| Structural highlights
Function
CM3A_CONKI Mu-conotoxins block voltage-gated sodium channels (Nav). This toxin potently blocks rNav1.2/SCN2A and rNav1.4/SCN4A. It also moderately blocks rNav1.1/SCN1A, rNav1.3/SCN3A, rNav1.5/SCN5A, mNav1.6/SCN8A, and rNav1.7/SCN9A. On rNav1.2/SCN2A, it produces a block that is only partially reversible. The block of SCN9A is modified when beta-subunits are coexpressed with the alpha subunit. Hence, blocks of channels containing beta-1 and beta-3 subunits are more potent (compared to channels without beta subunits), whereas blocks of channels containing beta-2 and beta-4 subunits are less potent (compared to channels without beta subunits).[1] [2] [3] [4] [5] [6] [7]
Publication Abstract from PubMed
In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of mu-conotoxin KIIIA, which was predicted originally to have a [C1-C9,C2-C15,C4-C16] disulfide pattern based on homology with closely related mu-conotoxins. The two major isomers of synthetic mu-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1-C15,C2-C9,C4-C16] disulfide connectivity, while the minor product adopts a [C1-C16,C2-C9,C4-C15] connectivity. Both of these peptides were potent blockers of Na(V)1.2 (K(d) values of 5 and 230 nM, respectively). The solution structure for mu-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1-C15,C2-C9,C4-C16] disulfide pattern; its structure was very similar to the mu-KIIIA structure calculated with the incorrect [C1-C9,C2-C15,C4-C16] disulfide pattern, with an alpha-helix spanning residues 7-12. In addition, the major folding isomers of mu-KIIIB, an N-terminally extended isoform of mu-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as mu-KIIIA, and both blocked Na(V)1.2 (K(d) values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic mu-KIIIA and mu-KIIIB folded in vitro is 1-5/2-4/3-6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of mu-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail's repertoire of active peptides.
Distinct Disulfide Isomers of mu-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels.,Khoo KK, Gupta K, Green BR, Zhang MM, Watkins M, Olivera BM, Balaram P, Yoshikami D, Bulaj G, Norton RS Biochemistry. 2012 Nov 28. PMID:23167564[8]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Bulaj G, West PJ, Garrett JE, Watkins M, Zhang MM, Norton RS, Smith BJ, Yoshikami D, Olivera BM. Novel conotoxins from Conus striatus and Conus kinoshitai selectively block TTX-resistant sodium channels. Biochemistry. 2005 May 17;44(19):7259-65. PMID:15882064 doi:http://dx.doi.org/10.1021/bi0473408
- ↑ Zhang MM, Green BR, Catlin P, Fiedler B, Azam L, Chadwick A, Terlau H, McArthur JR, French RJ, Gulyas J, Rivier JE, Smith BJ, Norton RS, Olivera BM, Yoshikami D, Bulaj G. Structure/function characterization of micro-conotoxin KIIIA, an analgesic, nearly irreversible blocker of mammalian neuronal sodium channels. J Biol Chem. 2007 Oct 19;282(42):30699-706. Epub 2007 Aug 27. PMID:17724025 doi:http://dx.doi.org/10.1074/jbc.M704616200
- ↑ Holford M, Zhang MM, Gowd KH, Azam L, Green BR, Watkins M, Ownby JP, Yoshikami D, Bulaj G, Olivera BM. Pruning nature: Biodiversity-derived discovery of novel sodium channel blocking conotoxins from Conus bullatus. Toxicon. 2009 Jan;53(1):90-8. doi: 10.1016/j.toxicon.2008.10.017. Epub 2008 Nov, 20. PMID:18950653 doi:http://dx.doi.org/10.1016/j.toxicon.2008.10.017
- ↑ Wilson MJ, Yoshikami D, Azam L, Gajewiak J, Olivera BM, Bulaj G, Zhang MM. mu-Conotoxins that differentially block sodium channels NaV1.1 through 1.8 identify those responsible for action potentials in sciatic nerve. Proc Natl Acad Sci U S A. 2011 Jun 21;108(25):10302-7. doi:, 10.1073/pnas.1107027108. Epub 2011 Jun 7. PMID:21652775 doi:http://dx.doi.org/10.1073/pnas.1107027108
- ↑ McArthur JR, Singh G, McMaster D, Winkfein R, Tieleman DP, French RJ. Interactions of key charged residues contributing to selective block of neuronal sodium channels by mu-conotoxin KIIIA. Mol Pharmacol. 2011 Oct;80(4):573-84. doi: 10.1124/mol.111.073460. Epub 2011 Jun , 27. PMID:21709136 doi:http://dx.doi.org/10.1124/mol.111.073460
- ↑ Van Der Haegen A, Peigneur S, Tytgat J. Importance of position 8 in mu-conotoxin KIIIA for voltage-gated sodium channel selectivity. FEBS J. 2011 Sep;278(18):3408-18. doi: 10.1111/j.1742-4658.2011.08264.x. Epub, 2011 Aug 24. PMID:21781281 doi:http://dx.doi.org/10.1111/j.1742-4658.2011.08264.x
- ↑ Zhang MM, Wilson MJ, Azam L, Gajewiak J, Rivier JE, Bulaj G, Olivera BM, Yoshikami D. Co-expression of Na(V)beta subunits alters the kinetics of inhibition of voltage-gated sodium channels by pore-blocking mu-conotoxins. Br J Pharmacol. 2013 Apr;168(7):1597-610. doi: 10.1111/bph.12051. PMID:23146020 doi:http://dx.doi.org/10.1111/bph.12051
- ↑ Khoo KK, Gupta K, Green BR, Zhang MM, Watkins M, Olivera BM, Balaram P, Yoshikami D, Bulaj G, Norton RS. Distinct Disulfide Isomers of mu-Conotoxins KIIIA and KIIIB Block Voltage-Gated Sodium Channels. Biochemistry. 2012 Nov 28. PMID:23167564 doi:http://dx.doi.org/10.1021/bi301256s
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