Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol

Corbin, John; Wang, Howard H.; Blanton, Michael P.

doi:10.1016/s0005-2736(98)00153-9

Cited by 76 publications

(61 citation statements)

References 39 publications

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“…1 A), and used it to photolabel nAChR ␣1. Previous studies have mapped the cholesterol-binding site of nAChR within the transmembrane domain (34,35), specifically within M1 and M4 (36). Thus, we expected CBPP to partition into the annulus of phospholipid and native cholesterol around the nAChR.…”

Section: Photolabeled Residues Identified Within the N-terminal Domainmentioning

confidence: 87%

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry

Leite

Blanton

Shahgholi

et al. 2003

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

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We characterized the differential accessibility of the nicotinic acetylcholine receptor α1 subunit in the open, closed, and desensitized states by using electrophysiology-coordinated photolabeling by several lipophilic probes followed by mass spectrometric analysis. Voltage-clamped oocytes expressing receptors were preincubated with one of the lipophilic probes and were continually exposed to acetylcholine; UV irradiation was applied during 500-ms pulses to + 40 or to -140 mV (which produced closed or ≈50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the β8–β9 loop of the acetylcholine-binding protein. In the closed state, probe incorporation was identified at several sites of the N-terminal domain within the conserved cysteine loop (residues 128–142), the cytoplasmic loop (M3–M4), and M4. The labeling pattern in the M4 region is consistent with previous results, further defining the lipid-exposed face of this transmembrane α-helix. These results show regions within the N-terminal domain that are involved in gating-dependent conformational shifts, confirm that the cysteine loop resides at or near the protein-membrane interface, and show that segments of the M3–M4 loop are near to the lipid bilayer

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Section: Photolabeled Residues Identified Within the N-terminal Domainmentioning

confidence: 87%

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry

Leite

Blanton

Shahgholi

et al. 2003

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

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“…Because various membrane proteins, e.g. receptors for oxytocin or acetylcholine, are endowed with specific binding sites for cholesterol (33)(34)(35), this raises the intriguing possibility that cholesterol may function as a second messenger to diacylglycerol and/or ceramide within the cytoplasmic membrane.…”

Section: Discussionmentioning

confidence: 99%

Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

Zitzer

Bittman

Verbicky

et al. 2001

Journal of Biological Chemistry

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Vibrio cholerae cytolysin (VCC) forms oligomeric pores in lipid bilayers containing cholesterol. Membrane permeabilization is inefficient if the sterol is embedded within bilayers prepared from phosphatidylcholine only but is greatly enhanced if the target membrane also contains ceramide. Although the enhancement of VCC action is stereospecific with respect to cholesterol, we show here that no such specificity applies to the two stereocenters in ceramide; all four stereoisomers of ceramide enhanced VCC activity in cholesterol-containing bilayers. A wide variety of ceramide analogs were as effective as D-erythro-ceramide, as was diacylglycerol, suggesting that the effect of ceramide exemplifies a general trend of lipids with a small headgroup to augment the activity of VCC. Incorporation of these cone-shaped lipids into cholesterol-containing bilayers also gave similar effects with streptolysin O, another cholesterol-specific but structurally unrelated cytolysin. In contrast, the activity of staphylococcal ␣-hemolysin, which does not share with the other toxins the requirement for cholesterol, was far less affected by the presence of lipids with a conical shape. The collective data indicate that sphingolipids and glycerolipids do not interact with the cytolysins specifically. Instead, lipids that have a conical molecular shape appear to effect a change in the energetic state of membrane cholesterol that in turn augments the interaction of the sterol with the cholesterol-specific cytolysins.To bacterial pore-forming cytolysins, cholesterol is a logical choice as a target molecule, because it confers specificity for animal as opposed to bacterial cell membranes. The specificity for cholesterol is shared between Vibrio cholerae cytolysin (VCC) 1 (1) and streptolysin O (SLO) (2). Otherwise, these toxins are not related, and the oligomeric pores they form are very different in size and morphology (1, 3). Although with SLO the sterol is already required in the initial event of membrane binding of the monomeric toxin (4), it only comes into play at the stage of oligomerization in the case of VCC (5, 6). When the sterol is incorporated into phosphatidylcholine (PC) bilayers at physiologically realistic concentrations (i.e. up to 40% by mol), these membranes do not become significantly sensitive to VCC. However, it was previously found that membrane susceptibility toward the cytolysin was greatly enhanced by inclusion of ceramide; free ceramide and monohexosyl ceramides proved similarly effective (7). A combined specificity for cholesterol and sphingolipids has previously been shown for the fusion protein of Semliki Forest virus. In that instance, the interaction with ceramide proved to be highly stereoselective (8 -10). Accordingly, we have examined the structural properties of the ceramide molecule responsible for the sensitization of membranes to VCC. To our surprise, no dependence on stereospecific features of ceramide could be detected. Membrane sensitization was readily achieved with a variety of synthetic ceramides...

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“…Chol covalently linked to a phospholipid in the bulk lipid is as effective as free Chol in stabilizing a conformationally competent nAChR, suggesting that the non-annular sites proposed by Jones and McNamee (4) are not critical (18). Only Chol interaction sites on the lipid-exposed surface of the nAChR were detected in a recent affinity labeling experiment (19). Although specific Chol binding sites on the nAChR are often invoked to explain the ability of Chol to enhance nAChR activity, both the loose structural requirements of the nAChR for neutral and anionic lipids and the lack of experimentally documented specific Chol binding sites are suggestive of an indirect effect of lipids on bulk properties of the lipid membrane.…”

Section: CMmentioning

confidence: 95%

Effect of Membrane Lipid Composition on the Conformational Equilibria of the Nicotinic Acetylcholine Receptor

Baenziger

Morris

Darsaut

et al. 2000

Journal of Biological Chemistry

140

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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 agonistinduced 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.

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Identifying the cholesterol binding domain in the nicotinic acetylcholine receptor with [125I]azido-cholesterol

Cited by 76 publications

References 39 publications

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry

Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry

Coupling of Cholesterol and Cone-shaped Lipids in Bilayers Augments Membrane Permeabilization by the Cholesterol-specific Toxins Streptolysin O and Vibrio cholerae Cytolysin

Effect of Membrane Lipid Composition on the Conformational Equilibria of the Nicotinic Acetylcholine Receptor

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