Allosteric transitions of <i>Torpedo</i> acetylcholine receptor in lipids, detergent and amphipols: molecular interactions vs. physical constraints

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Nicotinic acetylcholine receptors (AChRs) belong to a superfamily of oligomeric proteins that transduce electric signals across the cell membrane on binding of neurotransmitters. These receptors harbor a large extracellular ligand-binding domain directly linked to an ion-conducting channel-forming domain that spans the cell membrane 20 times and considerably extends into the cytoplasm. Thus far, none of these receptor channels has been crystallized in three dimensions. The crystallization of the AChR from Torpedo marmorata electric organs is challenged here in lipidic؊detergent matrices. Detergent-soluble AChR complexed with ␣-bungarotoxin (␣BTx), a polypeptidic competitive antagonist, was purified. The AChR-␣BTx complex was reconstituted in a lipidic matrix composed of monoolein bilayers that are structured in three dimensions. The ␣BTx was conjugated to a photo-stable fluorophore, enabling us to monitor the physical behavior of the receptor-toxin complex in the lipidic matrix under light stereomicroscope, and to freeze fracture regions containing the receptor-toxin complex for visualization under a transmission electron microscope. Conditions were established for forming 2D receptor-toxin lattices that are stacked in the third dimension. 3D AChR nanocrystals were thereby grown inside the highly viscous lipidic 3D matrix. Slow emulsification of the lipidic matrix converted these nanocrystals into 3D elongated thin crystal plates of micrometer size. The latter are stable in detergent-containing aqueous solutions and can currently be used for seeding and epitaxial growth, en route to crystals of appropriate dimensions for x-ray diffraction studies.nicotinic acetylcholine receptor ͉ crystallization ͉ structure T o date, the crystal structures of Ϸ50 integral membrane proteins have been determined at atomic resolution. This result is in stark contrast to their abundance, as they comprise one-third of all gene products. Inherent difficulties with highlevel expression, purification, and, particularly, crystallization of integral membrane proteins account for this situation (1). Receptor channels that selectively regulate transport of ions across the cell membrane are large membrane-spanning oligomers. Among them are the pentameric ligand-gated ion channels (also termed the Cys-loop receptors), which mediate and modulate fast cell-cell communication throughout the nervous system.The first and most extensively studied pentameric ligand-gated ion channel is the acetylcholine receptor (AChR; reviewed in refs. 2-4). The AChR isolated from the electric organs of Torpedo ray is available in milligram amounts suitable for crystallization experiments. It is a pseudosymmetrical glycoprotein of Ϸ290 kDa comprised of five subunits (2␣␤␥␦) and carries two ACh-binding sites at the ␣Ϫ␥ and ␣Ϫ␦ boundaries (2, 3). So far, it was possible to organize this oligomeric protein only in tubular 2D crystals within receptor-rich membrane fragments (5, 6). These crystals enabled the collection of electron diffraction data at 9-Å down to 4-Å r...

Section: Methodsmentioning

confidence: 87%

Electron microscopic evidence for nucleation and growth of 3D acetylcholine receptor microcrystals in structured lipid–detergent matrices

Paas

Cartaud

Recouvreur

et al. 2003

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“…In fact, these factors have been shown to affect the conformational dynamics of BtuB (the Escherichia coli outer-membrane transporter for vitamin B 12 ) (33)(34)(35) and the kinetics of the photocycle in bacteriorhodopsin (36), for example. In addition, of particular relevance to this article, the effect of membrane lipids on the relative occupancies of the closed, open and desensitized states of the AChR from Torpedo's electric organ has been well documented (3,37,38). Alternatively, it may well be that only one conformation of ELIC crystallizes under the tested conditions, in which case all efforts to crystallize specific functional states of the channel by appropriately tilting the free-energy landscape would be futile.…”

Section: Discussionmentioning

confidence: 99%

“…For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2,3). Similarly, mutations can also affect the conformational free-energy landscape of ion channels in such a way that the occupancy of a particular conformation is markedly favored or disfavored.…”

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confidence: 99%

Mutations that stabilize the open state of the Erwinia chrisanthemi ligand-gated ion channel fail to change the conformation of the pore domain in crystals

González-Gutiérrez

Lukk

Agarwal

et al. 2012

The determination of structural models of the various stable states of an ion channel is a key step toward the characterization of its conformational dynamics. In the case of nicotinic-type receptors, different structures have been solved but, thus far, these different models have been obtained from different members of the superfamily. In the case of the bacterial member ELIC, a cysteamine-gated channel from Erwinia chrisanthemi, a structural model of the protein in the absence of activating ligand (and thus, conceivably corresponding to the closed state of this channel) has been previously generated. In this article, electrophysiological characterization of ELIC mutants allowed us to identify pore mutations that slow down the time course of desensitization to the extent that the channel seems not to desensitize at all for the duration of the agonist applications (>20 min). Thus, it seems reasonable to conclude that the probability of ELIC occupying the closed state is much lower for the ligand-bound mutants than for the unliganded wild-type channel. To gain insight into the conformation adopted by ELIC under these conditions, we solved the crystal structures of two of these mutants in the presence of a concentration of cysteamine that elicits an intracluster open probability of >0.9. Curiously, the obtained structural models turned out to be nearly indistinguishable from the model of the wild-type channel in the absence of bound agonist. Overall, our findings bring to light the limited power of functional studies in intact membranes when it comes to inferring the functional state of a channel in a crystal, at least in the case of the nicotinicreceptor superfamily.electrophysiology | structure-function T he use of bacterial and archaeal ion channels as models of the structure and function of their eukaryotic counterparts has become one of the mainstays of ion-channel biophysics. It seems to us that this practice is particularly well justified whenever the use of noneukaryotic channels facilitates experiments that are otherwise too difficult or too cumbersome to perform. A clear case in point is the determination of structural models of these membrane proteins using X-ray crystallography.One of the main goals of direct structural approaches as applied to ion channels is to determine what these proteins look like in their different functional states. Assuming that all of these conformations can form well-diffracting crystals, the problem is reduced to finding the conditions under which the occupancy of each particular state is maximized. In general, for well-characterized ion channels, the experimental maneuvers that are needed to favor or disfavor at least some of these different conformations are known. For example, in the case of (wild-type) neurotransmitter-gated channels, the binding of the natural neurotransmitter strongly stabilizes the desensitized state (e.g., ref. 1), whereas the absence of neurotransmitter or the binding of a competitive antagonist keeps the channel mostly closed (2, 3). Similarly, mut...

“…nAChR was solubilized with CHAPS from postsynaptic membranes of Torpedo marmorata electric organs, purified, and trapped with a mixture of BA8-35 and A8-35 (see SI Text) as described in ref. 22 for nonfunctionalized A8-35. We have shown previously that A8-35-trapped nAChR, at variance with the CHAPS-solubilized receptor, binds a fluorescent analog of acetylcholine with kinetics comparable with that observed in native postsynaptic membranes (22).…”

Section: Binding Of a Fluorescent Toxin To An Immobilized Pharmacologmentioning

confidence: 99%

“…1A). Its use has been validated on a large panel of MPs (18), including bacteriorhodopsin (BR) (20), the nicotinic acetylcholine receptor (nAChR) (22), the cytochrome b 6 f and bc 1 complexes (17,18) and the transmembrane domain of Escherichia coli outer membrane protein A (tOmpA) (19,23). Of particular relevance to the present work is the fact that, although it is noncovalent, its association to MPs is strictly irreversible as long as it is not displaced by another surfactant (23,24).…”

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confidence: 99%

The use of amphipols as universal molecular adapters to immobilize membrane proteins onto solid supports

Charvolin

Perez

Rouvière

et al. 2009

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Because of the importance of their physiological functions, cell membranes represent critical targets in biological research. Membrane proteins, which make up Ϸ1/3 of the proteome, interact with a wide range of small ligands and macromolecular partners as well as with foreign molecules such as synthetic drugs, antibodies, toxins, or surface recognition proteins of pathogenic organisms. Whether it is for the sake of basic biomedical or pharmacological research, it is of great interest to develop tools facilitating the study of these interactions. Surface-based in vitro assays are appealing because they require minimum quantities of reagents, and they are suitable for multiplexing and high-throughput screening. We introduce here a general method for immobilizing functional, unmodified integral membrane proteins onto solid supports, thanks to amphipathic polymers called ''amphipols.'' The key point of this approach is that functionalized amphipols can be used as universal adapters to associate any membrane protein to virtually any kind of support while stabilizing its native state. The generality and versatility of this strategy is demonstrated by using 5 different target proteins, 2 types of supports (chips and beads), 2 types of ligands (antibodies and a snake toxin), and 2 detection methods (surface plasmon resonance and fluorescence microscopy).diagnostics ͉ drug discovery ͉ immobilization ͉ chips bioreactors