Muscarinic toxin 7 (MT7) is a mamba venom peptide that binds selectively to the M(1) muscarinic acetylcholine receptor. We have previously shown that the second (ECL2) and third (ECL3) extracellular loops of the M(1) receptor are critically involved in binding the peptide. In this study we used a mutagenesis approach on the M(5) subtype of the receptor family to find out if this possesses a similar structural architecture in terms of toxin binding as the M(1) receptor. An M(5) receptor construct (M(5)-E(175)Y(184)E(474)), mutated at the formerly deciphered critical residues on ECL2 and 3, gained the ability to bind MT7, but with rather low affinity as determined in a functional assay (apparent K(i) = 24 nM; apparent K(i) for M(1) = 0.5 nM). After screening for different domains and residues, we found a specific residue (P(179) to L in M(5)) in the middle portion of ECL2 that was necessary for high affinity binding of MT7 (M(5)-EL(179)YE, apparent K(i) = 0.5 nM). Mutation of P(179) to A confirmed a role for the leucine side chain in the binding of MT7. Together the results reveal new binding interactions between receptors and the MT7 peptide and strengthen the hypothesis that ECL2 sequence is of utmost importance for MT binding to muscarinic receptors.
Keywords: G protein-coupled receptor; acetylcholine receptor; ligand binding; muscarinic toxin.