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Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed ?M3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

ABSTRACT: The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coupled tryptophan scanning mutagenesis (FT-TrpScanM) approach to gain insight into the secondary structure of the ?M3 transmembrane domain of the Torpedo californica nAChR, to monitor conformational changes experienced by this domain during channel gating, and to identify which lipid-exposed positions are linked to the regulation of ion channel kinetics. The perturbations produced by periodic tryptophan substitutions along the ?M3 transmembrane domain were characterized by two-electrode voltage clamp and (125)I-labeled ?-bungarotoxin binding assays. The periodicity profiles and Fourier transform spectra of this domain revealed similar helical structures for the closed- and open-channel states. However, changes in the oscillation patterns observed between positions Val-299 and Val-304 during transition between the closed- and open-channel states can be explained by the structural effects caused by the presence of a bending point introduced by a Thr-Gly motif at positions 300-301. The changes in periodicity and localization of residues between the closed-and open-channel states could indicate a structural transition between helix types in this segment of the domain. Overall, the data further demonstrate a functional link between the lipid-exposed transmembrane domain and the nAChR gating machinery.

SUBMITTER: Caballero-Rivera D

PROVIDER: S-EPMC3225733 | biostudies-literature | 2011 Jul-Aug

REPOSITORIES: biostudies-literature

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Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

Caballero-Rivera Daniel D Cruz-Nieves Omar A OA Oyola-Cintrón Jessica J Torres-Núñez David A DA Otero-Cruz Jose D JD Lasalde-Dominicci José A JA

Channels (Austin, Tex.) 20110701 4

The nicotinic acetylcholine receptor (nAChR) is a member of a family of ligand-gated ion channels that mediate diverse physiological functions, including fast synaptic transmission along the peripheral and central nervous systems. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, a high-resolution atomic structure of the nAChR still remains elusive. In this study, we extended the Fourier transform coup ...[more]

PMID: 21785268

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Project description:The nicotinic acetylcholine receptor (nAChR) of Torpedo electric rays has been extensively characterized over the last three decades. However, high-resolution structural studies have been hampered by the lack of mechanistic molecular models that describe how detergents influence membrane protein stability and function. Furthermore, elucidation of the dynamic detergent-lipid-protein interactions of solubilized membrane proteins is a largely unexplored research field. This study examines the effects of nine detergents on: (1) nAChR-lipid composition (gas chromatography with flame ionization; GC-FID and/or mass selective detectors; GC-MSD), (2) stability and aggregation state (analytical size exclusion chromatography; A-SEC and electron microscopy; EM) and (3) ion channel function (planar lipid bilayers). Detergent solubilization of nAChR-enriched membranes did not result in significant native lipid depletion or destabilization. Upon purification, native lipid depletion occurred in all detergents, with lipid-analogue detergents CHAPS {(3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate}, FC-12 (n-dodecylphosphocholine) and sodium cholate (3alpha,7alpha,12alpha-trihydroxy-5beta-cholan-24-oic acid) maintaining stability and supporting ion channel function, and non-lipid-analogue detergents Cymal-6 (6-cyclohexyl-1-hexyl-beta-D-maltoside), DDM (n-dodecyl-beta-D-maltopyranoside), LDAO (lauryldimethylamine-N-oxide) and OG (n-octyl-beta-d-glucopyranoside) decreasing stability and significantly reducing or completely suppressing ion channel function. Anapoe-C(12)E(9 )(polyoxyethylene-[9]-dodecyl ether) and BigCHAP (N,N'-bis-[3-d-gluconamidopropyl] cholamide) retained residual amounts of native lipid, maintaining moderate stability and ion channel function compared to lipid-analogue detergents. Therefore, the nAChR can be stable and functional in lipid-analogue detergents or in detergents that retain moderate amounts of residual native lipids, but not in non-lipid-analogue detergents.

Project description:Based on the conformational dependence of the amide-I i.r. band, this paper explores the use of Fourier-transform i.r. spectroscopy methods to probe structural features of proteins present in native membranes from Torpedo highly enriched in acetylcholine receptor (AcChR). The interference of water absorbance on the amide-I spectral region has been eliminated through isotopic exchange by freeze-drying the membranes in the presence of trehalose to avoid protein denaturation induced by drying, followed by resuspension in deuterated water. AcChR-rich membrane samples prepared in such a way maintained an ability to undergo affinity-state transitions and to promote cation translocation in response to cholinergic agonists, which are functional characteristics of native untreated samples. The temperature-dependence of the i.r. spectrum indicates a massive loss of ordered protein structure, occurring at temperatures similar to those reported for thermal denaturation of the AcChR by differential scanning calorimetry and by thermal inactivation of alpha-bungarotoxin-binding sites on the AcChR [Artigues, Villar, Ferragut & Gonzalez-Ros (1987) Arch. Biochem. Biophys. 258, 33-41], thus suggesting that the observed i.r. spectral changes correspond to alterations in the structure of the AcChR protein. Furthermore, the presence of detergents as well as cholinergic agonists and antagonists produces spectral changes that are also consistent with the alterations in AcChR protein structure expected from previous calorimetric studies. In contrast with the information obtained by calorimetry, i.r. spectroscopy allows the contribution of secondary structural changes to be distinguished from the overall change in protein structure. Thus prolonged exposure to cholinergic agonists, which drives the AcChR protein into the desensitized state, produces only negligible alterations in the amide-I band shape, but increases substantially the thermal stability of the protein. This suggests that rearrangements in the tertiary or quaternary structure of the protein are more likely to occur than extensive changes in secondary structure as a consequence of AcChR desensitization.

Dataset Information

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed ?M3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

Publications

Fourier transform coupled tryptophan scanning mutagenesis identifies a bending point on the lipid-exposed δM3 transmembrane domain of the Torpedo californica nicotinic acetylcholine receptor.

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