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

Leite, John F. and Blanton, Michael P. and Shahgholi, Mona and Dougherty, Dennis A. and Lester, Henry A. (2003) Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: Electrophysiology-coordinated photochemistry and mass spectrometry. Proceedings of the National Academy of Sciences of the United States of America, 100 (22). pp. 13054-13059. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:LEIpnas03

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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.


Item Type:Article
Additional Information:Copyright © 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.
Subject Keywords:QUADRUPOLE ION-TRAP, ACETYLCHOLINE-RECEPTOR, NONCOMPETITIVE ANTAGONIST, ELECTROSPRAY-IONIZATION, VOLTAGE-DEPENDENCE, GABA(A) RECEPTOR, GLYCINE RECEPTOR, AGONIST BINDING, RESTING STATE, CHANNEL
Record Number:CaltechAUTHORS:LEIpnas03
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:LEIpnas03
Alternative URL:http://dx.doi.org/10.1073/pnas.2133028100
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:904
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:07 Nov 2005
Last Modified:14 Nov 2014 19:18

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