Allman, Karen; (1999) The agonist binding site of the M1 muscarinic acetylcholine receptors probed by scanning mutagenesis. Doctoral thesis (Ph.D), UCL (University College London).

Text out.pdf Download (10MB)

Abstract

The anchor point for the binding of the quaternary ammonium headgroup of acetylcholine to muscarinic acetylcholine receptors is known, but less is known about the interaction of other important moieties, particularly the side-chain of acetylcholine. The aim of this project was to identify residues in the M1 muscarinic acetylcholine receptor that may contact the side-chain methyl group of acetylcholine. Receptor models predict that residues in the sequence between Ile188 and Ala196 may be at a suitable distance from the aspartate residue which binds the headgroup. Substitution mutants were constructed of these residues. Mutation to Cys allowed probing with sulphydryl reagents including the alkylating acetylcholine analogue bromoacetylcholine and the group specific methanethiosulphonate (MTS) derivatives. Mutation to Ala or Gly allowed a complementary ligand side-chain / amino acid side-chain deletion strategy. Mutation of residues Ile188, Thr189, Thr192 and Ala196 caused significant decreases in acetylcholine binding. This led to a reduction in the potency of the functional response. There were small decreases in the binding of acetylcholine analogues carbachol and oxotremorine-M, but no significant effect on the affinity of the des-methyl analogue formylcholine. Interestingly, mutation of Phe190 to Cys resulted in a constitutively active receptor. The environment of this residue may change during receptor activation. The positively-charged MTSET reacted strongly with F190C, T192C and A193C, identifying these residues as accessible within the binding site crevice. Bromoacetylcholine interacted specifically with the T192C mutant receptor confirming the importance of this position. These results indicate that Ile188, Thr189, Thr192 and Ala196 contribute to the acetylcholine binding pocket, and that this section of TM V is α-helical. Ala193 is accessible in the binding pocket but not involved in acetylcholine binding. In addition to any involvement in a network of hydrogen bonds Thr192 is predicted to contact the acetylmethyl group of acetylcholine.

Download activity - last month

Export as CSVJSONXML

Download activity - last 12 months

Export as JSONXMLCSV

Downloads by country - last 12 months

Export as CSVJSONXML

Archive Staff Only

View Item