Cell Mol Neurobiol 1995 Aug 01;154:427-38. doi: 10.1007/bf02071878.

Mutations in the M1 region of the nicotinic acetylcholine receptor alter the sensitivity to inhibition by quinacrine.

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1. Site directed mutagenesis was used to alter the structure of Torpedo 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 induced by acetylcholine (ACh) were recorded by two electrode voltage clamp. 2. A series of mutations of a highly conserved Arg at position 209 of the alpha subunit of Torpedo californica nAChR revealed that positively charged amino acids are required for functional receptor expression. Mutation of Arg to Lys (alpha R209K) or His (alpha R209H) at position 209 shifted the EC50 for ACh slightly from 5 microM to 12 microM and 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 both 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 inhibition whereas mutations alpha I210A and alpha L212A did not have any effects. None of these mutations changed the sensitivity of nAChR to inhibition by a different noncompetitive inhibitor, chlorpromazine. 5. These findings support a hypothesis that the quinacrine binding site is located 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 beginning of M1 region might be beta sheet structure.

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