Marked difference in saxitoxin and tetrodotoxin affinity for the human nociceptive voltage-gated sodium channel (Na _v 1.7)

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.

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa V 1.7

“…Previous homology models of the Na V pore with STX bound have posited three key electrostatic interactions between (i) the five-membered ring guanidinium and E755, (ii) the six-membered ring guanidinium and D1532, and (iii) the C12-hydrated ketone and E758 (6,(17)(18)(19)(20). These contacts, defined by experimental comparison of STX and TTX affinity for site 1 mutants, enforce close packing between positions C10 and C11 on STX and DIII p-loop residues (Figs.…”

“…Amino acid substitution of DIII p-loop residues M1240 and D1241 has been found to significantly influence guanidinium toxin potency (6,8). In previous work from our laboratory, STX resistance in primate Na V 1.7 was noted and ascribed to a natural variation of two pore-forming amino acids at positions 1240 and 1241.…”

“…Previously reported docking models for STX have positioned C10 and C11 proximal to the pore loop of DIII (6,(17)(18)(19)(20). Accordingly, C10-Me STX 4 was tested against Na V 1.4 M1240T/D1241I in addition to other DIII mutant constructs (Fig.…”

“…Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na V isoforms with nanomolar affinity (5)(6)(7). Guanidinium toxins inhibit Na + influx through Na V s by occluding the outer pore above the ion selectivity filter (site 1).…”

“…The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11)(12)(13)(14)(15)(16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5,6,(21)(22)(23). The lack of structural information motivates a comprehensive, systematic study of toxin-protein interactions.…”