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. Mar-Apr 2012;6(2):111-23.
doi: 10.4161/chan.19540. Epub 2012 Mar 1.

Tryptophan Scanning Mutagenesis Reveals Distortions in the Helical Structure of the δM4 Transmembrane Domain of the Torpedo Californica Nicotinic Acetylcholine Receptor

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Tryptophan Scanning Mutagenesis Reveals Distortions in the Helical Structure of the δM4 Transmembrane Domain of the Torpedo Californica Nicotinic Acetylcholine Receptor

Daniel Caballero-Rivera et al. Channels (Austin). .
Free PMC article

Abstract

The lipid-protein interface is an important domain of the nicotinic acetylcholine receptor (nAChR) that has recently garnered increased relevance. Several studies have made significant advances toward determining the structure and dynamics of the lipid-exposed domains of the nAChR. However, there is still a need to gain insight into the mechanism by which lipid-protein interactions regulate the function and conformational transitions of the nAChR. In this study, we extended the tryptophan scanning mutagenesis (TrpScanM) approach to dissect secondary structure and monitor the conformational changes experienced by the δM4 transmembrane domain (TMD) of the Torpedo californica nAChR, and to identify which positions on this domain are potentially linked to the regulation of ion channel kinetics. The difference in oscillation patterns between the closed- and open-channel states suggests a substantial conformational change along this domain as a consequence of channel activation. Furthermore, TrpScanM revealed distortions along the helical structure of this TMD that are not present on current models of the nAChR. Our results show that a Thr-Pro motif at positions 462-463 markedly bends the helical structure of the TMD, consistent with the recent crystallographic structure of the GluCl Cys-loop receptor which reveals a highly bent TMD4 in each subunit. This Thr-Pro motif acts as a molecular hinge that delineates two gating blocks in the δM4 TMD. These results suggest a model in which a hinge-bending motion that tilts the helical structure is combined with a spring-like motion during transition between the closed- and open-channel states of the δM4 TMD.

Figures

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Figure 1. Sequence alignment of the δM4 transmembrane domain and functional response of wild type and δM4 mutant nAChRs. (A) Positions Leu-456 to Ile-472 were examined in this study. Positions Ser-457 and Met-465 (highlighted in yellow) were labeled as lipid-exposed using 125I-TID photolabeling affinity. Positions Met-458 and Phe-459 (highlighted in light blue) were labeled with 3H-DAF photolabeling affinity. Residues highlighted in red are conserved residues among all species. The numbers at the bottom indicate the position in the Torpedoδ subunit. (B) Representative families of macroscopic ionic current traces evoked by 1–300 μM ACh and recorded through voltage clamp. Bar scale: 5000 nA (abscissa), 5 sec (ordinate). (C) Concentration-response curves that were normalized to maximum ionic current for wild type-like (top), gain-of-function (middle) and loss-of-function (bottom) δM4 mutant nAChRs.
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Figure 2. Expression level profile for the δM4 nAChR mutants. 125I-Labeled α-BgTx binding assays were performed to determine nAChR expression levels in the plasmatic membrane. Each bar represents the mean level of nAChR expression (fmol) ± SEM *p < 0.05 compared with WT nAChR.
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Figure 3. Periodicity profiles for the Torpedo nAChR δM4 transmembrane domain. (A and B) Tryptophan periodicity profiles for the closed- and open-channel states, respectively. The values inside the boxes indicate the number of residues per helical turn between the adjacent maximums and minimums peaks.
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Figure 4. Helical net diagrams for the Torpedo nAChR δM4 transmembrane domain mutants in the open and closed-channel states. (A–D) Localization and amount of amino acids per helical turn between the adjacent maximum and minimum oscillatory peaks of the periodicity profiles for the Torpedo nAChR δM4 domain in the open-channel state (A and B) and the closed-channel state (C and D). The gray box delimits the area of the helix where the δM4 mutants that produced loss-of-function (blue circles) and gain-of-function (red circles) nAChRs are localized based on the data from the tryptophan periodicity profiles.

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