Importance of the conserved aromatic residues in the scorpion α-like toxin BmK M1. The hydrophobic surface region revisited

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 (Na_v1.5), and CD analysis. Our results show an eye-catching correlation between the LD₅₀ values in mice and the EC₅₀ values on Na_v1.5 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 β-sheet seem to be essential for the structure and function of the toxin. Trp³⁸ and Tyr⁴² (located in the β₂-sheet and in the loop between the β₂- and β₃-sheets, respectively) are most likely involved in the pharmacological function of the toxin.