68 results on '"Baenziger JE"'
Search Results
2. State-dependent binding of cholesterol and an anionic lipid to the muscle-type Torpedo nicotinic acetylcholine receptor.
- Author
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Ananchenko A, Gao RY, Dehez F, and Baenziger JE
- Subjects
- Animals, Torpedo metabolism, Phospholipids, Muscles metabolism, Phosphatidylcholines, Cholesterol metabolism, Receptors, Nicotinic metabolism
- Abstract
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. We also examine how the two lipids work synergistically to stabilize an agonist-responsive nAChR. We identify rapidly exchanging lipid binding sites, including both phospholipid sites with a high affinity for phosphatidic acid and promiscuous cholesterol binding sites in the grooves between adjacent transmembrane α-helices. A high affinity cholesterol site is confirmed in the inner leaflet framed by a key tryptophan residue on the MX α-helix. Our data provide insight into the dynamic nature of lipid-nAChR interactions and set the stage for a detailed understanding of the mechanisms by which lipids facilitate nAChR function at the neuromuscular junction., (© 2024. The Author(s).)
- Published
- 2024
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3. A release of local subunit conformational heterogeneity underlies gating in a muscle nicotinic acetylcholine receptor.
- Author
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Thompson MJ, Mansoub Bekarkhanechi F, Ananchenko A, Nury H, and Baenziger JE
- Subjects
- Ion Channel Gating physiology, Molecular Conformation, Receptors, Cholinergic metabolism, Muscles metabolism, Receptors, Nicotinic genetics, Receptors, Nicotinic metabolism
- Abstract
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. Notably, the analogous structures in non-α subunits adopt a locally active-like conformation in the apo state even though each L9' hydrophobic gate residue in each pore-lining M2 α-helix is closed. Agonist binding releases local conformational heterogeneity transitioning all five subunits into a conformationally symmetric open state. A release of conformational heterogeneity provides a framework for understanding allosteric communication in pentameric ligand-gated ion channels., (© 2024. The Author(s).)
- Published
- 2024
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4. Origin of acetylcholine antagonism in ELIC, a bacterial pentameric ligand-gated ion channel.
- Author
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Slobodyanyuk M, Banda-Vázquez JA, Thompson MJ, Dean RA, Baenziger JE, Chica RA, and daCosta CJB
- Subjects
- Acetylcholine, Cholinergic Antagonists, Binding Sites, Ligand-Gated Ion Channels genetics, Ligand-Gated Ion Channels chemistry, Receptors, Nicotinic genetics, Receptors, Nicotinic metabolism
- Abstract
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. What makes acetylcholine an agonist in an acetylcholine receptor context, and an antagonist in an ELIC context, is not known. Here we use available structures and statistical coupling analysis to identify residues in the ELIC agonist-binding site that contribute to agonism. Substitution of these ELIC residues for their acetylcholine receptor counterparts does not convert acetylcholine into an ELIC agonist, but in some cases reduces the sensitivity of ELIC to acetylcholine antagonism. Acetylcholine antagonism can be abolished by combining two substitutions that together appear to knock out acetylcholine binding. Thus, making the ELIC agonist-binding site more acetylcholine receptor-like, paradoxically reduces the apparent affinity for acetylcholine, demonstrating that residues important for agonist binding in one context can be deleterious in another. These findings reinforce the notion that although agonism originates from local interactions within the agonist-binding site, it is a global property with cryptic contributions from distant residues. Finally, our results highlight an underappreciated mechanism of antagonism, where agonists with appreciable affinity, but negligible efficacy, present as competitive antagonists., (© 2022. The Author(s).)
- Published
- 2022
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5. The molecular mechanism of snake short-chain α-neurotoxin binding to muscle-type nicotinic acetylcholine receptors.
- Author
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Nys M, Zarkadas E, Brams M, Mehregan A, Kambara K, Kool J, Casewell NR, Bertrand D, Baenziger JE, Nury H, and Ulens C
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Bungarotoxins metabolism, Elapidae, Humans, Muscles metabolism, Neurotoxins chemistry, Receptors, Nicotinic metabolism
- Abstract
Bites by elapid snakes (e.g. cobras) can result in life-threatening paralysis caused by venom neurotoxins blocking neuromuscular nicotinic acetylcholine receptors. Here, we determine the cryo-EM structure of the muscle-type Torpedo receptor in complex with ScNtx, a recombinant short-chain α-neurotoxin. ScNtx is pinched between loop C on the principal subunit and a unique hairpin in loop F on the complementary subunit, thereby blocking access to the neurotransmitter binding site. ScNtx adopts a binding mode that is tilted toward the complementary subunit, forming a wider network of interactions than those seen in the long-chain α-Bungarotoxin complex. Certain mutations in ScNtx at the toxin-receptor interface eliminate inhibition of neuronal α7 nAChRs, but not of human muscle-type receptors. These observations explain why ScNtx binds more tightly to muscle-type receptors than neuronal receptors. Together, these data offer a framework for understanding subtype-specific actions of short-chain α-neurotoxins and inspire strategies for design of new snake antivenoms., (© 2022. The Author(s).)
- Published
- 2022
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6. Distinct functional roles for the M4 α-helix from each homologous subunit in the heteropentameric ligand-gated ion channel nAChR.
- Author
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Thompson MJ, Domville JA, Edrington CH, Venes A, Giguère PM, and Baenziger JE
- Subjects
- Adult, Alanine, Humans, Membrane Lipids chemistry, Protein Conformation, alpha-Helical, Ligand-Gated Ion Channels chemistry, Receptors, Nicotinic metabolism
- Abstract
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. Although no critical interactions involving residues on M4 or in post-M4 were identified, we found that numerous mutations at the M4-M1/M3 interface altered the agonist-induced response. In addition, homologous mutations in M4 in different subunits were found to have different effects on channel function. The functional effects of multiple mutations either along M4 in one subunit or at homologous positions of M4 in different subunits were also found to be additive. Finally, when characterized in both Xenopus oocytes and human embryonic kidney 293T cells, select αM4 mutations displayed cell-specific phenotypes, possibly because of the different membrane lipid environments. Collectively, our data suggest different functional roles for the M4 α-helix in each heteromeric nAChR subunit and predict that lipid sensing involving M4 occurs primarily through the cumulative interactions at the M4-M1/M3 interface, as opposed to the alteration of specific interactions that are critical to channel function., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2022
- Full Text
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7. Recent Insight into Lipid Binding and Lipid Modulation of Pentameric Ligand-Gated Ion Channels.
- Author
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Ananchenko A, Hussein TOK, Mody D, Thompson MJ, and Baenziger JE
- Subjects
- Binding Sites, Lipids, Ligand-Gated Ion Channels chemistry, Ligand-Gated Ion Channels metabolism
- Abstract
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. Here, we update our evolving understanding of pLGIC-lipid interactions highlighting newly identified modes of lipid binding along with the mechanistic understanding derived from the new structural data.
- Published
- 2022
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8. Conformational transitions and ligand-binding to a muscle-type nicotinic acetylcholine receptor.
- Author
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Zarkadas E, Pebay-Peyroula E, Thompson MJ, Schoehn G, Uchański T, Steyaert J, Chipot C, Dehez F, Baenziger JE, and Nury H
- Subjects
- Animals, Binding Sites, Ligands, Muscles, Torpedo metabolism, Ligand-Gated Ion Channels metabolism, Receptors, Nicotinic metabolism
- Abstract
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. Using both a direct biochemical assay to define the conformational landscape and molecular dynamics simulations to assay flux through the pore, we correlate structures with functional states and elucidate the motions that lead to pore activation of a heteromeric nicotinic receptor. We highlight an underappreciated role for the complementary subunit in channel gating, establish the structural basis for the differential agonist affinities of α/δ versus α /γ sites, and explain why nicotine is less potent at muscle nicotinic receptors compared to neuronal ones., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)
- Published
- 2022
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9. IUPAB 2021 Symposium 13: ion channels and membrane transporters.
- Author
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Baenziger JE, Ananchenko A, Hussein TOK, and Mody D
- Abstract
Competing Interests: Conflict of interestThe authors declare no competing interests.
- Published
- 2021
- Full Text
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10. Author Correction: Ion channels as lipid sensors: from structures to mechanisms.
- Author
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Thompson MJ and Baenziger JE
- Published
- 2021
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11. Ion channels as lipid sensors: from structures to mechanisms.
- Author
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Thompson MJ and Baenziger JE
- Subjects
- Animals, Binding Sites, Cell Communication, Cell Membrane chemistry, Eukaryotic Cells chemistry, Eukaryotic Cells metabolism, G Protein-Coupled Inwardly-Rectifying Potassium Channels genetics, G Protein-Coupled Inwardly-Rectifying Potassium Channels metabolism, Humans, Lipid Bilayers chemistry, Mammals, Models, Molecular, Potassium Channels, Tandem Pore Domain genetics, Potassium Channels, Tandem Pore Domain metabolism, Potassium Channels, Voltage-Gated genetics, Potassium Channels, Voltage-Gated metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Interaction Domains and Motifs, Signal Transduction, Cell Membrane metabolism, G Protein-Coupled Inwardly-Rectifying Potassium Channels chemistry, Lipid Bilayers metabolism, Phosphatidylinositol Phosphates metabolism, Potassium Channels, Tandem Pore Domain chemistry, Potassium Channels, Voltage-Gated chemistry
- Abstract
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. Here, we describe how ion channel structures have evolved to respond to both lipid modulators and lipid activators to control the electrical activities of cells, highlighting diverse mechanisms and common themes.
- Published
- 2020
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12. Structural basis for the modulation of pentameric ligand-gated ion channel function by lipids.
- Author
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Thompson MJ and Baenziger JE
- Subjects
- Binding Sites, Humans, Ligand-Gated Ion Channels genetics, Lipids genetics, Membrane Lipids genetics, Models, Molecular, Protein Structure, Quaternary, Synapses metabolism, Ligand-Gated Ion Channels chemistry, Lipids chemistry, Membrane Lipids chemistry, Synapses genetics
- Abstract
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. Here, we review the literature describing the known functional effects of membrane lipids on different members of the pLGIC superfamily and highlight pLGIC structures that exhibit bound lipids. We discuss new insight into the mechanisms of pLGIC-lipid interactions that has been derived from these recent structures., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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13. The functional role of the αM4 transmembrane helix in the muscle nicotinic acetylcholine receptor probed through mutagenesis and coevolutionary analyses.
- Author
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Thompson MJ, Domville JA, and Baenziger JE
- Subjects
- Amino Acid Substitution, Animals, Hydrophobic and Hydrophilic Interactions, Ion Channel Gating, Lipids analysis, Models, Molecular, Mutagenesis, Protein Conformation, Receptors, Nicotinic chemistry, Receptors, Nicotinic genetics, Torpedo, Biological Evolution, Muscles metabolism, Receptors, Nicotinic metabolism
- Abstract
The activity of the muscle-type Torpedo 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. Ala substitutions for most αM4 residues, including those in clusters of polar residues at both the N and C termini, and deletion of up to 11 C-terminal residues had little impact on the agonist-induced response. Even Ala substitutions for coevolved pairs of residues at the interface between αM4 and the adjacent helices, αM1 and αM3, had little effect, although some impaired nAChR expression. On the other hand, Ala substitutions for Thr422 and Arg429 caused relatively large losses of function, suggesting functional roles for these specific residues. Ala substitutions for aromatic residues at the αM4-αM1/αM3 interface generally led to gains of function, as previously reported for the prokaryotic homolog, the Erwinia chrysanthemi ligand-gated ion channel (ELIC). The functional effects of individual Ala substitutions in αM4 were found to be additive, although not in a completely independent manner. Our results provide insight into the structural features of αM4 that are important. They also suggest how lipid-dependent changes in αM4 structure ultimately modify nAChR function., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Thompson et al.)
- Published
- 2020
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14. A lipid site shapes the agonist response of a pentameric ligand-gated ion channel.
- Author
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Hénault CM, Govaerts C, Spurny R, Brams M, Estrada-Mondragon A, Lynch J, Bertrand D, Pardon E, Evans GL, Woods K, Elberson BW, Cuello LG, Brannigan G, Nury H, Steyaert J, Baenziger JE, and Ulens C
- Subjects
- Animals, Ligands, Mutagenesis, Xenopus, Ion Channel Gating, Ion Channels metabolism, Lipids chemistry
- Abstract
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 GABA
A/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.- Published
- 2019
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15. An allosteric link connecting the lipid-protein interface to the gating of the nicotinic acetylcholine receptor.
- Author
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Domville JA and Baenziger JE
- Subjects
- Allosteric Regulation, Allosteric Site genetics, Cell Membrane metabolism, Humans, Ion Channel Gating physiology, Lipid-Linked Proteins physiology, Lipids chemistry, Lipids physiology, Models, Molecular, Protein Conformation, Protein Domains, Receptors, Nicotinic ultrastructure, Signal Transduction, Cell Membrane physiology, Receptors, Nicotinic genetics, Receptors, Nicotinic metabolism
- Abstract
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. To understand how changes at the lipid-protein interface influence gating, we examined how a mutation at position 418 on the lipid-facing surface of the outer most M4 transmembrane α-helix alters the energetic couplings between M4 and the remainder of the transmembrane domain. Human muscle nAChR is sensitive to mutations at position 418, with the Cys-to-Trp mutation resulting in a 16-fold potentiation in function that leads to a congenital myasthenic syndrome. Energetic coupling between M4 and the Cys-loop, a key structure implicated in gating, do not change with C418W. Instead, Trp418 and an adjacent residue couple energetically with residues on the M1 transmembrane α-helix, leading to a reorientation of M1 that stabilizes the open state. We thus identify an allosteric link connecting the lipid-protein interface of the nAChR to altered channel function.
- Published
- 2018
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16. Pentameric ligand-gated ion channels exhibit distinct transmembrane domain archetypes for folding/expression and function.
- Author
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Therien JP and Baenziger JE
- Subjects
- Alanine genetics, Amino Acid Sequence, Animals, Models, Molecular, Mutant Proteins chemistry, Mutation genetics, Protein Domains, Structure-Activity Relationship, Xenopus laevis, Ligand-Gated Ion Channels chemistry, Ligand-Gated Ion Channels metabolism, Protein Folding
- Abstract
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. Strikingly, disruption of most interactions in GLIC lead to either a reduction or a complete loss of expression and/or function, while analogous disruptions in ELIC often lead to gains in function. Structural comparisons suggest that GLIC and ELIC represent distinct transmembrane domain archetypes. One archetype, exemplified by GLIC, the glycine and GABA receptors and the glutamate activated chloride channel, has extensive aromatic contacts that govern M4-M1/M3 interactions and that are essential for expression and function. The other archetype, exemplified by ELIC and both the nicotinic acetylcholine and serotonin receptors, has relatively few aromatic contacts that are detrimental to function. These archetypes likely have evolved different mechanisms to balance the need for strong M4 "binding" to M1/M3 to promote folding/expression, and the need for weaker interactions that allow for greater conformational flexibility.
- Published
- 2017
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17. Functional characterization of two prokaryotic pentameric ligand-gated ion channel chimeras - role of the GLIC transmembrane domain in proton sensing.
- Author
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Hénault CM and Baenziger JE
- Subjects
- Animals, Chimera metabolism, Crystallography, X-Ray methods, Cysteamine metabolism, Histidine metabolism, Ion Channel Gating physiology, Ligands, Models, Molecular, Mutation genetics, Oocytes metabolism, Protein Domains physiology, Protons, Xenopus laevis metabolism, Ligand-Gated Ion Channels metabolism, Prokaryotic Cells metabolism
- Abstract
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. No significant improvements in expression/toxicity were observed with extensive loop substitutions at the ECD/TMD interface. Surprisingly, oocytes expressing EG-I9'A responded to both the ELIC agonist, cysteamine and the GLIC agonist, protons - the latter at pH values ≤4.0. The cysteamine- and proton-induced currents in EG-I9'A were inhibited by the GLIC TMD pore blocker, amantadine. The cysteamine-induced response of EG-I9'A was also inhibited by protons at pH values down to 4.5, but potentiated at lower pH values. Proton-induced gating at low pH was not abolished by mutation of an intramembrane histidine residue previously implicated in GLIC TMD function. We show that the TMD plays a major role governing the thermal stability of a pLGIC, and identify three distinct mechanisms by which agonists and protons influence the gating of the EG chimera. A structural basis for the impaired function of GE is suggested., (Copyright © 2016 Elsevier B.V. All rights reserved.)
- Published
- 2017
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18. Probing the structure of the uncoupled nicotinic acetylcholine receptor.
- Author
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Sun J, Comeau JF, and Baenziger JE
- Subjects
- Acetylcholine metabolism, Animals, Binding Sites physiology, Cell Membrane physiology, Cholesterol metabolism, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Models, Molecular, Protein Binding physiology, Protein Conformation, Spectroscopy, Fourier Transform Infrared methods, Torpedo metabolism, Receptors, Nicotinic chemistry, Receptors, Nicotinic metabolism
- Abstract
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. The measured K
d for acetylcholine binding to the uncoupled nAChR is similar to that for the resting state, confirming that the agonist binding site adopts a resting-state like conformation. Although both ethidium and crystal violet bind to the resting and desensitized channel pores with distinct affinities, no binding of either probe was detected to the uncoupled nAChR. Our data suggest that the transmembrane domain of the uncoupled nAChR adopts a conformation distinct from that of the resting and desensitized states. The lack of binding is consistent with a more constricted channel pore, possibly along the lines of what is observed in crystal structures of the prokaryotic homolog, ELIC., (Copyright © 2016 Elsevier B.V. All rights reserved.)- Published
- 2017
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19. The Role of Cholesterol in the Activation of Nicotinic Acetylcholine Receptors.
- Author
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Baenziger JE, Domville JA, and Therien JPD
- Subjects
- Animals, Humans, Receptors, Nicotinic chemistry, Cholesterol metabolism, Receptors, Nicotinic metabolism
- Abstract
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. In the absence of cholesterol and anionic lipids, the nAChR adopts an uncoupled conformation that binds agonist but does not undergo agonist-induced conformational transitions-unless the nAChR is located in a relatively thick lipid bilayer, such as that found in cholesterol-rich lipid rafts. We highlight different sites of cholesterol action, including the lipid-exposed M4 transmembrane α-helix. Cholesterol and other lipids likely alter function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These same interactions have been implicated in both the folding and trafficking of nAChRs to the cell surface. We evaluate the nature of cholesterol-nAChR interactions, considering the evidence supporting the roles of both direct binding to allosteric sites and cholesterol-induced changes in bulk membrane physical properties., (© 2017 Elsevier Inc. All rights reserved.)
- Published
- 2017
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20. The M4 Transmembrane α-Helix Contributes Differently to Both the Maturation and Function of Two Prokaryotic Pentameric Ligand-gated Ion Channels.
- Author
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Hénault CM, Juranka PF, and Baenziger JE
- Subjects
- Amino Acid Sequence, Models, Molecular, Molecular Sequence Data, Protein Structure, Quaternary, Protein Structure, Secondary, Receptors, Glycine genetics, Sequence Deletion, Cell Membrane metabolism, Protein Multimerization, Receptors, Glycine chemistry, Receptors, Glycine metabolism
- Abstract
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. M4-M1/M3 interactions appear to be optimized in GLIC with even subtle structural changes at this interface leading to detrimental effects. In contrast, mutations along the M4-M1/M3 interface of ELIC typically lead to gain-of-function phenotypes, suggesting that these interactions in ELIC are not optimized for channel function. In addition, no cluster of interacting residues involving the M4 C terminus, M3, and the β6-β7 loop was found, suggesting that the M4 C terminus plays little role in ELIC maturation or function. This study shows that M4 makes distinct contributions to the maturation and gating of these two closely related homologs, suggesting that GLIC and ELIC exhibit divergent features of channel function., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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21. The role of the M4 lipid-sensor in the folding, trafficking, and allosteric modulation of nicotinic acetylcholine receptors.
- Author
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Hénault CM, Sun J, Therien JP, daCosta CJ, Carswell CL, Labriola JM, Juranka PF, and Baenziger JE
- Subjects
- Allosteric Regulation, Animals, Brain metabolism, Humans, Neurons metabolism, Prokaryotic Cells chemistry, Protein Conformation, Protein Folding, Protein Transport, Structural Homology, Protein, Torpedo, Membrane Lipids chemistry, Membrane Lipids metabolism, Receptor, Muscarinic M4 chemistry, Receptor, Muscarinic M4 metabolism
- Abstract
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. Structural and functional data highlight a role for the lipid-exposed M4 transmembrane α-helix of each subunit in lipid sensing, and suggest that lipids influence gating by altering the binding of M4 to the adjacent transmembrane α-helices, M1 and M3. M4 has also been implicated in both the folding and trafficking of nAChRs to the cell surface, as well as in the potentiation of nAChR gating by neurosteroids. Here, we discuss the roles of M4 in the folding, trafficking, and allosteric modulation of nAChRs. We also consider the hypothesis that variable chemistry at the M4-M1/M3 transmembrane α-helical interface in different nAChR subunits governs the capacity for potentiation by activating lipids. This article is part of the Special Issue entitled 'The Nicotinic Acetylcholine Receptor: From Molecular Biology to Cognition'., (Copyright © 2014 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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22. Role of the Fourth Transmembrane α Helix in the Allosteric Modulation of Pentameric Ligand-Gated Ion Channels.
- Author
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Carswell CL, Hénault CM, Murlidaran S, Therien JPD, Juranka PF, Surujballi JA, Brannigan G, and Baenziger JE
- Subjects
- Allosteric Regulation, Humans, Models, Molecular, Molecular Dynamics Simulation, Protein Multimerization, Protein Structure, Secondary, Ligand-Gated Ion Channels chemistry, Ligand-Gated Ion Channels metabolism
- Abstract
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. Enhanced M4 interactions promote channel function while ineffective interactions reduce channel function. The interface chemistry governs the intrinsic strength of M4-M1/M3 inter-helical interactions, both influencing channel gating and imparting distinct susceptibilities to the potentiating effects of a lipid-facing M4 congenital myasthenic syndrome mutation. Through aromatic substitutions, functional studies, and molecular dynamics simulations, we elucidate a mechanism by which M4 modulates channel function., (Copyright © 2015 Elsevier Ltd. All rights reserved.)
- Published
- 2015
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23. Nicotinic acetylcholine receptor-lipid interactions: Mechanistic insight and biological function.
- Author
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Baenziger JE, Hénault CM, Therien JP, and Sun J
- Subjects
- Animals, Cell Membrane chemistry, Cell Membrane metabolism, Humans, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Membrane Lipids metabolism, Models, Molecular, Protein Binding, Receptors, Nicotinic metabolism, Membrane Lipids chemistry, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, Nicotinic chemistry
- Abstract
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. The uncoupled nAChR, however, does transition to activatable conformations in relatively thick lipid bilayers, such as those found in lipid rafts. In this review, we discuss current understanding of lipid-nAChR interactions in the context of increasingly available high resolution structural and functional data. These data highlight different sites of lipid action, including the lipid-exposed M4 transmembrane α-helix. Current evidence suggests that lipids alter nAChR function by modulating interactions between M4 and the adjacent transmembrane α-helices, M1 and M3. These interactions have also been implicated in both the folding and trafficking of nAChRs to the cell surface. We review current mechanistic understanding of lipid-nAChR interactions, and highlight potential biological roles for lipid-nAChR interactions in modulating the synaptic response. This article is part of a Special Issue entitled: Lipid-protein interactions., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
- Full Text
- View/download PDF
24. Intramembrane aromatic interactions influence the lipid sensitivities of pentameric ligand-gated ion channels.
- Author
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Carswell CL, Sun J, and Baenziger JE
- Subjects
- Animals, Cations, Cell Membrane metabolism, Crystallography, X-Ray, Hydrogen-Ion Concentration, Lipid Bilayers, Oocytes cytology, Protein Binding, Protein Structure, Secondary, Protein Structure, Tertiary, Proteolipids chemistry, Receptors, Nicotinic chemistry, Spectrophotometry, Infrared, Spectroscopy, Fourier Transform Infrared, Torpedo, Bacterial Proteins chemistry, Cyanobacteria chemistry, Erwinia chemistry, Ligand-Gated Ion Channels chemistry, Lipids chemistry
- Abstract
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., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
- Full Text
- View/download PDF
25. A distinct mechanism for activating uncoupled nicotinic acetylcholine receptors.
- Author
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daCosta CJ, Dey L, Therien JP, and Baenziger JE
- Subjects
- Hydrophobic and Hydrophilic Interactions, Kinetics, Protein Conformation, Receptors, Nicotinic chemistry, Receptors, Nicotinic metabolism
- Abstract
The ability of the nicotinic acetylcholine receptor (nAChR) to undergo conformational transitions is exquisitely sensitive to its surrounding lipid environment. Previous work has highlighted a conformational selection mechanism, whereby different lipids stabilize different proportions of activatable resting versus nonactivatable conformations. In the absence of anionic lipids and cholesterol, the nAChR adopts an uncoupled conformation, which binds agonist with resting state-like affinity but does not usually undergo agonist-induced conformational transitions. Very slow (minutes to hours) transitions from uncoupled to coupled (resting, open and/or desensitized) conformations, however, can occur in membranes with relatively thick hydrophobic cores. Increasing membrane hydrophobic thickness 'awakens' uncoupled nAChRs by reducing the large activation energy barrier (or barriers) leading to coupled states, thus allowing conformational transitions to occur on an experimentally tractable timescale. Lipids shape activity by modulating the relative proportions of activatable versus nonactivatable conformations and by controlling the transitions between uncoupled and coupled conformations.
- Published
- 2013
- Full Text
- View/download PDF
26. Gating of pentameric ligand-gated ion channels: structural insights and ambiguities.
- Author
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daCosta CJ and Baenziger JE
- Subjects
- Animals, Humans, Models, Molecular, Protein Interaction Domains and Motifs, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Subunits chemistry, Ion Channel Gating, Receptors, GABA chemistry, Receptors, Glycine chemistry, Receptors, Nicotinic chemistry, Receptors, Serotonin chemistry
- Abstract
Pentameric ligand-gated ion channels (pLGICs) mediate fast synaptic communication by converting chemical signals into an electrical response. Recently solved agonist-bound and agonist-free structures greatly extend our understanding of these complex molecular machines. A key challenge to a full description of function, however, is the ability to unequivocally relate determined structures to the canonical resting, open, and desensitized states. Here, we review current understanding of pLGIC structure, with a focus on the conformational changes underlying channel gating. We compare available structural information and review the evidence supporting the assignment of each structure to a particular conformational state. We discuss multiple factors that may complicate the interpretation of crystal structures, highlighting the potential influence of lipids and detergents. We contend that further advances in the structural biology of pLGICs will require deeper insight into the nature of pLGIC-lipid interactions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
27. Structural sensitivity of a prokaryotic pentameric ligand-gated ion channel to its membrane environment.
- Author
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Labriola JM, Pandhare A, Jansen M, Blanton MP, Corringer PJ, and Baenziger JE
- Subjects
- Protein Stability, Protein Structure, Quaternary, Structural Homology, Protein, Bacteria chemistry, Bacteria genetics, Bacteria metabolism, Bacterial Proteins chemistry, Bacterial Proteins genetics, Bacterial Proteins metabolism, Ion Channel Gating physiology, Ion Channels chemistry, Ion Channels genetics, Ion Channels metabolism
- Abstract
Although the activity of the nicotinic acetylcholine receptor (nAChR) is exquisitely sensitive to its membrane environment, the underlying mechanisms remain poorly defined. The homologous prokaryotic pentameric ligand-gated ion channel, Gloebacter ligand-gated ion channel (GLIC), represents an excellent model for probing the molecular basis of nAChR sensitivity because of its high structural homology, relative ease of expression, and amenability to crystallographic analysis. We show here that membrane-reconstituted GLIC exhibits structural and biophysical properties similar to those of the membrane-reconstituted nAChR, although GLIC is substantially more thermally stable. GLIC, however, does not possess the same exquisite lipid sensitivity. In particular, GLIC does not exhibit the same propensity to adopt an uncoupled conformation where agonist binding is uncoupled from channel gating. Structural comparisons provide insight into the chemical features that may predispose the nAChR to the formation of an uncoupled state.
- Published
- 2013
- Full Text
- View/download PDF
28. Molecular mechanisms of acetylcholine receptor-lipid interactions: from model membranes to human biology.
- Author
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Baenziger JE and daCosta CJB
- Abstract
Lipids are potent modulators of the Torpedo nicotinic acetylcholine receptor. Lipids influence nicotinic receptor function by allosteric mechanisms, stabilizing varying proportions of pre-existing resting, open, desensitized, and uncoupled conformations. Recent structures reveal that lipids could alter function by modulating transmembrane α-helix/α-helix packing, which in turn could alter the conformation of the allosteric interface that links the agonist-binding and transmembrane pore domains-this interface is essential in the coupling of agonist binding to channel gating. We discuss potential mechanisms by which lipids stabilize different conformational states in the context of the hypothesis that lipid-nicotinic receptor interactions modulate receptor function at biological synapses.
- Published
- 2013
- Full Text
- View/download PDF
29. Structural characterization and agonist binding to human α4β2 nicotinic receptors.
- Author
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daCosta CJ, Michel Sturgeon R, Hamouda AK, Blanton MP, and Baenziger JE
- Subjects
- HEK293 Cells, Humans, Protein Structure, Secondary, Protein Structure, Tertiary, Nicotinic Agonists chemistry, Receptors, Nicotinic chemistry
- Abstract
The Cys-loop receptor super-family of neurotransmitter-gated ion channels mediates fast synaptic transmission throughout the human nervous system. These receptors exhibit widely varying pharmacologies, yet their structural characterization has relied heavily on their homology with the naturally abundant muscle-type Torpedo nicotinic acetylcholine receptor. Here we examine for the first time the structure of a human α4β2 neuronal nicotinic acetylcholine receptor. We show that human α4β2 nicotinic receptors adopt a secondary/tertiary fold similar to that of the Torpedo nicotinic receptor with a large proportion of both α-helix and β-sheet, but exhibit a substantially increased thermal stability. Both receptors bind agonist, but with different patterns of agonist recognition - particularly in the nature of the interactions between aromatic residues and the agonist quaternary amine functional group. By comparing α4β2 and Torpedo receptors, we begin to delineate their structural similarities and differences., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2011
- Full Text
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30. 3D structure and allosteric modulation of the transmembrane domain of pentameric ligand-gated ion channels.
- Author
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Baenziger JE and Corringer PJ
- Subjects
- Ion Channel Gating physiology, Protein Conformation, Receptors, GABA-A metabolism, Receptors, Glycine metabolism, Receptors, Nicotinic metabolism, Allosteric Regulation physiology, Cell Membrane physiology, Ligand-Gated Ion Channels physiology
- Abstract
Pentameric ligand-gated ion channels mediate rapid chemo-electric signal transduction in animals. The active site of this family of proteins is their ion channel pore, which is located at the center of the transmembrane domain. The opening/closing motions of the channel pore are governed by the binding of neurotransmitter to the extracellular domain, but also by allosteric effectors acting within the transmembrane domain. Here, we review the structure of the transmembrane domain as well as its role in the allosteric modulation of pentameric ligand-gated ion channel function. We focus on two examples: the interactions of nicotinic ACh receptors with lipids, for which a novel "uncoupled" state has been proposed, and the interactions of GABA(A) and Glycine receptors with allosteric modulators, such as general anesthetics, ethanol and neurosteroids. We revisit these data in light of the recently solved X-ray structures of bacterial members of the family, which provide atomic-resolution insight into the structures of both the transmembrane domain and associated lipids., (Copyright © 2010 Elsevier Ltd. All rights reserved.)
- Published
- 2011
- Full Text
- View/download PDF
31. Phospholipase C activity affinity purifies with the Torpedo nicotinic acetylcholine receptor.
- Author
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Labriola JM, daCosta CJ, Wang S, Figeys D, Smith JC, Sturgeon RM, and Baenziger JE
- Subjects
- Animals, Chromatography, Affinity, Chromatography, Thin Layer, Diglycerides pharmacology, Ethylmaleimide pharmacology, Phosphatidic Acids metabolism, Sulfhydryl Reagents pharmacology, Cell Membrane metabolism, Receptors, Nicotinic isolation & purification, Receptors, Nicotinic metabolism, Torpedo metabolism, Type C Phospholipases isolation & purification, Type C Phospholipases metabolism
- Abstract
Nicotinic acetylcholine receptors mediate fast synaptic transmission by fluxing ions across the membrane in response to neurotransmitter binding. We show here that during affinity purification of the nicotinic acetylcholine receptor from Torpedo, phosphatidic acid, but not other anionic or zwitterionic phospholipids, is hydrolyzed to diacylglycerol. The phospholipase C activity elutes with the acetylcholine receptor and is inhibited by a lipid phosphate phosphohydrolase inhibitor, sodium vanadate, but not a phosphatidate phosphohydrolase inhibitor, N-ethylmaleimide. Further, the hydrolysis product of phosphatidic acid, diacylglycerol, enhances the functional capabilities of the acetylcholine receptor in the presence of anionic lipids. We conclude that a phospholipase C activity, which appears to be specific for phosphatidic acid, is associated with the nicotinic acetylcholine receptor. The acetylcholine receptor may directly or indirectly influence lipid metabolism in a manner that enhances its own function.
- Published
- 2010
- Full Text
- View/download PDF
32. Cations mediate interactions between the nicotinic acetylcholine receptor and anionic lipids.
- Author
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Sturgeon RM and Baenziger JE
- Subjects
- Binding Sites, Cations, Lipid Bilayers chemistry, Phospholipids chemistry, Receptors, Nicotinic chemistry
- Abstract
Interactions between the nicotinic acetylcholine receptor (nAChR) and phosphatidic acid (PA) are bidirectional in that membranes containing PA are effective at stabilizing an agonist-responsive nAChR, whereas incorporation of the nAChR into the same membranes leads to a substantial increase in lipid lateral packing density. A previous study suggested that the ability of PA to adopt a dianionic ionization state is key. We monitored the ionization state of PA in both reconstituted and protein-free membranes. In model membranes composed of PA and 3:2 (mol/mol) phosphatidylcholine (PC)/PA, the monoanionic-to-dianionic transition of PA was detected with a pKa of 8.7 and 6.5, respectively. In the reconstituted 3:2 PC/PA membranes, however, PA was stabilized in a monoanionic state at pH values up to 10. Although dianionic PA does not play a role in nAChR function, we found that both the stabilization of monoanionic PA and the concentration of other cations at the bilayer surface can account for changes in bilayer physical properties that are observed upon incorporation of the nAChR into 3:2 PC/PA membranes. A nAChR-induced concentration of cations at the bilayer surface likely mediates interactions between the nAChR and the anionic lipids in its membrane environment., (Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)
- Published
- 2010
- Full Text
- View/download PDF
33. Preparation of reconstituted acetylcholine receptor membranes suitable for AFM imaging of lipid-protein interactions.
- Author
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Vuong N, Baenziger JE, and Johnston LJ
- Subjects
- Calcium chemistry, Lipid Bilayers metabolism, Models, Molecular, Phosphatidylcholines chemistry, Protein Binding, Proteins metabolism, Proteolipids chemistry, Receptors, Nicotinic metabolism, Lipid Bilayers chemistry, Microscopy, Atomic Force, Proteins chemistry, Receptors, Nicotinic chemistry
- Abstract
The nicotinic acetylcholine receptor (nAChR) has been reconstituted in POPC vesicles at high lipid-protein (L/P) ratios for the preparation of supported lipid bilayers with a low protein density for studies of protein-lipid interactions using atomic force microscopy (AFM). Initial reconstitutions using a standard dialysis method with bulk L/P ratios ranging from 20:1 to 100:1 (w/w) gave heterogeneous samples that contained both empty vesicles and proteoliposomes with a range of L/P ratios. This is problematic because empty vesicles adsorb and rupture to form bilayer patches more rapidly than do protein-rich vesicles, resulting in the loss of protein during sample washing. Although it was not possible to find reconstitution conditions that gave homogeneous populations of vesicles with high L/P ratios, an additional freeze-thaw cycle immediately after dialysis did reproducibly yield a fraction of proteoliposomes with L/P ratios above 100:1. These proteoliposomes were separated by sucrose gradient centrifugation and used to prepare supported bilayers with well-separated individual receptors and minimal adsorbed proteoliposomes. AFM images of such samples showed many small features protruding from the bilayer surface. These features range in height from 1 to 5nm, consistent with the smaller intracellular domain of the protein exposed, and have lateral dimensions consistent with an individual receptor. Some bilayers with reconstituted protein also had a small fraction of higher features that are assigned to nAChR with the larger extracellular domain exposed and showed evidence for aggregation to give dimers or small oligomers. This work demonstrates the importance of using highly purified reconstituted membranes with uniform lipid-protein ratios for AFM studies of integral membrane protein-lipid interactions.
- Published
- 2010
- Full Text
- View/download PDF
34. Anionic lipids allosterically modulate multiple nicotinic acetylcholine receptor conformational equilibria.
- Author
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daCosta CJ, Medaglia SA, Lavigne N, Wang S, Carswell CL, and Baenziger JE
- Subjects
- Allosteric Site, Animals, Biophysics methods, Cell Membrane metabolism, Chromatography, Thin Layer methods, Electrochemistry methods, Models, Biological, Molecular Conformation, Spectrophotometry, Infrared methods, Torpedo, Anions metabolism, Lipid Bilayers chemistry, Lipids chemistry, Receptors, Nicotinic chemistry
- Abstract
Anionic lipids influence the ability of the nicotinic acetylcholine receptor to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. We show here that anionic lipids with relatively small headgroups, and thus the greatest ability to influence lipid packing/bilayer physical properties, are the most effective at stabilizing an agonist-activatable receptor. The differing abilities of anionic lipids to stabilize an activatable receptor stem from differing abilities to preferentially favor resting over both uncoupled and desensitized conformations. Anionic lipids thus modulate multiple acetylcholine receptor conformational equilibria. Our data suggest that both lipids and membrane physical properties act as classic allosteric modulators influencing function by interacting with and thus preferentially stabilizing different native acetylcholine receptor conformational states.
- Published
- 2009
- Full Text
- View/download PDF
35. A lipid-dependent uncoupled conformation of the acetylcholine receptor.
- Author
-
daCosta CJ and Baenziger JE
- Subjects
- Acetylcholine metabolism, Allosteric Regulation, Animals, Binding Sites, Bungarotoxins metabolism, Lipid Bilayers, Models, Molecular, Protein Conformation, Spectroscopy, Fourier Transform Infrared, Substrate Specificity, Cell Membrane metabolism, Phospholipids metabolism, Receptors, Nicotinic chemistry, Receptors, Nicotinic metabolism, Torpedo metabolism
- Abstract
Lipids influence the ability of Cys-loop receptors to gate open in response to neurotransmitter binding, but the underlying mechanisms are poorly understood. With the nicotinic acetylcholine receptor (nAChR) from Torpedo, current models suggest that lipids modulate the natural equilibrium between resting and desensitized conformations. We show that the lipid-inactivated nAChR is not desensitized, instead it adopts a novel conformation where the allosteric coupling between its neurotransmitter-binding sites and transmembrane pore is lost. The uncoupling is accompanied by an unmasking of previously buried residues, suggesting weakened association between structurally intact agonist-binding and transmembrane domains. These data combined with the extensive literature on Cys-loop receptor-lipid interactions suggest that the M4 transmembrane helix plays a key role as a lipid-sensor, translating bilayer properties into altered nAChR function.
- Published
- 2009
- Full Text
- View/download PDF
36. Structural characterization of the osmosensor ProP.
- Author
-
Sayeed WM and Baenziger JE
- Subjects
- Biophysical Phenomena, Deuterium Oxide, Escherichia coli genetics, Escherichia coli metabolism, Escherichia coli Proteins genetics, Escherichia coli Proteins metabolism, Hot Temperature, Monosaccharide Transport Proteins chemistry, Osmolar Concentration, Protein Denaturation, Protein Folding, Protein Structure, Secondary, Proteolipids, Spectroscopy, Fourier Transform Infrared, Symporters genetics, Symporters metabolism, Thermodynamics, Escherichia coli Proteins chemistry, Symporters chemistry
- Abstract
ProP, an osmoprotectant symporter from the major facilitator superfamily was expressed, purified and reconstituted into proteoliposomes that are amenable to structural characterization using infrared spectroscopy. Infrared spectra recorded in both (1)H(2)O and (2)H(2)O buffers reveal amide I band shapes that are characteristic of a predominantly alpha-helical protein, and that are similar to those recorded from the well-characterized homolog, lactose permease (LacY). Curve-fit analysis shows that ProP and LacY both exhibit a high alpha-helical content. Both proteins undergo extensive peptide hydrogen-deuterium exchange after exposure to (2)H(2)O, but are surprisingly thermally stable with denaturation temperatures greater than 60 degrees C. 25-30% of the peptide hydrogens in both ProP and LacY are resistant to exchange after 72 h in (2)H(2)O at 4 degrees C. Surprisingly, these exchange resistant peptide hydrogens exchange completely for deuterium at temperatures below those that lead to denaturation. Our results show that ProP adopts a highly alpha-helical fold similar to that of LacY, and that both transmembrane folds exhibit unusually high temperature-sensitive solvent accessibility. The results provide direct evidence that ProP adopts a structure consistent with other major facilitator superfamily members.
- Published
- 2009
- Full Text
- View/download PDF
37. Heterogeneity in the sn-1 carbon chain of platelet-activating factor glycerophospholipids determines pro- or anti-apoptotic signaling in primary neurons.
- Author
-
Ryan SD, Harris CS, Carswell CL, Baenziger JE, and Bennett SA
- Subjects
- Animals, Caspases metabolism, Cell Line, Cerebellum cytology, Cerebellum drug effects, Cerebellum metabolism, Gene Expression Regulation drug effects, Mice, Micelles, Neuroprotective Agents pharmacology, Neurotoxins chemistry, Neurotoxins toxicity, Phospholipid Ethers pharmacology, Platelet Activating Factor analogs & derivatives, Platelet Membrane Glycoproteins antagonists & inhibitors, Platelet Membrane Glycoproteins deficiency, Platelet Membrane Glycoproteins metabolism, Receptors, G-Protein-Coupled antagonists & inhibitors, Receptors, G-Protein-Coupled deficiency, Receptors, G-Protein-Coupled metabolism, Apoptosis drug effects, Carbon, Neurons cytology, Neurons drug effects, Platelet Activating Factor chemistry, Platelet Activating Factor toxicity, Signal Transduction drug effects
- Abstract
The platelet-activating factor (PAF) family of glycerophospholipids accumulates in damaged brain tissue following injury. Little is known about the role of individual isoforms in regulating neuronal survival. Here, we compared the neurotoxic and neuroprotective activities of 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (C16-PAF) and 1-O-octadecyl-2-acetyl-sn-glycero-3-phosphocholine (C18-PAF) in cerebellar granule neurons. We find that both C16-PAF and C18-PAF cause PAF receptor-independent death but signal through different pathways. C16-PAF activates caspase-7, whereas C18-PAF triggers caspase-independent death in PAF receptor-deficient neurons. We further show that PAF receptor signaling is either pro- or anti-apoptotic, depending upon the identity of the sn-1 fatty acid of the PAF ligand. Activation of the PAF G-protein-coupled receptor (PAFR) by C16-PAF stimulation is anti-apoptotic and inhibits caspase-dependent death. Activation of PAFR by C18-PAF is pro-apoptotic. These results demonstrate the importance of the long-chain sn-1 fatty acid in regulating PAF-induced caspase-dependent apoptosis, caspase-independent neurodegeneration, and neuroprotection in the presence or absence of the PAF receptor.
- Published
- 2008
- Full Text
- View/download PDF
38. Expression, purification, and structural characterization of CfrA, a putative iron transporter from Campylobacter jejuni.
- Author
-
Carswell CL, Rigden MD, and Baenziger JE
- Subjects
- Bacterial Proteins isolation & purification, Cloning, Molecular, Escherichia coli genetics, Gene Expression, Hot Temperature, Membrane Transport Proteins isolation & purification, Protein Denaturation, Protein Structure, Secondary, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins isolation & purification, Sequence Alignment, Sequence Homology, Amino Acid, Spectroscopy, Fourier Transform Infrared, Bacterial Proteins chemistry, Bacterial Proteins genetics, Campylobacter jejuni genetics, Campylobacter jejuni metabolism, Iron metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins genetics
- Abstract
The gene for the Campylobacter ferric receptor (CfrA), a putative iron-siderophore transporter in the enteric food-borne pathogen Campylobacter jejuni, was cloned, and the membrane protein was expressed in Escherichia coli, affinity purified, and then reconstituted into model lipid membranes. Fourier transform infrared spectra recorded from the membrane-reconstituted CfrA are similar to spectra that have been recorded from other iron-siderophore transporters and are highly characteristic of a beta-sheet protein (approximately 44% beta-sheet and approximately 10% alpha-helix). CfrA undergoes relatively extensive peptide hydrogen-deuterium exchange upon exposure to (2)H(2)O and yet is resistant to thermal denaturation at temperatures up to 95 degrees C. The secondary structure, relatively high aqueous solvent exposure, and high thermal stability are all consistent with a transmembrane beta-barrel structure containing a plug domain. Sequence alignments indicate that CfrA contains many of the structural motifs conserved in other iron-siderophore transporters, including the Ton box, PGV, IRG, RP, and LIDG motifs of the plug domain. Surprisingly, a homology model reveals that regions of CfrA that are expected to play a role in enterobactin binding exhibit sequences that differ substantially from the sequences of the corresponding regions that play an essential role in binding/transport by the E. coli enterobactin transporter, FepA. The sequence variations suggest that there are differences in the mechanisms used by CfrA and FepA to interact with bacterial siderophores. It may be possible to exploit these structural differences to develop CfrA-specific therapeutics.
- Published
- 2008
- Full Text
- View/download PDF
39. Lipid composition alters drug action at the nicotinic acetylcholine receptor.
- Author
-
Baenziger JE, Ryan SE, Goodreid MM, Vuong NQ, Sturgeon RM, and daCosta CJ
- Subjects
- Anesthetics, Local metabolism, Anesthetics, Local pharmacology, Animals, Dose-Response Relationship, Drug, Inhibitory Concentration 50, Kinetics, Lipid Bilayers chemistry, Protein Conformation, Receptors, Nicotinic drug effects, Receptors, Nicotinic isolation & purification, Receptors, Nicotinic metabolism, Spectroscopy, Fourier Transform Infrared, Tetracaine metabolism, Tetracaine pharmacology, Torpedo, Cholesterol chemistry, Lipids chemistry, Phosphatidic Acids chemistry, Phosphatidylcholines chemistry, Receptors, Nicotinic chemistry
- Abstract
We tested the hypothesis that membrane lipid composition influences drug action at membrane proteins by studying local anesthetic action at the nicotinic acetylcholine receptor (nAChR). Infrared difference spectra show that concentrations of tetracaine consistent with binding to the ion channel (<50 microM) stabilize a resting-like state when the nAChR is reconstituted into phosphatidylcholine membranes containing the anionic lipid, phosphatidic acid, but have no effect on the nAChR reconstituted into membranes lacking phosphatidic acid, either in the presence or absence of cholesterol. Concentrations of tetracaine above 200 microM lead to neurotransmitter site binding in all membranes. In the presence of phosphatidic acid, cholesterol, or both, neurotransmitter site binding leads to the formation of quaternary amine-aromatic interactions between tetracaine and binding site tyrosine/tryptophan residues and the stabilization of a desensitized state. One interpretation suggested by lipid partitioning studies is that phosphatidic acid enhances tetracaine action at the channel pore by increasing the partitioning of tetracaine into the lipid bilayer, thereby enhancing access to the transmembrane pore. However, subtle membrane-dependent variations in the vibrations of tyrosine and tryptophan residues, and agonist analog binding studies indicate that the structures of the agonist-bound neurotransmitter sites of the nAChR in membranes lacking both phosphatidic acid and cholesterol differ from the structures of the agonist-desensitized neurotransmitter sites in the presence of both lipids. Lipid action at the nAChR thus involves more than a simple modulation of the equilibrium between resting and desensitized states.
- Published
- 2008
- Full Text
- View/download PDF
40. The net orientation of nicotinic receptor transmembrane alpha-helices in the resting and desensitized states.
- Author
-
Hill DG and Baenziger JE
- Subjects
- Animals, Protein Structure, Tertiary physiology, Spectroscopy, Fourier Transform Infrared, Torpedo metabolism, Lipid Bilayers chemistry, Membrane Lipids chemistry, Receptors, Nicotinic chemistry
- Abstract
The net orientation of nicotinic acetylcholine receptor transmembrane alpha-helices has been probed in both the activatable resting and nonactivatable desensitized states using linear dichroism Fourier-transform infrared spectroscopy. Infrared spectra recorded from reconstituted nicotinic acetylcholine receptor membranes after 72 h exposure to (2)H2O exhibit an intense amide I component band near 1655 cm(-1) that is due predominantly to hydrogen-exchange-resistant transmembrane peptides in an alpha-helical conformation. The measured dichroism of this band is 2.37, suggesting a net tilt of the transmembrane alpha-helices of roughly 40 degrees from the bilayer normal, although this value overestimates the tilt angle because the measured dichroism at 1655 cm(-1) also reflects the dichroism of overlapping amide I component bands. Significantly, no change in the net orientation of the transmembrane alpha-helices is observed upon agonist binding. In fact, the main changes in structure and orientation detected upon desensitization involve highly solvent accessible regions of the polypeptide backbone. Our data are consistent with a capping of the ligand binding site by the solvent accessible C-loop with little change in the structure of the transmembrane domain in the desensitized state. Changes in structure at the interface between the ligand-binding and transmembrane domains may uncouple binding from gating.
- Published
- 2006
- Full Text
- View/download PDF
41. Role of glycosylation and membrane environment in nicotinic acetylcholine receptor stability.
- Author
-
daCosta CJ, Kaiser DE, and Baenziger JE
- Subjects
- Animals, Crystallography methods, Feasibility Studies, Glycosylation, Protein Conformation, Cell Membrane chemistry, Crystallization methods, Membrane Lipids chemistry, Receptors, Nicotinic chemistry, Torpedo metabolism
- Abstract
The effects of glycosylation and membrane environment on the structural stability of the nicotinic acetylcholine receptor (nAChR) from Torpedo have been investigated to improve our understanding of factors that influence eukaryotic membrane protein crystallization. Gel shift assays and carbohydrate-specific staining show that the deglycosylation enzyme, Endo F1, removes at least 50% of membrane-reconstituted nAChR glycosylation. The extent of deglycosylation with Endo F1 increases upon detergent solubilization. Removal of between 60-100% of high mannose moieties from the nAChR has no effect on nAChR secondary structure, stability, or flexibility. Deglycosylation does not influence either agonist binding or the ability of the nAChR to undergo agonist-induced conformational change. In contrast, nAChR structural stability, flexibility, and function are all negatively influenced by simple changes in reconstituted membrane lipid composition. Our results suggest that deglycosylation may represent a feasible approach for enhancing the crystallizability of the nAChR. Our data also demonstrate that the dependence of nAChR structural stability on lipid environment may represent a significant obstacle to nAChR crystallization. Some membrane proteins may have evolved complex interactions with their lipid environments. Understanding the complexity of these interactions may be essential for devising an appropriate strategy for the crystallization of some membrane proteins.
- Published
- 2005
- Full Text
- View/download PDF
42. Phosphatidic acid and phosphatidylserine have distinct structural and functional interactions with the nicotinic acetylcholine receptor.
- Author
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daCosta CJ, Wagg ID, McKay ME, and Baenziger JE
- Subjects
- Animals, Calcium chemistry, Carbon chemistry, Cations, Hydrogen chemistry, Kinetics, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Lipid Metabolism, Lipids chemistry, Phosphatidylserines metabolism, Protein Binding, Protein Conformation, Receptors, Nicotinic metabolism, Spectrophotometry, Spectroscopy, Fourier Transform Infrared, Structure-Activity Relationship, Temperature, Torpedo, Phosphatidic Acids metabolism, Phosphatidylserines chemistry, Receptors, Nicotinic chemistry
- Abstract
Bilayers containing phosphatidylcholine (PC) and the anionic lipid phosphatidic acid (PA) are particularly effective at stabilizing the nicotinic acetylcholine receptor (nAChR) in a functional conformation that undergoes agonist-induced conformational change. The physical properties of PC membranes containing PA are also substantially altered upon incorporation of the nAChR. To test whether or not the negative charge of PA is responsible for this "bi-directional coupling," the nAChR was reconstituted into membranes composed of PC with varying levels of the net negatively charged lipid phosphatidylserine (PS). In contrast to PA, increasing levels of PS in PC membranes do not stabilize an increasing proportion of nAChRs in a functional resting conformation, nor do they slow nAChR peptide hydrogen exchange kinetics. Incorporation of the nAChR had little effect on the physical properties of the PC/PS membranes, as monitored by the gel-to-liquid crystal phase transition temperatures of the bilayers. These results show that a net negative charge alone is not sufficient to account for the unique interactions that occur between the nAChR and PC/PA membranes. Incorporation of the receptor into PC/PS membranes, however, did lead to an altered head group conformation of PS possibly by recruiting divalent cations to the membrane surface. The results show that the nAChR has complex and unique interactions with both PA and PS. The interactions between the nAChR and PS may be bridged by divalent cations, such as calcium.
- Published
- 2004
- Full Text
- View/download PDF
43. A rapid method for assessing lipid:protein and detergent:protein ratios in membrane-protein crystallization.
- Author
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daCosta CJ and Baenziger JE
- Subjects
- Amides chemistry, Filtration methods, Membrane Proteins analysis, Micelles, Muramidase analysis, Phosphatidylcholines analysis, Protein Structure, Secondary, Solubility, Spectroscopy, Fourier Transform Infrared, Surface-Active Agents analysis, Crystallization methods, Membrane Proteins chemistry, Muramidase chemistry, Phosphatidylcholines chemistry, Surface-Active Agents chemistry
- Abstract
A simple procedure for rapidly measuring lipid:protein ratios and detergent concentrations at different stages of the solubilization, purification and crystallization of membrane proteins has been developed. Fourier-transform infrared spectra recorded from 10 micro l aliquots of solution using a single-bounce diamond-attenuated total reflectance apparatus exhibit characteristic bands arising from the vibrations of lipid, protein and detergent. Lipid:protein molar ratios as low as 5:1 (for a protein with a molecular weight of 300 kDa) are determined by comparing the ratio of the integrated intensity of the lipid ester carbonyl band near 1740 cm(-1) with the protein amide I band near 1650 cm(-1). Detergent concentrations at levels well below the critical micellar concentration of most detergents are determined by comparing the integrated intensities of the detergent vibrations, particularly in the 1200-1000 cm(-1) region, with a standard curve. Protein amide I band-shape analysis provides insight into the effects of detergents on protein secondary structure. The importance of monitoring detergent concentration changes during simple procedures, such as the concentration of a membrane protein by ultrafiltration, is demonstrated. This analytical tool has been used to rapidly establish protocols for minimizing lipid and detergent levels in solubilized membrane-protein samples.
- Published
- 2003
- Full Text
- View/download PDF
44. Dissecting the chemistry of nicotinic receptor-ligand interactions with infrared difference spectroscopy.
- Author
-
Ryan SE, Hill DG, and Baenziger JE
- Subjects
- Amines chemistry, Animals, Arginine, Esters chemistry, Hydrogen Bonding, Models, Chemical, Torpedo genetics, Torpedo metabolism, Water chemistry, Ligands, Receptors, Nicotinic chemistry, Receptors, Nicotinic metabolism
- Abstract
The physical interactions that occur between the nicotinic acetylcholine receptor from Torpedo and the agonists carbamylcholine and tetramethylamine have been studied using both conventional infrared difference spectroscopy and a novel double-ligand difference technique. The latter was developed to isolate vibrational bands from residues in a membrane receptor that interact with individual functional groups on a small molecule ligand. The binding of either agonist leads to an increase in vibrational intensity at frequencies centered near 1663, 1655, 1547, 1430, and 1059 cm(-1) indicating that both induce a conformational change from the resting to the desensitized state. Vibrational shifts near 1580, 1516, 1455, 1334, and between 1300 and 1400 cm(-1) are assigned to structural perturbations of tyrosine and possibly both tryptophan and charged carboxylic acid residues upon the formation of receptor-quaternary amine interactions, with the relatively intense feature near 1516 cm(-1) indicating a key role for tyrosine. Other vibrational bands suggest the involvement of additional side chains in agonist binding. Two side-chain vibrational shifts from 1668 and 1605 cm(-1) to 1690 and 1620 cm(-1), respectively, could reflect the formation of a hydrogen bond between the ester carbonyl of carbamylcholine and an arginine residue. The results demonstrate the potential of the double-ligand difference technique for dissecting the chemistry of membrane receptor-ligand interactions and provide new insight into the nature of nicotinic receptor-agonist interactions.
- Published
- 2002
- Full Text
- View/download PDF
45. Lipid-protein interactions at the nicotinic acetylcholine receptor. A functional coupling between nicotinic receptors and phosphatidic acid-containing lipid bilayers.
- Author
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daCosta CJ, Ogrel AA, McCardy EA, Blanton MP, and Baenziger JE
- Subjects
- Animals, Protein Conformation, Receptors, Nicotinic physiology, Spectroscopy, Fourier Transform Infrared, Lipid Bilayers chemistry, Phosphatidic Acids chemistry, Receptors, Nicotinic chemistry
- Abstract
The structural and functional properties of reconstituted nicotinic acetylcholine receptor membranes composed of phosphatidyl choline either with or without cholesterol and/or phosphatidic acid have been examined to test the hypothesis that receptor conformational equilibria are modulated by the physical properties of the surrounding lipid environment. Spectroscopic and chemical labeling data indicate that the receptor in phosphatidylcholine alone is stabilized in a desensitized-like state, whereas the presence of either cholesterol or phosphatidic acid favors a resting-like conformation. Membranes that effectively stabilize a resting-like state exhibit a relatively large proportion of non-hydrogen-bonded lipid ester carbonyls, suggesting a relatively tight packing of the lipid head groups and thus a well ordered membrane. Functional reconstituted membranes also exhibit gel-to-liquid crystal phase transition temperatures that are higher than those of nonfunctional reconstituted membranes composed of phosphatidylcholine alone. Significantly, incorporation of the receptor into phosphatidic acid-containing membranes leads to a dramatic increase in both the lateral packing densities and the gel-to-liquid crystal phase transition temperatures of the reconstituted lipid bilayers. These results suggest a functional link between the nicotinic acetylcholine receptor and the physical properties of phosphatidic acid-containing membranes that could underlie the mechanism by which this lipid preferentially enhances receptor function.
- Published
- 2002
- Full Text
- View/download PDF
46. Structure of the pore-forming transmembrane domain of a ligand-gated ion channel.
- Author
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Méthot N, Ritchie BD, Blanton MP, and Baenziger JE
- Subjects
- Animals, Circular Dichroism, Endopeptidase K chemistry, Ion Channels chemistry, Ligands, Pronase chemistry, Protein Structure, Secondary, Protein Structure, Tertiary, Spectroscopy, Fourier Transform Infrared, Torpedo, Receptors, Nicotinic chemistry
- Abstract
The structure of the pore-forming transmembrane domain of the nicotinic acetylcholine receptor from Torpedo has been investigated by infrared spectroscopy. Treatment of affinity-purified receptor with either Pronase or proteinase K digests the extramembranous domains (roughly 75% of the protein mass), leaving hydrophobic membrane-imbedded peptides 3-6 kDa in size that are resistant to peptide (1)H/(2)H exchange. Infrared spectra of the transmembrane domain preparations exhibit relatively sharp and symmetric amide I and amide II band contours centered near 1655 and 1545 cm(-)1, respectively, in both (1)H(2)O and (2)H(2)O. The amide I band is very similar to the amide I bands observed in the spectra of alpha-helical proteins, such as myoglobin and bacteriorhodopsin, that lack beta structure and exhibit much less beta-sheet character than is observed in proteins with as little as 20% beta sheet. Curve-fitting estimates 75-80% alpha-helical character, with the remaining peptides likely adopting extended and/or turn structures at the bilayer surface. Infrared dichroism spectra are consistent with transmembrane alpha-helices oriented perpendicular to the bilayer surface. The evidence strongly suggests that the transmembrane domain of the nicotinic receptor, the most intensively studied ligand-gated ion channel, is composed of five bundles of four transmembrane alpha-helices.
- Published
- 2001
- Full Text
- View/download PDF
47. A conformational intermediate between the resting and desensitized states of the nicotinic acetylcholine receptor.
- Author
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Ryan SE, Blanton MP, and Baenziger JE
- Subjects
- Animals, Azirines metabolism, Dose-Response Relationship, Drug, Models, Biological, Nicotinic Antagonists metabolism, Phencyclidine metabolism, Photoaffinity Labels metabolism, Proadifen metabolism, Protein Conformation, Receptors, Nicotinic metabolism, Spectroscopy, Fourier Transform Infrared, Receptors, Nicotinic chemistry
- Abstract
The structural changes induced in the nicotinic acetylcholine receptor by two noncompetitive channel blockers, proadifen and phencyclidine, have been studied by infrared difference spectroscopy and using the conformationally sensitive photoreactive noncompetitive antagonist 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine. Simultaneous binding of proadifen to both the ion channel pore and neurotransmitter sites leads to the loss of positive markers near 1663, 1655, 1547, 1430, and 1059 cm(-)(1) in carbamylcholine difference spectra, suggesting the stabilization of a desensitized conformation. In contrast, only the positive markers near 1663 and 1059 cm(-)(1) are maximally affected by the binding of either blocker to the ion channel pore suggesting that the conformationally sensitive residues vibrating at these two frequencies are stabilized in a desensitized-like conformation, whereas those vibrating near 1655 and 1430 cm(-)(1) remain in a resting-like state. The vibrations at 1547 cm(-)(1) are coupled to those at both 1663 and 1655 cm(-)(1) and thus exhibit an intermediate pattern of band intensity change. The formation of a structural intermediate between the resting and desensitized states in the presence of phencyclidine is further supported by the pattern of 3-(trifluoromethyl)-3-m-([(125)I]iodophenyl)diazirine photoincorporation. In the presence of phencyclidine, the subunit labeling pattern is distinct from that observed in either the resting or desensitized conformations; specifically, there is a concentration-dependent increase in the extent of photoincorporation into the delta-subunit. Our data show that domains of the nicotinic acetylcholine receptor interconvert between the resting and desensitized states independently of each other and suggest a revised model of channel blocker action that involves both low and high affinity agonist binding conformational intermediates.
- Published
- 2001
- Full Text
- View/download PDF
48. Effect of membrane lipid composition on the conformational equilibria of the nicotinic acetylcholine receptor.
- Author
-
Baenziger JE, Morris ML, Darsaut TE, and Ryan SE
- Subjects
- Animals, Cholesterol chemistry, Electric Organ metabolism, Kinetics, Membrane Lipids chemistry, Membranes, Artificial, Models, Molecular, Phosphatidic Acids chemistry, Protein Conformation, Receptors, Nicotinic drug effects, Receptors, Nicotinic metabolism, Spectroscopy, Fourier Transform Infrared, Torpedo, Cholesterol pharmacology, Membrane Lipids pharmacology, Phosphatidic Acids pharmacology, Receptors, Nicotinic chemistry
- Abstract
The effects of cholesterol (Chol) and an anionic lipid, dioleoylphosphatidic acid (DOPA) on the conformational equilibria of the nicotinic acetylcholine receptor (nAChR) have been investigated using Fourier transform infrared difference spectroscopy. The difference between spectra recorded in the presence and absence of agonist from the nAChR reconstituted into 3:1:1 egg phosphatidylcholine (EPC)/DOPA/Chol membranes exhibits positive and negative bands that serve as markers of the structural changes associated with the resting to desensitized conformational change. These markers are absent in similar difference spectra recorded from the nAChR reconstituted into EPC membranes lacking both Chol and DOPA, indicating that the nAChR cannot undergo conformational change in response to agonist binding. When low levels of either Chol or DOPA up to 25 mol % of the total lipid are included in the EPC membranes, the markers suggest the predominant stabilization of a conformation that is a structural intermediate between the resting and desensitized states. At higher levels of either Chol or DOPA, the nAChR is stabilized in a conformation that is capable of undergoing agonist-induced desensitization, although DOPA appears to be required for the nAChR to adopt a conformation fully equivalent to that found in native and 3:1:1 EPC/DOPA/Chol membranes. The ability of these two structurally diverse lipids, as well as others (Ryan, S. E., Demers, C. N., Chew, J. P., Baenziger, J. E. (1996) J. Biol. Chem. 271, 24590-24597), to modulate the functional state of the nAChR suggests that lipids act on the nAChR via an indirect effect on some physical property of the lipid bilayer. The data also suggest that anionic lipids are essential to stabilize a fully functional nAChR. We propose that membrane fluidity modulates the relative populations of nAChRs in the resting and desensitized states but that subtle structural changes in the presence of anionic lipids are essential for full activity.
- Published
- 2000
- Full Text
- View/download PDF
49. Internal dynamics of the nicotinic acetylcholine receptor in reconstituted membranes.
- Author
-
Baenziger JE, Darsaut TE, and Morris ML
- Subjects
- Animals, Cholesterol chemistry, Deuterium Oxide chemistry, Dihydroxyphenylalanine chemistry, Kinetics, Membrane Fluidity, Peptides chemistry, Phosphatidylcholines chemistry, Protein Conformation, Spectroscopy, Fourier Transform Infrared, Torpedo, Membrane Lipids chemistry, Receptors, Nicotinic chemistry
- Abstract
The structure and 1H/2H exchange kinetics of affinity-purified nAChR reconstituted into egg phosphatidylcholine membranes with increasing levels of either dioleoylphosphatidic acid (DOPA) or cholesterol (Chol) have been examined using infrared spectroscopy. All spectra of the reconstituted nAChR membranes recorded after 72 h in 2H2O exhibit comparable amide I band shapes, suggesting a similar secondary structure for the nAChR in each lipid environment. Increasing levels of either DOPA or Chol, however, lead to an increasing intensity of the amide II band, indicating a decreasing proportion of nAChR peptide hydrogens that have exchanged for deuterium. Spectra recorded as a function of time after exposure of the nAChR to 2H2O show that the presence of either lipid slows down the 1H/2H exchange of those peptide hydrogens that normally exchange on the minutes to hours time scale. The slowing of peptide 1H/2H exchange correlates with both an increasing ability of the nAChR to undergo agonist-induced conformational change [Baenziger, J. E., Morris, M.-L., Darsaut, T. E., and Ryan, S. E. (1999) in preparation] and possibly a decreasing membrane fluidity. Our data suggest that lipid composition dependent changes in nAChR peptide 1H/2H exchange kinetics reflect altered internal dynamics of the nAChR. Lipids may influence protein function by changing the internal dynamics of integral membrane proteins.
- Published
- 1999
- Full Text
- View/download PDF
50. A structure-based approach to nicotinic receptor pharmacology.
- Author
-
Ryan SE and Baenziger JE
- Subjects
- Animals, Binding Sites, Binding, Competitive drug effects, Carbachol metabolism, Dibucaine pharmacology, Lidocaine pharmacology, Neurotransmitter Agents metabolism, Prilocaine pharmacology, Protein Conformation drug effects, Receptors, Nicotinic drug effects, Receptors, Nicotinic metabolism, Spectroscopy, Fourier Transform Infrared, Tetracaine pharmacology, Torpedo, Anesthetics, Local pharmacology, Receptors, Nicotinic chemistry
- Abstract
Infrared difference spectroscopy has been used to examine the structural effects of local anesthetic (LA) binding to the nicotinic acetylcholine receptor (nAChR). Several LAs induce subtle changes in the vibrational spectrum of the nAChR over a range of concentrations consistent with their reported nAChR-binding affinities. At concentrations of the desensitizing LAs prilocaine and lidocaine consistent with their binding to the ion channel pore, the vibrational changes suggest the stabilization of an intermediate conformation that shares structural features in common with both the resting and desensitized states. Higher concentrations of prilocaine and lidocaine, as well as the LA dibucaine, lead to additional binding to the neurotransmitter-binding site, the formation of physical interactions (most notably cation-tyrosine interactions) between LAs and neurotransmitter-binding-site residues, and the subsequent formation of a presumed desensitized nAChR. Although concentrations of the LA tetracaine consistent with binding to the ion channel pore elicit a reversed pattern of spectral changes suggestive of a resting state-like nAChR, higher concentrations also lead to neurotransmitter site binding and desensitization. Our results suggest that LAs stabilize multiple conformations of the nAChR by binding to at least two conformationally sensitive LA-binding sites. The spectra also reveal subtle differences in the strengths of the physical interactions that occur between LAs and binding-site residues. These differences correlate with LA potency at the nAChR.
- Published
- 1999
- Full Text
- View/download PDF
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