Probing the structure of the nicotinic acetylcholine receptor ion channel with the uncharged photoactivable compound -3H-diazofluorene.
The uncharged photoactivable probe 2-[3H]diazofluorene ([3H]DAF) was used to examine structural changes in the Torpedo californica nicotinic acetylcholine receptor (AChR) ion channel induced by agonists. Photoincorporation of [3H]DAF into the AChR consisted of the following two components: a nonspecific component consistent with incorporation into residues situated at the lipid-protein interface, and a specific component, inhibitable by noncompetitive antagonists and localized to the M2 hydrophobic segments of AChR subunits. The nonspecific [3H]DAF incorporation was characterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled residues reinforce the conclusion that the M4 segments are organized as transmembrane alpha-helices with a common "face" of each helix in contact with lipid. Within the M2 segments, in the absence of agonist [3H]DAF specifically labeled homologous residues betaVal-261 and deltaVal-269, with incorporation into deltaVal-269 at a 5-fold greater efficiency than into betaVal-261. This observation, coupled with the lack of detectable incorporation into alpha-M2 including the homologous alphaVal-255, indicates that within the resting channel [3H]DAF is bound with its photoreactive diazo group oriented toward deltaVal-269. In the presence of agonist, there is an approximately 90% reduction in the labeling of betaVal-261 and deltaVal-269 accompanied by specific incorporation into residues (betaLeu-257, betaAla-258, deltaSer-262, and deltaLeu-265) situated 1 or 2 turns of an alpha-helix closer to the cytoplasmic end of the M2 segments. The results provide a further characterization of agonist-induced rearrangements of the M2 (ion channel) domain of the AChR.
Blanton
M P
MP
Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Dangott
L J
LJ
Raja
S K
SK
Lala
A K
AK
Cohen
J B
JB
D000345:Affinity Labels; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003979:Diazonium Compounds / Q000378:metabolism*; D004557:Electric Organ / Q000378:metabolism*; D005449:Fluorenes / Q000378:metabolism*; D007473:Ion Channels / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D017433:Protein Structure, Secondary; D011978:Receptors, Nicotinic / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014101:Torpedo; D014316:Tritium; D014357:Trypsin
D000345:Affinity Labels; D003979:Diazonium Compounds; D005449:Fluorenes; D007473:Ion Channels; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011978:Receptors, Nicotinic; D014316:Tritium; C052770:2-diazofluorene; D014357:Trypsin