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.
Footnotes
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This work was supported by AG06170 and AG12976.
- Abbreviations:
- [3H]NMS
- [3H]N-methylscopolamine
- PCR
- polymerase chain reaction
- RACE
- rapid amplification of cDNA ends
- MG
- minimal glycerol media
- MM
- minimal methanol media
- BSA
- bovine serum albumin
- CHO
- Chinese hamster ovary
- HPLC
- high-performance liquid chromatography
- Received February 23, 2001.
- Accepted June 22, 2001.
- The American Society for Pharmacology and Experimental Therapeutics
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