Identification of sites of incorporation in the nicotinic acetylcholine receptor of a photoactivatible general anesthetic

Abstract

Most general anesthetics including long chain aliphatic alcohols act as noncompetitive antagonists of the nicotinic acetylcholine receptor (nAChR). To locate the sites of interaction of a long chain alcohol with the Torpedo nAChR, we have used the photoactivatible alcohol 3-[3H]azioctanol, which inhibits the nAChR and photo-incorporates into nAChR subunits. At 1 and 275 μM, 3-[3H]azioctanol photoincorporated into nAChR subunits with increased incorporation in the α-subunit in the desensitized state. The incorporation into the α-subunit was mapped to two large proteolytic fragments. One fragment of ∼20 kDa (αV8-20), containing the M1, M2, and M3 transmembrane segments, showed enhanced incorporation in the presence of agonist whereas the other of ∼10 kDa (αV8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label. Within αV8-20, the primary site of incorporation was αGlu-262 at the C-terminal end of αM2, labeled preferentially in the desensitized state. The incorporation at αGlu-262 approached saturation between 1 μpM, with ∼6% labeled, and 275 μM, with ∼30% labeled. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at ∼1% of the levels in αGlu-262. Therefore, the primary binding site of 3-azioctanol is within the ion channel with additional lower affinity interactions within the agonist binding site and at the protein-lipid interface.