Marked difference in saxitoxin and tetrodotoxin affinity for the human nociceptive voltage-gated sodium channel (Nav1.7) [corrected]

Abstract

Human nociceptive voltage-gated sodium channel (Na(v)1.7), a target of significant interest for the development of antinociceptive agents, is blocked by low nanomolar concentrations of (-)-tetrodotoxin(TTX) but not (+)-saxitoxin (STX) and (+)-gonyautoxin-III (GTX-III). These findings question the long-accepted view that the 1.7 isoform is both tetrodotoxin- and saxitoxin-sensitive and identify the outer pore region of the channel as a possible target for the design of Na(v)1.7-selective inhibitors. Single- and double-point amino acid mutagenesis studies along with whole-cell electrophysiology recordings establish two domain III residues (T1398 and I1399), which occur as methionine and aspartate in other Na(v) isoforms, as critical determinants of STX and gonyautoxin-III binding affinity. An advanced homology model of the Na(v) pore region is used to provide a structural rationalization for these surprising results.

Fig. 1.

Whole-cell voltage-clamp electrophysiology measurements of STX and TTX against rNa_v1.4 and hNa_v1.7. (A) Langmuir isotherms of STX and TTX binding to recombinant rNa_v1.4 and hNa_v1.7 expressed in CHO cells; error bars represent SD for n ≥ 3. (B and C) Representative normalized current vs. time plots of hNa_v1.7 varying TTX and STX concentrations, which highlight the reduced potency of STX for inhibition of hNa_v1.7. Additional normalized current vs. time plots for STX binding are provided in Fig. S1.

Fig. 2.

Homology model-derived images highlighting differences between rNa_v1.4 and hNa_v1.7. STX bound in site 1 of the α-subunit of the homology model of rNa_v1.4 is viewed from (A) above and (B) the side of the pore. The four domains of the pore are shown in cartoon representations and colored green (domain I), cyan (domain II), magenta (domain III), and yellow (domain IV). In B, residues comprising the DEKA selectivity filter are displayed as space-filling models; atoms are colored red (oxygen), blue (nitrogen), white (hydrogen), yellow (sulfur), and green (STX carbons) or white (protein carbons). In C–H, STX is docked into rNa_v1.4 (C) and the identical pose in hNa_v1.7 (D) to highlight the steric differences of the M1240T and D1241I variations. In C and D, STX is colored similarly as in A and B. The molecular surface of the protein is in white, with M1240 (C) or T1398 (D) shown in yellow and D1241 (C) or I1399 (D) in red. Electrostatic potential surfaces of rNa_v1.4 (E), hNa_v1.7 T1398M-I1399D (F), rNa_v1.4 M1240T-D1241I (G), hNa_v1.7 (H); depicted range is from −20 (red) to +5 kT (blue). In each image, residues 1,240 (1,398) and 1,241 (1,399) are displayed as stick figures.

Fig. 3.

Electrostatic potential surfaces from −4 (red) to 4 kT (blue) of the toxins (A) TTX, (B) STX, and (C) GTX-III highlighting the differences in charge distributions between the molecules.