PROBING THE STRUCTURE OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR ION-CHANNEL WITH THE UNCHARGED PHOTOACTIVABLE COMPOUND [H-3]DIAZOFLUORENE

The uncharged photoactivable probe 2-[H-3]diazofluorene ([H-3]DAF) was used to examine structural changes in the Torpedo californica nicotin ic acetylcholine receptor (AChR) ion channel induced by agonists. Phot oincorporation of [H-3]DAF into the AChR consisted of the following tw o components: a nonspecific component consistent with incorporation in to residues situated at the lipid-protein interface, and a specific co mponent, inhibitable by noncompetitive antagonists and localized to th e M2 hydrophobic segments of AChR subunits. The nonspecific [3H]DAF in corporation was characterized in the M4 segment of each AChR subunit. The observed distribution and periodicity of labeled residues reinforc e the conclusion that the M4 segments are organized as transmembrane a -helices with a common ''face'' of each helix in contact with lipid, W ithin the M2 segments, in the absence of agonist [H-3]DAF specifically labeled homologous residues beta Val-261 and delta Val-269, with inco rporation into beta Val-269 at a 5-fold greater efficiency than into b eta Val-261. This observation, coupled with the lack of detectable inc orporation into alpha-M2 including the homologous alpha Val-255, indic ates that within the resting channel [H-3]DAF is bound with its photor eactive diazo group oriented toward delta Val-269. In the presence of agonist, there is an similar to 90% reduction in the labeling of beta Val-261 and delta Val-269 accompanied by specific incorporation into r esidues (beta Leu-257, beta Ala-258, delta Ser-262, and delta Leu-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.