The Role of Individual Disulfide Bonds of μ-Conotoxin GIIIA in the Inhibition of NaV1.4

Abstract

μ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel Na_V1.4. 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 Na_V1.4, 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 Na_V1.4 was assayed by whole cell patch-clamp on rNa_V1.4, heterologously expressed in HEK293 cells. The corresponding IC₅₀ 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 IC₅₀ measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of Na_V1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10-Cys21 is the most important for the Na_V1.4 potency.

Keywords: GIIIA; NaV1.4; disulfide bond; electrophysiology; μ-conotoxin.

Figure 1

Amino acid sequence and disulfide bonds of μ-conotoxin GIIIA. *: amidated C-terminus; O: trans-4-hydroxyproline.

Figure 2

Synthesis and oxidation strategy for GIIIA-3. GIIIA-3 was chemically synthesized using two sets of Cys thiol-protecting groups. Cys3 and Cys15 were protected with trityl groups, while Cys4 and Cys20 were protected with Acm groups. A two-step oxidation protocol was used to selectively fold the linear peptide. Air oxidation was applied to form the disulfide bond Cys3–Cys15, and iodine oxidation was used to remove Acm protection and close the second disulfide bond Cys4–Cys20. * denotes C-terminal amide.

Figure 3

HPLC elution profiles and MALDI-TOF mass spectra of GIIIA and GIIIA-3. Final folded peptides were dissolved in 0.1% TFA, then applied to a C18 analytical column and eluted with a linear gradient of 0%–30% ACN in 0.1% TFA at 1 mL/min over 30 min. Peaks were collected for MALDI-TOF-MS analysis. The MS data (MH⁺, 2607.9 for GIIIA, 2546.2 for GIIIA-3) were consistent with the calculated mass, indicating that the peptides were correctly synthesized.

Figure 4

Effect of GIIIA and its analogues on rNa_V1.4 expressed in HEK293 cells. Sodium currents were elicited by a depolarizing step from a holding potential of −80 mV to a test voltage of −10 mV from HEK293 cells expressing rNa_V1.4. Representative current traces are shown in the absence (Control) and presence of (A) 100 nM GIIIA; (B) 5 μM GIIIA-1, GIIIA-2, GIIIA-3.

Figure 5

Concentration–response curves obtained for the inhibition of rNa_V1.4-mediated Na⁺ currents by GIIIA (◆), GIIIA-1(●), GIIIA-2 (★) and GIIIA-3 (╋). Corresponding IC₅₀ 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. Values are mean ± SEM from three to five separate cells.