Krajewski_2001_Mol.Pharmacol_60_725

m1-Toxin1 binds specifically and irreversibly to M(1) muscarinic receptors and can slow the dissociation of [(3)H]N-methylscopolamine ([(3)H]NMS) from these receptors. Yet only 7 of its 65 amino acids are not conserved in six other mamba toxins that bind reversibly to M(2)-M(5) muscarinic receptors. Two of these seven residues (Phe(38), Lys(65)) were mutated to corresponding residues of the other toxins (Ile(38), Glu(65)), to evaluate amino acids in m1-toxin1 that confer its remarkable affinity and specificity. The cDNA for m1-toxin1 was cloned from venom gland mRNA using polymerase chain reaction (PCR)-based techniques. Its nucleotide sequence is remarkably similar to those of other short-chain neurotoxins. The cDNAs for mutant toxins Phe(38) to Ile(38) (F38I) and Lys(65) to Glu(65) (K65E) were constructed by PCR-based techniques. Each cDNA was expressed in yeast, and the toxins were purified from yeast media by cation-exchange and reversed phase chromatography. Recoveries were 40 to 152 microg/l. Recombinant m1-toxin1 was identical to the native toxin (observed mass: 7471 Da; irreversible blockade of [(3)H]NMS binding to cloned M(1) receptors at 25 degrees C; no blockade of M(2)-M(5) receptors; 6-fold slowing of [(3)H]NMS dissociation at 37 degrees C). F38I also bound specifically to M(1) receptors, but reversibly and without effect on NMS dissociation. Thus, Phe(38) contributes to the stability of toxin-receptor complexes, but not to M(1)-selectivity. K65E bound selectively and irreversibly to unliganded M(1) receptors but did not slow NMS dissociation. It is suggested that the C-terminal Lys(65) of m1-toxin1 may contact an outer loop of the M(1) receptor.