A BINDING SITE FOR THE VOLATILE ANESTHETIC HALOTHANE WITHIN THE NICOTINIC ACETYLCHOLINE RECEPTOR IDENTIFIED BY PHOTOAFFINITY LABELING.
David C. Chiara1 , Roderic G. Eckenhoff2 , Lawrence J. Dangott1 , Jonathan B. Cohen1,
1Department of Neurobiology, Harvard Medical School, 220 Longwood Avenue, Boston, Massachusetts 02115, 2Department of Anesthesia, University of Pennsylvania, Philadelphia, PA
The volatile anesthetic halothane inhibits nicotinic acetylcholine receptor (nAChR) and potentiates GABAA receptor responses at similar concentrations. For [14C]halothane equilibrated with Torpedo nAChR-rich membranes, UV irradiation at 254 nm results in covalent incorporation into each nAChR subunit. Sites of [14C]halothane incorporation were identified by N-terminal sequencing of labeled fragments purified by SDS-PAGE and reversed-phase HPLC from proteolytic digests of isolated subunits. Digestion of the d-subunit with endoproteinase Lys-C generated 2 14C-labeled fragments, one containing the membrane spanning segment dM1 and the other dM2. No 14C-labeled amino acids were detected within dM2. Sequence analysis of the dM1 fragment revealed [14C]halothane incorporation in dTyr-212 preceding M1 and in dTyr-228 within M1. The labeling at dTyr-228 was ~2-fold higher in the presence of the agonist carbamylcholine, whereas the volatile anesthetic isoflurane reduced the labeling by ~50%. In contrast, the labeling at dTyr-212 was unaffected by either carbamylcholine or isoflurane. Since halothane interacts directly in a pharmacologically sensitive manner with the nAChR in a region of primary structure within M1 that has been previously identified as contributing to the lumen of the ion channel, this interaction may contribute to nAChR inhibition.