Abstract

m1-Toxin1 binds specifically and irreversibly to M₁muscarinic receptors and can slow the dissociation of [³H]N-methylscopolamine ([³H]NMS) from these receptors. Yet only 7 of its 65 amino acids are not conserved in six other mamba toxins that bind reversibly to M₂-M₅ muscarinic receptors. Two of these seven residues (Phe³⁸, Lys⁶⁵) were mutated to corresponding residues of the other toxins (Ile³⁸, Glu⁶⁵), 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 Phe³⁸ to Ile³⁸ (F38I) and Lys⁶⁵ to Glu⁶⁵ (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 μg/l. Recombinant m1-toxin1 was identical to the native toxin (observed mass: 7471 Da; irreversible blockade of [³H]NMS binding to cloned M₁ receptors at 25°C; no blockade of M₂-M₅ receptors; 6-fold slowing of [³H]NMS dissociation at 37°C). F38I also bound specifically to M₁ receptors, but reversibly and without effect on NMS dissociation. Thus, Phe³⁸ contributes to the stability of toxin-receptor complexes, but not to M₁-selectivity. K65E bound selectively and irreversibly to unliganded M₁ receptors but did not slow NMS dissociation. It is suggested that the C-terminal Lys⁶⁵ of m1-toxin1 may contact an outer loop of the M₁ receptor.