Journal of Biological Chemistry
Volume 278, Issue 47, 21 November 2003, Pages 46805-46813
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Protein Structure and Folding
Structural Basis for Tetrodotoxin-resistant Sodium Channel Binding by μ-Conotoxin SmIIIA*

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SmIIIA is a new μ-conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other μ-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 Arg14 (using a consensus numbering system), which plays a key role in sodium channel binding, are similar to those in other μ-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 μ-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other μ-conotoxins suggests that the affinity of SmIIIA for TTX-resistant channels is influenced by the Trp15 side chain, which is unique to SmIIIA. Arg17, which replaces Lys in the other μ-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 C-terminal 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.

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*

This work was supported in part by National Institute of General Medical Sciences Grant GM 48677 (to B. M. O.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. S1 and Tables SI–SIII.