Structural basis for tetrodotoxin-resistant sodium channel binding by mu-conotoxin SmIIIA.
SmIIIA is a new micro-conotoxin isolated recently from Conus stercusmuscarum. Although it shares several biochemical characteristics with other micro-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 micro-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 micro-conotoxins GIIIA and GIIIB. Comparison of the surfaces of SmIIIA with other micro-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 micro-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.
Keizer
David W
DW
The Walter & Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3050, Australia.
West
Peter J
PJ
Lee
Erinna F
EF
Yoshikami
Doju
D
Olivera
Baldomero M
BM
Bulaj
Grzegorz
G
Norton
Raymond S
RS
D000200:Action Potentials / Q000187:drug effects; D000595:Amino Acid Sequence; D000818:Animals; D001369:Axons / Q000737:chemistry; D020916:Conotoxins / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D008978:Mollusk Venoms / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D009479:Neuropeptides / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011898:Ranidae; D011993:Recombinant Fusion Proteins; D012908:Snails; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
D020916:Conotoxins; D008978:Mollusk Venoms; D009479:Neuropeptides; D011993:Recombinant Fusion Proteins; D015222:Sodium Channels