The Role of Individual Disulfide Bonds of μ-Conotoxin GIIIA in the Inhibition of Na1.4.

μ-Conotoxin GIIIA, a peptide toxin isolated from , preferentially blocks the skeletal muscle sodium channel Na1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of Na1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against Na1.4 was assayed by whole cell patch-clamp on rNa1.4, heterologously expressed in HEK293 cells. The corresponding IC values were 0.069 ± 0.005 μM for GIIIA, 2.1 ± 0.3 μM for GIIIA-1, 3.3 ± 0.2 μM for GIIIA-2, and 15.8 ± 0.8 μM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3-Cys15, Cys4-Cys20 and Cys10-Cys21, respectively). Other analogues were not active enough for IC measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of Na1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10-Cys21 is the most important for the Na1.4 potency.