the nAchR.

Absolute binding affinities of phospholipids to Erwinia ligand-gated ion channel (ELIC) by streamlined alchemical free energy perturbations (SAFEP).

An increasing number of cryo-EM structures of membrane proteins contain putative lipid binding sites with partially resolved lipid densities. In principle, these densities might be identified by computing the binding free energies of candidate ligands by alchemical free energy perturbation (FEP). In practice, however, these systems have several challenging features including lipid flexibility, system heterogeneity, and low binding affinity. Each of these confounds the distinction between the bound and unbound state: flexible molecules have large accessible volumes in conformation space; heterogeneous environments complicate the unbound state; and open, low-affinity sites mean that a “bound” ligand may still have considerably higher entropy than more in a more traditional, high-affinity binding pocket. Our SAFEP method seeks to address these problems by defining a single collective variable to define the bound state. This distance-from-bound configuration (DBC) metric reliably classifies ligand-protein conformations as “bound” or “unbound.” SAFEP has been successfully applied to the computation of absolute binding affinities of cholesterol to three GPCRs and relative binding affinities of phospholipids to two mutants of ELIC based on structural data. We have extended SAFEP to the calculation of absolute binding affinities of phospholipids. This has required additional sampling of each state and refinement to the collective variables used, especially in the unbound state. While this is somewhat more computationally expensive than computing relative free energies, absolute affinities make functional questions more accessible and mitigate the error propagation inherent to relative binding affinities. It is our hope that these methods will aid in the interpretation of similar systems.

Open-channel structure of a pentameric ligand-gated ion channel reveals a mechanism of leaflet-specific phospholipid modulation

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are sensitive to their lipid environment. The mechanism of phospholipid modulation of any pLGIC is not well understood. We demonstrate that the model pLGIC, ELIC (Erwinia ligand-gated ion channel), is positively modulated by the anionic phospholipid, phosphatidylglycerol, from the outer leaflet of the membrane. To explore the mechanism of phosphatidylglycerol modulation, we determine a structure of ELIC in an open-channel conformation. The structure shows a bound phospholipid in an outer leaflet site, and structural changes in the phospholipid binding site unique to the open-channel. In combination with streamlined alchemical free energy perturbation calculations and functional measurements in asymmetric liposomes, the data support a mechanism by which an anionic phospholipid stabilizes the activated, open-channel state of a pLGIC by specific, state-dependent binding to this site.

Polyunsaturated fatty acids inhibit a pentameric ligand‐gated ion channel through one of two specific binding sites

Polyunsaturated fatty acids (PUFAs) inhibit pentameric ligand-gated ion channels (pLGICs), including the GABAAreceptor (GABAAR) and nicotinic acetylcholine receptor (nAchR). PUFA inhibition of pLGICs is thought to occur through an allosteric mechanism mediated by direct binding to specific sites. However, the sites of PUFA binding, and those sites that mediate channel inhibition are not fully elucidated. We investigated fatty acid binding sites in the prototypic pLGIC, Erwinia ligand-gated ion channel (ELIC), using a novel fatty acid photolabeling reagent, KK-242. The PUFA, docosahexaenoic acid (DHA), inhibited ELIC channel responses, similar to the effect reported in the GABAAR and nAchR. KK-242 also produced an inhibitory effect in ELIC, and photolabeled two binding sites in the outer transmembrane domain (TMD). In contrast, the commercially-available fatty acid photolabeling reagent, pacFA, did not photolabel ELIC. Fatty acid binding to these photolabeled sites was specific for DHA over palmitic acid (PA), and DHA preferentially bound to these sites in the agonist-bound state. Coarse-grained simulations corroborated the selectivity of polyunsaturated fatty acid binding to these sites. Cysteines introduced in the fatty acid binding sites of the outer TMD were efficiently modified by hexadecyl-methanethiosulfonate (hMTS), while inner TMD sites were not accessible to hMTS, possibly due to the presence of tightly bound phospholipids. hMTS modification of only one of these binding sites recapitulated the inhibitory effect of fatty acids on ELIC function. The results demonstrate that PUFAs occupy multiple sites in the outer TMD of ELIC, but that a single intrasubunit binding site mediates PUFA inhibition of ELIC. Additionally, KK-242 is a useful photolabeling reagent for the identification of fatty acid binding sites in other membrane proteins with advantages over currently available reagents.

Fatty Acids Inhibit a Pentameric Ligand-Gated Ion Channel through One of Two Binding Sites

Pentameric ligand-gated ion channels (pLGICs) mediate synaptic transmission and are inhibited by fatty acids. To investigate the structural determinants of fatty acid inhibition of pLGICs, we developed a new photolabeling reagent, KK-242, for the identification of fatty acid binding sites. by giant liposome patch-clamping, fatty acids inhibit the model pLGIC, Erwinia ligand-gated ion channel (ELIC), decreasing peak response to agonist and increasing the rate and extent of current decay. Polyunsaturated fatty acids (PUFAs) are more efficacious than saturated fatty acids. This modulatory effect is similar to previous reports of fatty acid inhibition of the GABA(A) receptor and nicotinic acetylcholine receptor, and is consistent with a model where fatty acids stabilize a pre-activated state. KK-242 also inhibits ELIC. KK-242 photolabels two fatty acid binding sites per subunit, which are also binding sites for docosahexaenoic acid and palmitic acid. To probe the functional significance of these sites, a cysteine was introduced to each site and modified with hexadecyl-methanethiosulfonate (MTS). Modification of one site with hexadecyl-MTS recapitulates the inhibitory effect of fatty acids on ELIC function. The results show that fatty acids bind to two sites, and inhibit ELIC by binding to just one of these sites. KK-242 may be a useful tool for identification of fatty acid binding sites in other membrane proteins.

Charge Reduction of Membrane Proteins in Native Mass Spectrometry Using Alkali Metal Acetate Salts

Native mass spectrometry (MS) provides the capacity to monitor membrane protein complexes and noncovalent binding of ligands and lipids to membrane proteins. The charge states produced by native MS of membrane proteins often result in gas-phase protein unfolding or loss of noncovalent interactions. In an effort to reduce the charge of membrane proteins, we examined the utility of alkali metal salts as a charge-reducing agent. Low concentrations of alkali metal salts caused marked charge reduction in the membrane protein, Erwinia ligand-gated ion channel (ELIC). The charge-reducing effect only occurred for membrane proteins and was detergent-dependent, being most pronounced in long polyethylene glycol (PEG)-based detergents such as C10E5 and C12E8. On the basis of these results, we propose a mechanism for alkali metal charge reduction of membrane proteins. Addition of low concentrations of alkali metals may provide an advantageous approach for charge reduction of detergent-solubilized membrane proteins by native MS.

Modulation of the Erwinia ligand-gated ion channel (ELIC) and the 5-HT3 receptor via a common vestibule site.

Pentameric ligand-gated ion channels (pLGICs) or Cys-loop receptors are involved in fast synaptic signaling in the nervous system. Allosteric modulators bind to sites that are remote from the neurotransmitter binding site, but modify coupling of ligand binding to channel opening. In this study, we developed nanobodies (single domain antibodies), which are functionally active as allosteric modulators, and solved co-crystal structures of the prokaryote (Erwinia) channel ELIC bound either to a positive or a negative allosteric modulator. The allosteric nanobody binding sites partially overlap with those of small molecule modulators, including a vestibule binding site that is not accessible in some pLGICs. Using mutagenesis, we extrapolate the functional importance of the vestibule binding site to the human 5-HT3 receptor, suggesting a common mechanism of modulation in this protein and ELIC. Thus we identify key elements of allosteric binding sites, and extend drug design possibilities in pLGICs with an accessible vestibule site.

Direct binding of phosphatidylglycerol at specific sites modulates desensitization of a ligand-gated ion channel.

Pentameric ligand-gated ion channels (pLGICs) are essential determinants of synaptic transmission, and are modulated by specific lipids including anionic phospholipids. The exact modulatory effect of anionic phospholipids in pLGICs and the mechanism of this effect are not well understood. Using native mass spectrometry, coarse-grained molecular dynamics simulations and functional assays, we show that the anionic phospholipid, 1-palmitoyl-2-oleoyl phosphatidylglycerol (POPG), preferentially binds to and stabilizes the pLGIC, Erwinia ligand-gated ion channel (ELIC), and decreases ELIC desensitization. Mutations of five arginines located in the interfacial regions of the transmembrane domain (TMD) reduce POPG binding, and a subset of these mutations increase ELIC desensitization. In contrast, a mutation that decreases ELIC desensitization, increases POPG binding. The results support a mechanism by which POPG stabilizes the open state of ELIC relative to the desensitized state by direct binding at specific sites.

A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.

Phospholipids are key components of cellular membranes and are emerging as important functional regulators of different membrane proteins, including pentameric ligand-gated ion channels (pLGICs). Here, we take advantage of the prokaryote channel ELIC (Erwinia ligand-gated ion channel) as a model to understand the determinants of phospholipid interactions in this family of receptors. A high-resolution structure of ELIC in a lipid-bound state reveals a phospholipid site at the lower half of pore-forming transmembrane helices M1 and M4 and at a nearby site for neurosteroids, cholesterol or general anesthetics. This site is shaped by an M4-helix kink and a Trp-Arg-Pro triad that is highly conserved in eukaryote GABAA/C and glycine receptors. A combined approach reveals that M4 is intrinsically flexible and that M4 deletions or disruptions of the lipid-binding site accelerate desensitization in ELIC, suggesting that lipid interactions shape the agonist response. Our data offer a structural context for understanding lipid modulation in pLGICs.

Direct Lipid Binding in a Pentameric Ligand-Gated Ion Channel Assessed by Native Mass Spectrometry

Pentameric ligand-gated ion channels (pLGICs) such as the nicotinic acetylcholine receptor or the prokaryotic homologue, Erwinia ligand-gated ion channel (ELIC), require anionic phospholipids for agonist-induced channel activation. Whether this requirement is mediated by direct lipid binding events to specific sites is not known, and understanding this phenomenon is essential for the development of lipophilic allosteric modulators of pLGICs. We used native mass spectrometry (MS) coupled with thermal stability and liposome flux assays to examine direct binding of phospholipids to ELIC and the functional effects of binding. ELIC from E. coli membranes co-purify with bound phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) lipids, showing selectivity for PG lipids. Native MS analysis of de-lipidated ELIC using exogenous phospholipids reveal that anionic phospholipids such as PG directly bind to ELIC with higher affinity than PE or phosphatidylcholine (PC). By correlating the stoichiometry of binding with lipid concentration, we determine that two bound PG lipids per subunit are required to stabilize ELIC from thermal denaturation. To identify the sites of PG binding, single and double mutations of five arginine residues at the transmembrane domain interfacial region were generated. Analysis of these mutants reveal two clusters of positively charged residues at both extracellular and intracellular leaflets that are essential for PG binding and thermal stabilization. Ongoing single particle cryo-EM studies of ELIC reconstituted in nanodiscs of defined lipid composition is anticipated to corroborate these sites of lipid binding, and may reveal the structure of uncoupled and activated/desensitized ELIC channels induced by these lipid-binding events.

Probing Proline Residues in the Prokaryotic Ligand-Gated Ion Channel, ELIC.

Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate pentameric ligand-gated ion channels (pLGICs) and has proven to be a valuable model for understanding the structure and function of this important protein family. There is nevertheless still a question about whether molecular details can be accurately extrapolated from this protein to those found in eukaryotes. Here we explore the role of proline residues (Pros) in ELIC by creating mutant receptors, expressing them in Xenopus laevis oocytes, and using whole-cell voltage-clamp electrophysiology to monitor channel activity. In contrast to eukaryotic pLGICs, proline-to-alanine (Pro-to-Ala) substitution in ELIC mostly resulted in gain of function, and even altering highly conserved Pro residues in M1 and the M2-M3 loop did not ablate function. These substitutions also mostly resulted in ablation of the modulation by Ca2+ observed in wild-type receptors. Substitution of the Pro in the "Cys loop", however, did result in nonfunctional receptors. Probing this residue with noncanonical amino acids revealed a requirement for a substituted amine at this position, as well as a general preference for Pro analogues with greater intrinsic cis biases. We propose there is likely a cis bond at the apex of the Cys loop in this protein, which is consistent with some, but not all, findings from other pLGICs. Overall, the data show that the roles of proline residues are less critical in ELIC than in other pLGICs, supporting other studies that suggest caution must be applied in using data from this prokaryotic receptor to understand molecular details of eukaryotic pLGIC receptor function.

Probing Molecular Interactions in Erwinia Ligand-Gated Ion Channel (ELIC)

The Erwinia ligand-gated ion channel (ELIC) is a bacterial pentameric ligand ion channel (pLGIC) originally identified in the enterobacterium Erwinia chrysanthemii. ELIC is closely related to a large class of neurotransmitter-gated ion channels (Cys-loop receptors) that underpin fast synaptic transmission in vertebrates and whose malfunction underlies a range of neurological disorders. As structures of Cys-loop receptors have lagged well behind those from prokaryotic pLGICs, ELIC, whose structure was solved in 2008, has proved to be an attractive model system. Here we explore molecular interactions in ELIC revealed by its structure. Of particular interest are intra-protein cation-π interactions, of which there are potentially six in each ELIC subunit: R48-W66; R99-F95; R105-Y38; R141-F142; R199-W160; and R301-W224. To probe these, the amino acids in each pair were swopped, using site directed mutagenesis, in addition to creating mutants with single amino acid changes to Ala and its pair. ELIC function was then probed following expression in HEKT cells using a membrane potential sensitive dye in a Flexstation. WT ELIC revealed GABA concentration dependent increases in fluorescence with a pEC50 of 2.97 + 0.01 (M, n > 6). The mutations had a range of effects from ablation of responses with all substituted amino acids (e.g. R105-Y38) to wild type responses with all substituted amino acids (e.g. R48-W66). The nature and importance of the interactions formed by these amino acids in ELIC, and whether the data can be extrapolated to better understand vertebrate Cys loop receptors, will be discussed.

Intramembrane Aromatic Interactions Influence the Lipid Sensitivities of Pentameric Ligand-gated Ion Channels

Although the Torpedo nicotinic acetylcholine receptor (nAChR) reconstituted into phosphatidylcholine (PC) membranes lacking cholesterol and anionic lipids adopts a conformation where agonist binding is uncoupled from channel gating, the underlying mechanism remains to be defined. Here, we examine the mechanism behind lipid-dependent uncoupling by comparing the propensities of two prokaryotic homologs, Gloebacter and Erwinia ligand-gated ion channel (GLIC and ELIC, respectively), to adopt a similar uncoupled conformation. Membrane-reconstituted GLIC and ELIC both exhibit folded structures in the minimal PC membranes that stabilize an uncoupled nAChR. GLIC, with a large number of aromatic interactions at the interface between the outermost transmembrane α-helix, M4, and the adjacent transmembrane α-helices, M1 and M3, retains the ability to flux cations in this uncoupling PC membrane environment. In contrast, ELIC, with a level of aromatic interactions intermediate between that of the nAChR and GLIC, does not undergo agonist-induced channel gating, although it does not exhibit the expected biophysical characteristics of the uncoupled state. Engineering new aromatic interactions at the M4-M1/M3 interface to promote effective M4 interactions with M1/M3, however, increases the stability of the transmembrane domain to restore channel function. Our data provide direct evidence that M4 interactions with M1/M3 are modulated during lipid sensing. Aromatic residues strengthen M4 interactions with M1/M3 to reduce the sensitivities of pentameric ligand-gated ion channels to their surrounding membrane environment.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16′A-, F16′D-, F16′S-, and F16′T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16′ is small. T6′S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16′S, indicating that the inhibitor binds at position 6′, as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16′ is taken into consideration.

Characterising the Ion Selectivity and Calcium Permeability of the Erwinia Ligand-Gated Ion Channel

The Erwinia ligand-gated ion channel (ELIC) is a bacterial GABA-gated cation-selective channel that shares structural homology with vertebrate Cys-loop receptors[1]. Here we investigated the effects of Ca2+ on the functional properties of ELIC using wild-type and mutant receptors. Receptors were expressed in Xenopus oocytes and characterised using two electrode voltage-clamp electrophysiology. GABA concentration-response curves were recorded in ND96 buffer with 0, 1.8, 5 or 10 mM Ca2+. The data show that Ca2+ alters the rate of apparent desensitization and the GABA EC50. To probe ion selectivity the external NaCl concentration was altered and the effects on reversal potential measured by clamping cells between −60 and +100 mV in 10 mV steps. These data showed that ELIC is predominantly cation selective as previously reported[2]. The −1' (E229) residue has been identified as a critical region for controlling cation selectivity in Cys-loop receptors[3][4]. Consistent with this, an E229A mutation in ELIC decreased cation selectivity. When E229A was combined with a mutation at the N20' position (E229A/N250R) ELIC became predominantly anion selective, similar to experiments described for the 5-HT3 receptor[4]. This mutant eliminated the effects of Ca2+ on ELIC, demonstrating that Ca2+ effects are mediated either in the channel or in the intracellular domain.[1] Hilf & Dutzler., 2008, Nature, 452 (7185) 375-9.[2] Zimmermann et al., 2011 PLoS Biol, 9 (6).[3] Galzi et al., 1992 Nature, 359 (6395) 500-5.[4] Thompson & Lummis, 2003 Br J Pharmacol, 140 359-65.

The pharmacological profile of ELIC, a prokaryotic GABA-gated receptor

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of vertebrate Cys-loop ligand-gated ion channels. It is activated by GABA, and this property, combined with its structural similarity to GABAA and other Cys-loop receptors, makes it potentially an excellent model to probe their structure and function. Here we characterise the pharmacological profile of ELIC, examining the effects of compounds that could activate or inhibit the receptor. We confirm that a range of amino acids and classic GABAA receptor agonists do not elicit responses in ELIC, and we show the receptor can be at least partially activated by 5-aminovaleric acid and γ-hydroxybutyric acid, which are weak agonists. A range of GABAA receptor non-competitive antagonists inhibit GABA-elicited ELIC responses including α-endosulfan (IC50 = 17 μM), dieldrin (IC50 = 66 μM), and picrotoxinin (IC50 = 96 μM) which were the most potent. Docking suggested possible interactions at the 2′ and 6′ pore-lining residues, and mutagenesis of these residues supports this hypothesis for α-endosulfan. A selection of compounds that act at Cys-loop and other receptors also showed some efficacy at blocking ELIC responses, but most were of low potency (IC50 > 100 μM). Overall our data show that a number of compounds can inhibit ELIC, but it has limited pharmacological similarity to GLIC and to Cys-loop receptors.