Assessing the Lipid Requirements of the<i>Torpedo californica</i>Nicotinic Acetylcholine Receptor

Hamouda, Ayman K.; Sanghvi, Mitesh; Sauls, Daniel; Machu, Tina K.; Blanton, Michael P.

doi:10.1021/bi052281z

Cited by 75 publications

(105 citation statements)

References 35 publications

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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%

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

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Discussionmentioning

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

Proc. Natl. Acad. Sci. U.S.A.

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“…Torpedo nAChR-rich membranes at 1 mg/ml protein were solubilized in 1% sodium cholate in vesicle dialysis buffer (VDB; 100 mM NaCl, 0.1 mM EDTA, 0.02% NaN 3 , 10 mM MOPS, pH 7.5) and treated with 0.1 mM diisopropylfluorophosphate after insoluble material was pelleted by centrifugation (91,000 ϫ g for 1 h). The nAChR was affinity-purified on a bromoacetylcholine bromide-derivatized Affi-Gel 10 column and then reconstituted into lipid vesicles composed of dioleoyl phosphatidic acid/dioleoyl phosphatidylcholine/cholesterol (at a molar ratio of 3:1:1), as described (23,24). The lipid/nAChR ratio was adjusted to molar ratio of 400:1.…”

Section: Materials-[mentioning

confidence: 99%

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Srivastava¹,

Hamouda²,

Pandhare³

et al. 2009

Journal of Biological Chemistry

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Nicotinic acetylcholine receptor (nAChR) agonists, such as epibatidine and its molecular derivatives, are potential therapeutic agents for a variety of neurological disorders. In order to identify determinants for subtype-selective agonist binding, it is important to determine whether an agonist binds in a common orientation in different nAChR subtypes. To compare the mode of binding of epibatidine in a muscle and a neuronal nAChR, we photolabeled Torpedo ␣ 2 ␤␥␦ and expressed human ␣4␤2

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“…Direct interaction of these molecules with the nAChR was proposed to account for the requisite specific lipid composition for receptor functions. However, several lines of experimental evidence have later emerged to an alternate mechanism that CHOL and/or PA may act on the nAChR via an indirect effect on some physical properties of the lipid bilayer 5,10,15 . Both CHOL and PA are able to increase the lateral packing density of the lipid bilayers, which could lead to slow nAChR internal motion 5,16 .…”

Section: Introductionmentioning

confidence: 99%

Molecular Dynamics Simulations of Ternary Membrane Mixture: Phosphatidylcholine, Phosphatidic Acid, and Cholesterol

et al. 2007

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A ternary mixture of 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl phosphatidic acid (POPA), and cholesterol (CHOL) works effectively for a functional conformation of nicotinic acetylcholine receptor (nAChR) that can undergo agonist-induced conformation changes, but POPC alone can stabilize only a desensitized state of nAChR. To gain insights into the lipid mixture that have strong impact to nAChR functions, we performed more than 50-ns all atom molecular dynamic (MD) simulations at 303 K on a fully hydrated bilayer consisting of 240 POPC, 80 POPA, and 80 CHOL (3:1:1). The MD simulation revealed various interactions between different types of molecular pairs that ultimately regulated lipid organization. The heterogeneous interactions among three different constituents resulted in a broad spectrum of lipid properties, including extensive distribution of average area per lipids and varied lipid ordering as a function of lipid closeness to CHOL. Higher percentage of POPA than POPC had close association with CHOL, which coincided with relatively higher ordering of POPA molecules in their acyl chains near lipid head groups. Lower fraction of gauche dihedrals was also found in the same region of POPA. Although the CHOL molecules had the effects on the enhancement of surrounding lipid order, relatively low lipid order parameters and high fraction of gauche bonds were observed in the ternary mixture. Collectively, these results suggest that the dynamical structure of the ternary system could be determinant for a functional nAChR.

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Assessing the Lipid Requirements of theTorpedo californicaNicotinic Acetylcholine Receptor

Cited by 75 publications

References 35 publications

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

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

[3H]Epibatidine Photolabels Non-equivalent Amino Acids in the Agonist Binding Site of Torpedo and α4β2 Nicotinic Acetylcholine Receptors

Molecular Dynamics Simulations of Ternary Membrane Mixture: Phosphatidylcholine, Phosphatidic Acid, and Cholesterol

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