Structural Basis for Tetrodotoxin-resistant Sodium Channel Binding by μ-Conotoxin SmIIIA

Abstract

SmIIIA is a new -conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other -conotoxins (the arrangement of cysteine residues and a conserved arginine believed to interact with residues near the channel pore), it has several distinctive features, including the absence of hydroxyproline, and is the first specific antagonist of tetrodotoxin-resistant voltage-gated sodium channels to be characterized. It therefore represents a potentially useful tool to investigate the functional roles of these channels. We have determined the three-dimensional structure of SmIIIA in aqueous solution. Consistent with the absence of hydroxyprolines, SmIIIA adopts a single conformation with all peptide bonds in the trans configuration. The spatial orientations of several conserved Arg and Lys side chains, including Arg 14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other -conotoxins but the N-terminal regions differ, reflecting the trans conformation for the peptide bond preceding residue 8 in SmIIIA, as opposed to the cis conformation in -conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other -conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp 15 side chain, which is unique to SmIIIA. Arg 17 , which replaces Lys in the other -conotoxins, may also be important. Consistent with these inferences from the structure, assays of two chimeras of SmIIIA and PIIIA in which their N-and Cterminal halves were recombined, indicated that residues in the C-terminal half of SmIIIA confer affinity for tetrodotoxin-resistant sodium channels in the cell bodies of frog sympathetic neurons. SmIIIA and the chimera possessing the C-terminal half of SmIIIA also inhibit tetrodotoxin-resistant sodium channels in the postganglionic axons of sympathetic neurons, as indicated by their inhibition of C-neuron compound action potentials that persist in the presence of tetrodotoxin.Polypeptide toxins typically interact with their target receptors with high potency and exquisite selectivity and as such are valuable tools in elucidating the physiological functions of their targets and in probing the size and shape of their cognate binding sites. Toxins from the genus Conus have been especially valuable in this respect, and, of the various classes of conotoxin that have been characterized to date (1, 2), the -conotoxins represent a particularly good example. Their targets are the voltage-gated sodium channels (VGSCs), 1 which are responsible for the influx of sodium ions during action potentials in excitable tissues.Three families of conotoxins target VGSCs, causing either inhibition (-and O-conotoxins) or delayed inactivation (␦-conotoxins), but to date a detailed understanding of their interactions with the channel has been achieved only in the case of the -conotoxins. These toxins bind to Site 1 on VGSCs, one of several toxin binding sites identified on these channels ...

Structural and Functional Diversities among μ-Conotoxins Targeting TTX-resistant Sodium Channels

“…We used PCR primers specific for the M-superfamily (23) and identified four new µ-conotoxins from the venom of fishhunting cone snails Conus consors, C. catus, and C. magus ( Figure 2). The predicted translation products based on DNA sequences (Table 1, group I) share considerable sequence similarity in the two C-terminal loops, which were previously postulated to be important for the recognition of TTXresistant sodium channels (15). Sequence comparison identified three major groups of µ-conotoxins: (I) CIIIA, CnIIIA, CnIIIB, and MIIIA; (II) KIIIA, SIIIA, and SmIIIA; and (III) PIIIA, GIIIA, GIIIB, and GIIIC.…”

“…Recently, our group discovered three new µ-conotoxins, namely SmIIIA, SIIIA, and KIIIA that blocked TTX-resistant (TTXr) 1 channels and had different potencies against TTXsensitive channels (14)(15)(16). Although 1 µM SmIIIA blocked TTX-sensitive action potentials in skeletal muscle by 70%, 1 µM of either KIIIA or SIIIA had hardly any effect (16).…”

NMR of conotoxins: structural features and an analysis of chemical shifts of post-translationally modified amino acids

“…N-terminal pyroglutamic acid is seen in -conotoxins such as PIIIA and SmIIIA 36,76 for which the proton chemical shifts are given in Table 2. A detailed analysis is not meaningful with such a small number of data sets, but the chemical shifts are very similar to those found in Glu and Gln, except for the amide chemical shift, which is upfield of the values given for Glu and Gln in the BMRB.…”

Structure/Function Characterization of μ-Conotoxin KIIIA, an Analgesic, Nearly Irreversible Blocker of Mammalian Neuronal Sodium Channels

“…1). Structural and functional studies on peptides in this group to date suggest that amino acid residues in the C-terminal region of these peptides, including Trp and His (see Table 1), are important for function (19,22).…”

“…1). Structural and functional studies on peptides in this group to date suggest that amino acid residues in the C-terminal region of these peptides, including Trp and His (see Table 1), are important for function (19,22).It is widely believed that TTX-r sodium channels are potential molecular targets for analgesic therapy (23,24). In the peripheral nerves of mammals, there are two TTX-r subtypes, Na v 1.8 and Na v 1.9; a variety of data suggest that these may participate in pain signaling.…”