Vol. 60, Issue 4, 725-731, October 2001

Site-Directed Mutagenesis of m1-Toxin1: Two Amino Acids Responsible for Stable Toxin Binding to M₁ Muscarinic Receptors

Jeffrey L. Krajewski, Ian M. Dickerson, and Lincoln T. Potter

Department of Molecular and Cellular Pharmacology (J.L.K., L.T.P.), Department of Physiology and Biophysics, and Neuroscience Program (I.M.D.), University of Miami School of Medicine, Miami, Florida

m1-Toxin1 binds specifically and irreversibly to M₁ muscarinic 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.