Journal of Biological Chemistry vol:278 issue:26 pages:24125-31
About one-third of the amino acid residues conserved in all scorpion long chain Na+ channel toxins are aromatic residues, some of which constitute the so-called "conserved hydrophobic surface." At present, in-depth structure-function studies of these aromatic residues using site-directed mutagenesis are still rare. In this study, an effective yeast expression system was used to study the role of seven conserved aromatic residues (Tyr5, Tyr14, Tyr21, Tyr35, Trp38, Tyr42, and Trp47) from the scorpion toxin BmK M1. Using site-directed mutagenesis, all of these aromatic residues were individually substituted with Gly in association with a more conservative substitution of Phe for Tyr5, Tyr14, Tyr35, or Trp47. The mutants, which were expressed in Saccharomyces cerevisiae S-78 cells, were then subjected to a bioassay in mice, electrophysiological characterization on cloned Na+ channels (Nav1.5), and CD analysis. Our results show an eye-catching correlation between the LD50 values in mice and the EC50 values on Nav1.5 channels in oocytes, indicating large mutant-dependent differences that emphasize important specific roles for the conserved aromatic residues in BmK M1. The aromatic side chains of the Tyr5, Tyr35, and Trp47 cluster protruding from the three-stranded beta-sheet seem to be essential for the structure and function of the toxin. Trp38 and Tyr42 (located in the beta2-sheet and in the loop between the beta2- and beta3-sheets, respectively) are most likely involved in the pharmacological function of the toxin.