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Published October 28, 2003 | Published + Erratum
Journal Article Open

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

Abstract

We characterized the differential accessibility of the nicotinic acetylcholine receptor all 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 approximate to50% open receptors, respectively). In the open state, there was specific probe incorporation within the N-terminal domain at residues that align with the beta8-beta9 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 a-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.

Additional Information

© 2003 by the National Academy of Sciences. Edited by William A. Catterall, University of Washington School of Medicine, Seattle, WA, and approved August 22, 2003 (received for review May 16, 2003). This paper was submitted directly (Track II) to the PNAS office. This work was supported by grants from the National Institutes of Health (NS11756, NS34407, NS35786, and NRSA) to J.F.L.

Errata

Proceedings of the National Academy of Sciences Dec 2003, 100 (26) 16144; DOI: 10.1073/pnas.2637015100

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Published - LEIpnas03.pdf

Erratum - 16144.full.pdf

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August 22, 2023
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