State-dependent binding of cholesterol and an anionic lipid to the muscle-type Torpedo nicotinic acetylcholine receptor.

The ability of the Torpedo nicotinic acetylcholine receptor (nAChR) to undergo agonist-induced conformational transitions requires the presence of cholesterol and/or anionic lipids. Here we use recently solved structures along with multiscale molecular dynamics simulations to examine lipid binding to the nAChR in bilayers that have defined effects on nAChR function. We examine how phosphatidic acid and cholesterol, lipids that support conformational transitions, individually compete for binding with phosphatidylcholine, a lipid that does not.

A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor.

Synaptic receptors respond to neurotransmitters by opening an ion channel across the post-synaptic membrane to elicit a cellular response. Here we use recent Torpedo acetylcholine receptor structures and functional measurements to delineate a key feature underlying allosteric communication between the agonist-binding extracellular and channel-gating transmembrane domains. Extensive mutagenesis at this inter-domain interface re-affirms a critical energetically coupled role for the principal α subunit β1-β2 and M2-M3 loops, with agonist binding re-positioning a key β1-β2 glutamate/valine to facilitate the outward motions of a conserved M2-M3 proline to open the channel gate.

Origin of acetylcholine antagonism in ELIC, a bacterial pentameric ligand-gated ion channel.

ELIC is a prokaryotic homopentameric ligand-gated ion channel that is homologous to vertebrate nicotinic acetylcholine receptors. Acetylcholine binds to ELIC but fails to activate it, despite bringing about conformational changes indicative of activation. Instead, acetylcholine competitively inhibits agonist-activated ELIC currents.

The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors.

Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors.

Recent Insight into Lipid Binding and Lipid Modulation of Pentameric Ligand-Gated Ion Channels.

Pentameric ligand-gated ion channels (pLGICs) play a leading role in synaptic communication, are implicated in a variety of neurological processes, and are important targets for the treatment of neurological and neuromuscular disorders. Endogenous lipids and lipophilic compounds are potent modulators of pLGIC function and may help shape synaptic communication. Increasing structural and biophysical data reveal sites for lipid binding to pLGICs.

Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR.

The outermost lipid-exposed α-helix (M4) in each of the homologous α, β, δ, and γ/ε subunits of the muscle nicotinic acetylcholine receptor (nAChR) has previously been proposed to act as a lipid sensor. However, the mechanism by which this sensor would function is not clear. To explore how the M4 α-helix from each subunit in human adult muscle nAChR influences function, and thus explore its putative role in lipid sensing, we functionally characterized alanine mutations at every residue in αM4, βM4, δM4, and εM4, along with both alanine and deletion mutations in the post-M4 region of each subunit.

Conformational transitions and ligand-binding to a muscle-type nicotinic acetylcholine receptor.

Fast synaptic communication requires receptors that respond to the presence of neurotransmitter by opening an ion channel across the post-synaptic membrane. The muscle-type nicotinic acetylcholine receptor from the electric fish, Torpedo, is the prototypic ligand-gated ion channel, yet the structural changes underlying channel activation remain undefined. Here we use cryo-EM to solve apo and agonist-bound structures of the Torpedo nicotinic receptor embedded in a lipid nanodisc.

Ion channels as lipid sensors: from structures to mechanisms.

Ion channels play critical roles in cellular function by facilitating the flow of ions across the membrane in response to chemical or mechanical stimuli. Ion channels operate in a lipid bilayer, which can modulate or define their function. Recent technical advancements have led to the solution of numerous ion channel structures solubilized in detergent and/or reconstituted into lipid bilayers, thus providing unprecedented insight into the mechanisms underlying ion channel-lipid interactions.

The functional role of the αM4 transmembrane helix in the muscle nicotinic acetylcholine receptor probed through mutagenesis and coevolutionary analyses.

The activity of the muscle-type nicotinic acetylcholine receptor (nAChR) is highly sensitive to lipids, but the underlying mechanisms remain poorly understood. The nAChR transmembrane α-helix, M4, is positioned at the perimeter of each subunit in direct contact with lipids and likely plays a central role in lipid sensing. To gain insight into the mechanisms underlying nAChR lipid sensing, we used homology modeling, coevolutionary analyses, site-directed mutagenesis, and electrophysiology to examine the role of the α-subunit M4 (αM4) in the function of the adult muscle nAChR.

Structural basis for the modulation of pentameric ligand-gated ion channel function by lipids.

Pentameric ligand-gated ion channels (pLGICs) play a central role in synaptic communication and are implicated in a plethora of neurological disorders leading to human disease. Membrane lipids are known to modulate pLGIC function, but the mechanisms underlying their effects are poorly understood. Recent structures reveal sites for the binding of membrane lipids to pLGICs, thus providing a structural basis for interpreting functional data on pLGIC-lipid interactions.

An allosteric link connecting the lipid-protein interface to the gating of the nicotinic acetylcholine receptor.

The mechanisms underlying lipid-sensing by membrane proteins is of considerable biological importance. A unifying mechanistic question is how a change in structure at the lipid-protein interface is translated through the transmembrane domain to influence structures critical to protein function. Gating of the nicotinic acetylcholine receptor (nAChR) is sensitive to its lipid environment.

The Role of Cholesterol in the Activation of Nicotinic Acetylcholine Receptors.

Cholesterol is a potent modulator of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Here, we review current understanding of the mechanisms underlying cholesterol-nAChR interactions in the context of increasingly available high-resolution structural and functional data. Cholesterol and other lipids influence function by conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable vs nonactivatable conformations, as well as influencing the rates of transitions between conformational states.

Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.

Although transmembrane helix-helix interactions must be strong enough to drive folding, they must still permit the inter-helix movements associated with conformational change. Interactions between the outermost M4 and adjacent M1 and M3 α-helices of pentameric ligand-gated ion channels have been implicated in folding and function. Here, we evaluate the role of different physical interactions at this interface in the function of two prokaryotic homologs, GLIC and ELIC.

Probing the structure of the uncoupled nicotinic acetylcholine receptor.

In the absence of activating anionic lipids and cholesterol, the nicotinic acetylcholine receptor (nAChR) from Torpedo adopts an uncoupled conformation that does not usually gate open in response to agonist. The uncoupled conformation binds both agonists and non-competitive channel blockers with a lower affinity than the desensitized state, consistent with both the extracellular agonist-binding and transmembrane channel-gating domains individually adopting resting-state like conformations. To test this hypothesis, we characterized the binding of the agonist, acetylcholine, and two fluorescent channel blockers, ethidium and crystal violet, to resting, desensitized and uncoupled nAChRs in reconstituted membranes.

Functional characterization of two prokaryotic pentameric ligand-gated ion channel chimeras - role of the GLIC transmembrane domain in proton sensing.

With the long-term goal of using a chimeric approach to dissect the distinct lipid sensitivities and thermal stabilities of the pentameric ligand-gated ion channels (pLGIC), GLIC and ELIC, we constructed chimeras by cross-combining their extracellular (ECD) and transmembrane (TMD) domains. As expected, the chimera formed between GLIC-ECD and ELIC-TMD (GE) responded to protons, the agonist for GLIC, but not cysteamine, the agonist for ELIC, although GE exhibited a 25-fold decrease in proton-sensitivity relative to wild type. The chimera formed between ELIC-ECD and the GLIC-TMD (EG) was usually toxic, unless it contained a pore-lining Ile9'Ala gain-of-function mutation.

The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels.

The role of the outermost transmembrane α-helix in both the maturation and function of the prokaryotic pentameric ligand-gated ion channels, GLIC and ELIC, was examined by Ala scanning mutagenesis, deletion mutations, and mutant cycle analyses. Ala mutations at the M4-M1/M3 interface lead to loss-of-function phenotypes in GLIC, with the largest negative effects occurring near the M4 C terminus. In particular, two aromatic residues at the M4 C terminus form a network of π-π and/or cation-π interactions with residues on M3 and the β6-β7 loop that is essential for both maturation and function.

Role of the Fourth Transmembrane α Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels.

The gating of pentameric ligand-gated ion channels is sensitive to a variety of allosteric modulators that act on structures peripheral to those involved in the allosteric pathway leading from the agonist site to the channel gate. One such structure, the lipid-exposed transmembrane α helix, M4, is the target of lipids, neurosteroids, and disease-causing mutations. Here we show that M4 interactions with the adjacent transmembrane α helices, M1 and M3, modulate pLGIC function.

Nicotinic acetylcholine receptor-lipid interactions: Mechanistic insight and biological function.

Membrane lipids are potent modulators of the nicotinic acetylcholine receptor (nAChR) from Torpedo. Lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of activatable versus non-activatable conformations, as well as influencing the transitions between these conformational states. Of note, some membranes stabilize an electrically silent uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions.

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.

The role of the M4 lipid-sensor in the folding, trafficking, and allosteric modulation of nicotinic acetylcholine receptors.

With the availability of high resolution structural data, increasing attention has focused on the mechanisms by which drugs and endogenous compounds allosterically modulate nicotinic acetylcholine receptor (nAChR) function. Lipids are potent modulators of the nAChR from Torpedo. Membrane lipids influence nAChR function by both conformational selection and kinetic mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations, as well as influencing the transitions between these conformational states.