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Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating.

ABSTRACT: The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings and Fourier analyses to characterize the functional role and structural aspects of the betaM4 transmembrane domain of the Torpedo AChR. We performed tryptophan replacements, from residues L438 through F455, along the betaM4 transmembrane domain. Expression levels of mutants F439W-G450W and F452W-I454W produced peak currents similar to or lower than those in wild-type (WT). Tryptophan substitutions at positions L438 and T451 led to a deficiency in either subunit expression or receptor assembly. Mutations L440W, V442W, C447W and S453W produced a gain-of-function response. Mutation F455W produced a loss of ion channel function. The periodicity profile of the normalized expression level (closed state) and EC(50) (open state) revealed a minor conformational change of 0.4 residues/turn of the betaM4 domain. These findings suggest that a minor movement of the betaM4 domain occurs during channel activation.

SUBMITTER: Diaz-De Leon R

PROVIDER: S-EPMC4835820 | biostudies-literature | 2008 Nov-Dec

REPOSITORIES: biostudies-literature

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Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating.

Díaz-De León Rosedelma R Otero-Cruz José David JD Torres-Nuñez David Abner DA Casiano Anette A Lasalde-Dominicci José Antonio JA

Channels (Austin, Tex.) 20081106 6

The nicotinic acetylcholine receptor (nAChR) is a ligand-gated ion channel protein that mediates fast excitatory synaptic transmission in the peripheral and central nervous systems. Changes in the structure and function of the AChR can lead to serious impairment of physiological processes. In this study, we combined site-directed mutagenesis, radioligand binding assays, electrophysiological recordings and Fourier analyses to characterize the functional role and structural aspects of the betaM4 t ...[more]

PMID: 19066450

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Project description:Previous cysteine scanning studies of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have identified several transmembrane segments (TMs), including TM1, 3, 6, 9, and 12, as structural components of the pore. Some of these TMs such as TM6 and 12 may also be involved in gating conformational changes. However, recent results on TM1 seem puzzling in that the observed reactive pattern was quite different from those seen with TM6 and 12. In addition, whether TM1 also plays a role in gating motions remains largely unknown. Here, we investigated CFTR's TM1 by applying methanethiosulfonate (MTS) reagents from both cytoplasmic and extracellular sides of the membrane. Our experiments identified four positive positions, E92, K95, Q98, and L102, when the negatively charged MTSES was applied from the cytoplasmic side. Intriguingly, these four residues reside in the extracellular half of TM1 in previously defined CFTR topology; we thus extended our scanning to residues located extracellularly to L102. We found that cysteines introduced into positions 106, 107, and 109 indeed react with extracellularly applied MTS probes, but not to intracellularly applied reagents. Interestingly, whole-cell A107C-CFTR currents were very sensitive to changes of bath pH as if the introduced cysteine assumes an altered pKa-like T338C in TM6. These findings lead us to propose a revised topology for CFTR's TM1 that spans at least from E92 to Y109. Additionally, side-dependent modifications of these positions indicate a narrow region (L102-I106) that prevents MTS reagents from penetrating the pore, a picture similar to what has been reported for TM6. Moreover, modifications of K95C, Q98C, and L102C exhibit strong state dependency with negligible modification when the channel is closed, suggesting a significant rearrangement of TM1 during CFTR's gating cycle. The structural implications of these findings are discussed in light of the crystal structures of ABC transporters and homology models of CFTR.

Dataset Information

Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating.

Publications

Tryptophan scanning of the acetylcholine receptor's betaM4 transmembrane domain: decoding allosteric linkage at the lipid-protein interface with ion-channel gating.

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