Cooccupancy of the Outer Vestibule of Voltage-Gated Sodium Channels by μ-Conotoxin KIIIA and Saxitoxin or Tetrodotoxin

Abstract

of the outer vestibule of voltage-gated sodium channels by -conotoxin KIIIA and saxitoxin or tetrodotoxin. J Neurophysiol 104: 88 -97, 2010. First published April 2, 2010 doi:10.1152/jn.00145.2010. The guanidinium alkaloids tetrodotoxin (TTX) and saxitoxin (STX) are classic ligands of voltage-gated sodium channels (VGSCs). Like TTX and STX, -conotoxin peptides are pore blockers but with greater VGSC subtype selectivity. -Conotoxin KIIIA blocks the neuronal subtype Na V 1.2 with nanomolar affinity and we recently discovered that KIIIA and its mutant with one fewer positive charge, KIIIA[K7A], could act synergistically with TTX in a ternary peptide·TTX·Na V complex. In the complex, the peptide appeared to trap TTX in its normal binding site such that TTX could not readily dissociate from the channel until the peptide had done so; in turn, the presence of TTX accelerated the rate at which peptide dissociated from the channel. In the present study we examined the inhibition of Na V 1.2, exogenously expressed in Xenopus oocytes, by STX (a divalent cation) and its sulfated congener GTX2/3 (with a net ϩ1 charge). Each could form a ternary complex with KIIIA and Na V 1.2, as previously found with TTX (a monovalent cation), but only when STX or GTX2/3 was added before KIIIA. The KIIIA·alkaloid·Na V complex was considerably less stable with STX than with either GTX2/3 or TTX. In contrast, ternary KIIIA[K7A]·alkaloid·Na V complexes could be formed with either order of ligand addition and were about equally stable with STX, GTX2/3, or TTX. The most parsimonious interpretation of the overall results is that the alkaloid and peptide are closely apposed in the ternary complex. The demonstration that two interacting ligands ("syntoxins") occupy adjacent sites raises the possibility of evolving a much more sophisticated neuropharmacology of VGSCs. I N T R O D U C T I O NVoltage-gated sodium channels (VGSCs) are responsible for the upstroke of action potentials in excitable cells. Tetrodotoxin (TTX) and saxitoxin (STX) are guanidinium alkaloids (Fig. 1A) that block six of the nine isoforms of the pore-bearing ␣-subunit of mammalian VGSCs with K d values in the nanomolar range (Catterall et al. 2005). -Conotoxins, which also block VGSCs, are peptides of 16 to 25 residues, including six cysteines that form a characteristic disulfide framework (see Fig. 1B) (Terlau and Olivera 2004). In contrast to the promiscuity of TTX and STX, GIIIA, the best-studied -conotoxin, is significantly more potent against the skeletal muscle subtype of channel Na V 1.4 (half-maximal inhibitory concentration [IC 50 ] ϭ 19 nM), than other TTX-sensitive subtypes, such as brain We recently discovered -conotoxins KIIIA and SIIIA, which block Na V 1.2 with higher affinity than Na V 1.4 (Bulaj et al. 2005;Yao et al. 2008;Zhang et al. 2007). Single-channel analyses of brain VGSCs in planar lipid bilayers showed that KIIIA produced only a partial block (Zhang et al. 2007). In saturating concentrations of KIIIA or its mutant KIIIA[K7A], a residual sodium curre...

μ-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve

“…The block of Na V 1.7 by KIIIA appears to have approximately a 90% efficacy. We presume KIIIA's "leaky" block of Na V 1.7 occurs by a mechanism similar to that hypothesized for KIIIA's incomplete block of Na V 1.2 (16,17), and studies are under way to investigate this further.…”

“…KIIIA blocks Na V 1.2 with 95% efficacy, as previously described in some detail (16,17; also see ref. 18).…”

Design of Bioactive Peptides from Naturally Occurring μ-Conotoxin Structures

“…Thereby, the pore of the channel is blocked electrostatically or sterically (41). However, this toxin-channel interaction could allow some Na ϩ ions or even tetrodotoxin to sneak by, causing a residual current (12,16,24,40). A small residual current was similarly observed for derivatives of GIIIA at one single position (Arg 13 ).…”

“…They affect Na v s by plugging into the pore analogously to the guanidinium toxins tetrodotoxin and saxitoxin. However, they do not target precisely the same binding area (12). Other contact points on the outer channel vestibule are possibly also essential with respect to their binding (12)(13)(14)(15)(16)(17).…”

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin μO§-GVIIJ

“…We had shown previously that -KIIIA and its analogs block rNa V 1.2 and compete for, and can even co-occupy, site 1 with the guanidinium alkaloids tetrodotoxin, saxitoxin, and saxitoxin congeners (9,10,33). In the present experiments, we employed -PIIIA[R14Q] and rNa V 1.4.…”