Pentameric Ligand-gated Ion Channels Research Articles

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.

A Single Mutation in the Outer Lipid-Facing Helix of a Pentameric Ligand-Gated Ion Channel Affects Channel Function Through a Radially-Propagating Mechanism.

Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic transmission and are crucial drug targets. Their gating mechanism is triggered by ligand binding in the extracellular domain that culminates in the opening of a hydrophobic gate in the transmembrane domain. This domain is made of four α-helices (M1 to M4). Recently the outer lipid-facing helix (M4) has been shown to be key to receptor function, however its role in channel opening is still poorly understood. It could act through its neighboring helices (M1/M3), or via the M4 tip interacting with the pivotal Cys-loop in the extracellular domain. Mutation of a single M4 tyrosine (Y441) to alanine renders one pLGIC—the 5-HT3A receptor—unable to function despite robust ligand binding. Using Y441A as a proxy for M4 function, we here predict likely paths of Y441 action using molecular dynamics, and test these predictions with functional assays of mutant receptors in HEK cells and Xenopus oocytes using fluorescent membrane potential sensitive dye and two-electrode voltage clamp respectively. We show that Y441 does not act via the M4 tip or Cys-loop, but instead connects radially through M1 to a residue near the ion channel hydrophobic gate on the pore-lining helix M2. This demonstrates the active role of the M4 helix in channel opening.

Physiological role for GABAA receptor desensitization in the induction of long-term potentiation at inhibitory synapses

GABAA receptors (GABAARs) are pentameric ligand-gated ion channels distributed throughout the brain where they mediate synaptic and tonic inhibition. Following activation, these receptors undergo desensitization which involves entry into long-lived agonist-bound closed states. Although the kinetic effects of this state are recognised and its structural basis has been uncovered, the physiological impact of desensitization on inhibitory neurotransmission remains unknown. Here we describe an enduring form of long-term potentiation at inhibitory synapses that elevates synaptic current amplitude for 24 h following desensitization of GABAARs in response to agonist exposure or allosteric modulation. Using receptor mutants and allosteric modulators we demonstrate that desensitization of GABAARs facilitates their phosphorylation by PKC, which increases the number of receptors at inhibitory synapses. These observations provide a physiological relevance to the desensitized state of GABAARs, acting as a signal to regulate the efficacy of inhibitory synapses during prolonged periods of inhibitory neurotransmission.

A cationic lipid site at the outward transmembrane face of a pentameric ligand-gated ion channel

A cationic lipid site at the outward transmembrane face of a pentameric ligand-gated ion channel

Markov State Models of Proton- and Gate-Dependent Activation in a Pentameric Ligand-Gated Ion Channel

Markov State Models of Proton- and Gate-Dependent Activation in a Pentameric Ligand-Gated Ion Channel

Markov State Models of Proton- and Pore-Dependent Activation in a Pentameric Ligand-Gated Ion Channel

Markov State Models of Proton- and Pore-Dependent Activation in a Pentameric Ligand-Gated Ion Channel

C-2-Linked Dimeric Strychnine Analogues as Bivalent Ligands Targeting Glycine Receptors.

Strychnine is the prototypic antagonist of glycine receptors, a family of pentameric ligand-gated ion channels. Recent high-resolution structures of homomeric glycine receptors have confirmed the presence of five orthosteric binding sites located in the extracellular subunit interfaces of the receptor complex that are targeted by strychnine. Here, we report the synthesis and extensive pharmacological evaluation of bivalent ligands composed of two strychnine pharmacophores connected by appropriate spacers optimized toward simultaneous binding to two adjacent orthosteric sites of homomeric α1 glycine receptors. In all bivalent ligands, the two strychnine units were linked through C-2 by amide spacers of various lengths ranging from 6 to 69 atoms. Characterization of the compounds in two functional assays and in a radioligand binding assay indicated that compound 11a, with a spacer consisting of 57 atoms, may be capable of bridging the homomeric α1 GlyRs by simultaneous occupation of two adjacent strychnine-binding sites. The findings are supported by docking experiments to the crystal structure of the homomeric glycine receptor. Based on its unique binding mode, its relatively high binding affinity and antagonist potency, and its slow binding kinetics, the bivalent strychnine analogue 11a could be a valuable tool to study the functional properties of glycine receptors.

Asymmetric opening of the homopentameric 5-HT3A serotonin receptor in lipid bilayers

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, ‘coupled’ state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.

State-dependent protein-lipid interactions of a pentameric ligand-gated ion channel in a neuronal membrane.

Pentameric ligand-gated ion channels (pLGICs) are receptor proteins that are sensitive to their membrane environment, but the mechanism for how lipids modulate function under physiological conditions in a state dependent manner is not known. The glycine receptor is a pLGIC whose structure has been resolved in different functional states. Using a realistic model of a neuronal membrane coupled with coarse-grained molecular dynamics simulations, we demonstrate that some key lipid-protein interactions are dependent on the receptor state, suggesting that lipids may regulate the receptor’s conformational dynamics. Comparison with existing structural data confirms known lipid binding sites, but we also predict further protein-lipid interactions including a site at the communication interface between the extracellular and transmembrane domain. Moreover, in the active state, cholesterol can bind to the binding site of the positive allosteric modulator ivermectin. These protein-lipid interaction sites could in future be exploited for the rational design of lipid-like allosteric drugs.

Structural Investigation into Gating and Modulation of Alpha1 Glycine Receptor in Lipid Nanodiscs

Glycine receptors (GlyR) mediate fast chemical transmission of synaptic signals in the central and peripheral nervous system. GlyR are anionic members of pentameric ligand gated ion channels and are central players in motor coordination and pain perception. Potentiation of GlyR activity results in dampening pain signals in the spinal cord making these receptors an attractive target for pain therapy. Here, we present Cryo-EM structures of the full-length alpha1 GlyR reconstituted into lipid nanodiscs that are captured in multiple functional states that include, the unliganded state (closed) and glycine-bound conformations (open and desensitized). in addition, we present conformations of GlyR in various ligand-bound forms that provide insights into the mechanisms underlying allosteric modulation. The functional state assignments were validated by molecular dynamics simulations. A comparison of these states reveals global conformational changes underlying GlyR channel gating and modulation.

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.

The Role of the Outer Lipid-Facing Helix in Cationic Plgics is Dependent on the Cellular Context

The mechanism by which ligand binding causes channel opening in pentameric ligand-gated ion channels (pLGICs) is not yet fully understood. The outer, lipid-facing helix (M4) and the lipid bilayer in which the channel is embedded have emerged as factors that affect channel opening, though details remain to be determined. Alanine mutations in the M4 helix of anionic pLGICs M4 are neutral or detrimental to channel opening, but there has so far been no clear pattern in cationic pLGICs. Alanine mutations in the α7 nicotinic acetylcholine receptor (nAChR) M4 are generally beneficial to channel opening, but are neutral or ablate function 5-HT3A M4. This apparent discrepancy in the importance of M4 led us to assess its role in a third cationic pLGIC, the α4β2 nAChR, using fluorescence and two-electrode voltage clamp to elucidate the determinants of M4 requirement. When expressed in HEK cells, 9 alanine mutations along the length of the M4, of residues of varying charge, size, and aromaticity, ablated function. However, when expressed in Xenopus oocytes, 7 of these 9 were fully functional. Thus the cellular environment contributes to determining whether certain M4 residues are required for function, and we suggest the composition of the lipid bilayer may be of major importance here.

Probing Allosteric Networks in Ligand-Gated Ion Channel Gating by Electrophysiology and Molecular Dynamics in the Model Receptor GLIC

In all kingdoms of life, pentameric ligand-gated ion channels mediate electrical activity in response to chemical stimuli including pH, neurotransmitters, alcohols, and a wide range of pharmacological agents. Recent biochemical and structural studies in this family of receptors have identified putative binding sites for various agonists and modulators, but leave open questions as to the mechanisms of coupling between ligand binding and pore opening. Here, we used voltage-clamp electrophysiology and molecular dynamics to investigate transmembrane gating determinants in GLIC, a structurally accessible model channel from cyanobacteria. As measured by two-electrode voltage-clamp electrophysiology, mutations in the pore or at the subunit interface enhanced proton sensitivity, but had opposing effects on ethanol potentiation. Molecular dynamics simulations indicated these mutations enhanced gating by different mechanisms, primarily involving either extracellular blooming or transmembrane electrostatic networks. Ethanol flooding in closed and open states substantiated the presence of multiple modulatory binding sites, with mutations modifying state-dependent occupancies in different regions. Modifications to both closed- and open-state models were required for stable extended simulations, highlighting tractable uncertainties in experimental structures. Finally, comparative modeling of eukaryotic family members provided a rationale for differential sensitivities to allosteric modulators. These results reveal distinct networks influencing gating and modulation in the pentameric ligand-gated ion channel family, processes likely critical to elucidating channel evolution and designing novel therapeutics.

Solution Structure of the Gloeobacter Violaceus Ligand-Gated Ion Channel Probed by Small-Angle Neutron Scattering

Pentameric ligand-gated ion channels transform chemical signals into electrical ones, a process during which they undergo subtle conformational cycling resulting in rapid, reversible gating of an intrinsic transmembrane pore. The pH-gated bacterial channel GLIC has proved a valuable model system for this receptor family, in part due to its accessibility to X-ray crystallography - which has provided structures of closed, open, and intermediate conformations. However, such models raise critical questions as to whether the X-ray structures represent actual functional states, and if the cycle includes additional conformations such as highly contracted or expanded states. Small-angle neutron scattering (SANS) offers an approach to characterizing solution-phase structure, increasingly applicable to biomolecular problems on this scale. Here we characterized GLIC by SANS, in combination with inline size-exclusion chromatography (SEC), match-out deuterated detergents, and computational modeling of multiple putative structural models. SEC-SANS under resting conditions (pH 7) enabled the characterization of non-aggegated species which fit predictions from resting-state models, particularly following unrestrained molecular dynamics simulation of X-ray structures. Curves collected under activating conditions (pH 3) best fit a mixture of resting- and open-state models, whereas they diverged from expanded-pore models. in addition to supporting less-expanded models of the open state, this work demonstrates that SEC-SANS, in combination with molecular modeling and simulations, can distinguish solution-phase functional states of an ion channel. As neutron source brilliance increases and inline SEC-SANS set-ups become increasingly accessible, such methods will offer valuable tools for the elucidation of receptor conformational cycling and pharmaceutical development.

Molecular Simulations of Hydrophobic Gating of Pentameric Ligand Gated Ion Channels: Insights into Water and Ions.

Ion channels are proteins which form gated nanopores in biological membranes. Many channels exhibit hydrophobic gating, whereby functional closure of a pore occurs by local dewetting. The pentameric ligand gated ion channels (pLGICs) provide a biologically important example of hydrophobic gating. Molecular simulation studies comparing additive vs polarizable models indicate predictions of hydrophobic gating are robust to the model employed. However, polarizable models suggest favorable interactions of hydrophobic pore-lining regions with chloride ions, of relevance to both synthetic carriers and channel proteins. Electrowetting of a closed pLGIC hydrophobic gate requires too high a voltage to occur physiologically but may inform designs for switchable nanopores. Global analysis of ∼200 channels yields a simple heuristic for structure-based prediction of (closed) hydrophobic gates. Simulation-based analysis is shown to provide an aid to interpretation of functional states of new channel structures. These studies indicate the importance of understanding the behavior of water and ions within the nanoconfined environment presented by ion channels.

Recombinant expression and purification of pentameric ligand-gated ion channels for Cryo-EM structural studies.

Pentameric ligand-gated ion channels (pLGICs) are central players in synaptic neurotransmission and are targets to a range of drugs used to treat neurological disorders and pain. pLGICs are intrinsically dynamic membrane proteins that upon stimulation by neurotransmitters, undergo global conformational changes across multiple domains spanning a distance of over 165Å. The inter-domain flexibility, a feature crucial for their function as signal transducers in chemical synapses, has been problematic in the efforts toward determining high-resolution structures. Earlier structural studies tackled this issue with a variety of strategies that included partial truncation of flexible domains and the use of antibodies and small-molecule inhibitors to restrict domain movement. With the recent advances in cryo-electron microscopy and single-particle analysis, many of these limitations have been overcome. Here, we describe the methods used in the recombinant expression and purification of full-length constructs of two members of the pentameric ligand-gated ion channel family and the approaches used for capturing multiple conformations in cryo-EM imaging.

Purification of a native nicotinic receptor.

Nicotinic acetylcholine receptors are members of the Cys-loop superfamily of pentameric ligand-gated ion channels. The electric organ of the Torpedo ray is extraordinarily rich in an acetylcholine receptor that is homologous to the human nicotinic receptor found at the neuromuscular junction. Due to this abundant natural source in the fish and the relatively accessible preparation of the neuromuscular junction (compared to a central synapse), this muscle-type receptor and specifically the fish receptors have long been used as the prototype for study of nicotinic receptors. However, an absence of structural detail at high resolution has limited the chemical interpretation of this archetypal nicotinic receptor. One of the main concerns in preparing receptor for high resolution structural analysis was its documented sensitivity to particular detergents and requirements for specific lipids in order to maintain function after reconstitution in a membrane. Here, we present methods for purifying native nicotinic receptor from Torpedo electric tissue that maintains functionality after reconstitution and that is amenable to high resolution structural analysis. The specific developments we describe include detergent exchange during purification, inclusion of specific lipids during purification and for nanodisc reconstitution, and synthesis of a new affinity reagent for rapid isolation of receptors.

M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor.

Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1-M4), with M2 lining the pore, then M1 and M3, and with M4 outermost and adjacent to the membrane lipids. Despite its remote location, M4 contributes both to receptor assembly and gating in pLGICs where it has been examined. This study probes the role of M4 residues in the α4β2 nAChR using site-directed mutagenesis to individually mutate each residue to alanine, followed by expression in HEK293 cells and then characterization using membrane potential sensitive dye and radioligand binding. Two of the resulting mutant receptors showed altered EC50s, while 13 were nonfunctional, although coexpression with the chaperones RIC3 and nAChO resulted in 4 of these responding to agonist. Of the remaining 9, radioligand binding with epibatidine showed that 8 were expressed, suggesting these residues may play a role in channel opening. These data differ from similar studies in other pLGIC, where few or no Ala mutants in M4 ablate function, and they suggest that the α4β2 nAChR M4 may play a more significant role than in related receptors.

Structure, Function and Physiology of 5-Hydroxytryptamine Receptors Subtype 3.

5-hydroxytryptamine receptor subtype 3 (5-HT3R) is a pentameric ligand-gated ion channel (pLGIC) involved in neuronal signaling. It is best known for its prominent role in gut-CNS signaling though there is growing interest in its other functions, particularly in modulating non-serotonergic synaptic activity. Recent advances in structural biology have provided mechanistic understanding of 5-HT3R function and present new opportunities for the field. This chapter gives a broad overview of 5-HT3R from a physiological and structural perspective and then discusses the specific details of ion permeation, ligand binding and allosteric coupling between these two events. Biochemical evidence is summarized and placed within a physiological context. This perspective underscores the progress that has been made as well as outstanding challenges and opportunities for future 5-HT3R research.

Proline Residues Contribute to Efficient GABAp Receptor Function.

GABAp receptors are homomeric pentameric ligand-gated ion channels (pLGICs) and are useful for probing the molecular details of the mechanism of action in this important protein family. Here, we explore the role of proline (Pro) residues by creating mutant receptors, expressing them in HEK293 cells, and using fluorescent membrane potential sensitive dye to monitor receptor activity. The data revealed that 3 of the Pro-to-alanine substitutions resulted in nonfunctional receptors (one in the Cys-loop, one in loop A and one in the β2-β3 loop), 7 resulted in increased EC50 values, and the remaining 13 resulted in receptors with properties similar to WT receptors. Further exploration of the nonfunctional mutants using expression in Xenopus laevis oocytes and whole-cell voltage-clamp electrophysiology, incorporating both canonical and noncanonical amino acids, revealed that the Pro in the Cys-loop had a preference for analogues with a high intrinsic cis bias, the Pro in loop A required a ring, and the β2-β3 loop Pro contributes to expression. The data from the Cys-loop Pro are consistent with those from other pLGICs, while those of the loop A Pro and some of the other Pros surprisingly differ. Thus, overall, the data clarify the roles of many of the Pros in the GABAp receptor and also suggest that caution must be applied in using data from one receptor to understand molecular details of all pLGICs.