Site-directed mutagenesis of m1-toxin1: Two amino acids responsible for stable toxin binding to M-1 muscarinic receptors

m1-Toxin1 binds specifically and irreversibly to M-1 muscarinic receptors a nd can slow the dissociation of [H-3]N-methyiscopolamine ([H-3]NMS) from th ese receptors. Yet only 7 of its 65 amino acids are not conserved in six ot her 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 re sidues of the other toxins (Ile(38), Glu(65)), to evaluate amino acids in m 1-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 th ose of other short-chain neurotoxins. The cDNAs, for mutant toxins Phe(38) to Ile(38) (F381) and Lys(65) to Glu(65) (K65E) were constructed by PCR-bas ed techniques. Each cDNA was expressed in yeast, and the toxins were purifi ed from yeast media by cation-exchange and reversed phase chromatography. R ecoveries were 40 to 152 mug/l. Recombinant m1-toxin1 was identical to the native toxin (observed mass: 7471 Da; irreversible blockade of [H-3]NMS bin ding to cloned M-1 receptors at 25 degreesC; no blockade of M-2-M-5 recepto rs; 6-fold slowing of [H-3]NMS dissociation at 37 degreesC). F381 also boun d specifically to M-1 receptors, but reversibly and without effect on NMS d issociation. Thus, Phe(38) contributes to the stability of toxin-receptor c omplexes, but not to M-1-selectivity. K65E bound selectively and irreversib ly to unliganded M-1 receptors but did not slow NMS dissociation. It is sug gested that the C-terminal Lys(65) of m1-toxin1 may contact an outer loop o f the M-1 receptor.