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

Most general anesthetics including long chain aliphatic alcohols act as non competitive antagonists of the nicotinic acetylcholine receptor (nAChR), To locate the sites of interaction of a long chain alcohol with the Torpedo n AChR, we have used the photoactivatible alcohol 3-[H-3]azioctanol, which in hibits the nAChR and photoincorporates into nAChR subunits. At 1 and 275 mu M, 3-[H-3]azioctanol photoincorporated into nAChR subunits with increased incorporation in the rw-subunit in the desensitized state. The incorporatio n into the cu-subunit was mapped to two large proteolytic fragments. One fr agment of similar to 20 kDa (alpha V8-20), containing the M1, M2, and M3 tr ansmembrane segments, showed enhanced incorporation in the presence of agon ist whereas the other of similar to 10 kDa (alpha V8-10), containing the M4 transmembrane segment, did not show agonist-induced incorporation of label . Within alpha V8-20, the primary site of incorporation was alpha Glu-262 a t the C-terminal end of alpha M2, labeled preferentially in the desensitize d state. The incorporation at alpha Glu-262 approached saturation between 1 mu M, with similar to 6% labeled, and 215 mu M, With similar to 30% labele d. Low level incorporation was seen in residues at the agonist binding site and the protein-lipid interface at similar to 1% of the levels in alpha Gl u-262. Therefore, the primary binding site of 3-azioctanol is within the io n channel with additional lower affinity interactions within the agonist bi nding site and at the protein-lipid interface.