Conformation-dependent hydrophobic photolabeling of the nicotinic receptor: electrophysiology-coordinated photochemistry and mass spectrometry.
We characterized the differential accessibility of the nicotinic acetylcholine receptor alpha1 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 approximately 50% 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 alpha-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.
Leite
John F
JF
Divisions of Biology and Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA. jleite@caltech.edu
Blanton
Michael P
MP
Shahgholi
Mona
M
Dougherty
Dennis A
DA
Lester
Henry A
HA
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004594:Electrophysiology / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D021122:Protein Subunits / Q000737:chemistry; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D021122:Protein Subunits; D011978:Receptors, Nicotinic; D011994:Recombinant Proteins