MUTATIONS IN THE M1 REGION OF THE NICOTINIC ACETYLCHOLINE-RECEPTOR ALTER THE SENSITIVITY TO INHIBITION BY QUINACRINE

1. Site directed mutagenesis was used to alter the structure of Torped o californica nicotinic acetylcholine receptor (nAChR) and to identify amino acid residues which contribute to noncompetitive inhibition by quinacrine. Mutant receptors were expressed in Xenopus laevis oocytes injected with in vitro synthesized mRNA and the whole cell currents in duced by acetylcholine (ACh) were recorded by two electrode voltage cl amp. 2. A series of mutations of a highly conserved Arg at position 20 9 of the alpha subunit of Torpedo californica nAChR revealed that posi tively charged amino acids are required for functional receptor expres sion. Mutation of Arg to Lys (alpha R209K) or His (alpha R209H) at pos ition 209 shifted the EC(50) for ACh slightly from 5 mu M to 12 mu M a nd increased the normalized maximal channel activity 8.5- and 3.2-fold , respectively. 3. These mutations altered the sensitivity of nAChR to noncompetitive inhibition by quinacrine. The extent of inhibition of ion channel function by quinacrine was decreased as pH increased in bo th wild type and mutant nAChR suggesting that the doubly charged form of quinacrine was responsible for the inhibition. 4. Further mutations at different positions of the alpha subunit suggest the contribution of Pro and Tyr residues at positions 211 and 213 to quinacrine inhibit ion whereas mutations alpha I210A and alpha L212A did not have any eff ects, None of these mutations changed the sensitivity of nAChR to inhi bition by a different noncompetitive inhibitor, chlorpromazine. 5. The se findings support a hypothesis that the quinacrine binding site is l ocated in the lumen of the ion channel. In addition, the quantitative effect of point mutations at alternate positions on the sensitivity of quinacrine inhibition suggests that the secondary structure at the be ginning of M1 region might be beta sheet structure.