Neuronally Selective μ-Conotoxins from Conus striatus Utilize an α-Helical Motif to Target Mammalian Sodium Channels
2008; Elsevier BV; Volume: 283; Issue: 31 Linguagem: Inglês
10.1074/jbc.m802852200
ISSN1083-351X
AutoresChristina I. Schroeder, Jenny Ekberg, Katherine J. Nielsen, Denise A. Adams, Marion Loughnan, Linda Thomas, David J. Adams, Paul F. Alewood, Richard J. Lewis,
Tópico(s)Receptor Mechanisms and Signaling
Resumoμ-Conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of μ-conotoxins SIIIA and SIIIB by 125I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced 125I-TIIIA from native rat brain Nav1.2 (IC50 values 10 and 5 nm, respectively) and muscle Nav1.4 (IC50 values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Nav1.2 and Nav1.4. An alanine scan of SIIIA-(2–20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2–20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2–20) had significantly enhanced neuronal affinity (IC50 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2–20) showed greatest Nav1.2 versus 1.4 selectivity (136-fold). 1H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and anα-helical motif across residues 11–16 that is not found in larger μ-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller μ-conotoxin pharmacophore. μ-Conotoxins are small peptide inhibitors of muscle and neuronal tetrodotoxin (TTX)-sensitive voltage-gated sodium channels (VGSCs). Here we report the isolation of μ-conotoxins SIIIA and SIIIB by 125I-TIIIA-guided fractionation of milked Conus striatus venom. SIIIA and SIIIB potently displaced 125I-TIIIA from native rat brain Nav1.2 (IC50 values 10 and 5 nm, respectively) and muscle Nav1.4 (IC50 values 60 and 3 nm, respectively) VGSCs, and both inhibited current through Xenopus oocyte-expressed Nav1.2 and Nav1.4. An alanine scan of SIIIA-(2–20), a pyroglutamate-truncated analogue with enhanced neuronal activity, revealed residues important for affinity and selectivity. Alanine replacement of the solvent-exposed Trp-12, Arg-14, His-16, Arg-18 resulted in large reductions in SIIIA-(2–20) affinity, with His-16 replacement affecting structure. In contrast, [D15A]SIIIA-(2–20) had significantly enhanced neuronal affinity (IC50 0.65 nm), while the double mutant [D15A/H16R]SIIIA-(2–20) showed greatest Nav1.2 versus 1.4 selectivity (136-fold). 1H NMR studies revealed that SIIIA adopted a single conformation in solution comprising a series of turns and anα-helical motif across residues 11–16 that is not found in larger μ-conotoxins. The structure of SIIIA provides a new structural template for the development of neuronally selective inhibitors of TTX-sensitive VGSCs based on the smaller μ-conotoxin pharmacophore. Voltage-gated sodium channels (VGSC) 3The abbreviations used are: VGSC, voltage-gated sodium channel; ACN, acetonitrile; NMR, nuclear magnetic resonance spectroscopy; RP-HPLC, reversed-phase high performance liquid chromatography; STX, saxitoxin; TTX, tetrodotoxin; RMSD, root mean-square deviation; DRG, dorsal root ganglion. 3The abbreviations used are: VGSC, voltage-gated sodium channel; ACN, acetonitrile; NMR, nuclear magnetic resonance spectroscopy; RP-HPLC, reversed-phase high performance liquid chromatography; STX, saxitoxin; TTX, tetrodotoxin; RMSD, root mean-square deviation; DRG, dorsal root ganglion. are crucial for the excitability of nerve and muscle cells (1Catterall W.A. Neuron. 2000; 26: 13-25Abstract Full Text Full Text PDF PubMed Scopus (1702) Google Scholar). Nine different VGSC isoforms (Nav1.1–1.9) have been identified and characterized as either tetrodotoxin-sensitive (TTX-S) or tetrodotoxin-resistant (TTX-R) (2French R.J. Terlau H. Curr. Med. Chem. 2004; 11: 3053-3064Crossref PubMed Scopus (49) Google Scholar, 3Catterall W.A. Goldin A.L. Waxman S.G. Pharmacol. Rev. 2005; 57: 397-409Crossref PubMed Scopus (1054) Google Scholar). Of these isoforms, the TTX-R Nav1.8 and 1.9 are implicated in neuropathic pain states (4Kalso E. Curr. Pharm. Des. 2005; 11: 3005-3011Crossref PubMed Scopus (71) Google Scholar, 5Wood J.N. Boorman J.P. Okuse K. Baker M.D. J. Neurobiol. 2004; 61: 55-71Crossref PubMed Scopus (306) Google Scholar), the TTX-S Nav1.7 is implicated in allodynia and neuropathic pain (6Luy Y.S. Park S.K. Chung K. Chung J.M. Brain Res. 2000; 871: 98-103Crossref PubMed Scopus (101) Google Scholar, 7Boucher T.J. Okuse K. Bennett D.L. Munson J.B. Wood J.N. Science. 2000; 290: 124-127Crossref PubMed Scopus (455) Google Scholar, 8Nassar M.A. Stirling L.C. Forlani G. Baker M.D. Matthews E.A. Dickenson A.H. Wood J.N. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 12706-12711Crossref PubMed Scopus (520) Google Scholar, 9Fertleman C.R. Baker M.D. Parker K.A. Moffatt S. Elmslie F.V. Abrahamsen B. Ostman J. Klugbauer N. Wood J.N. Gardiner R.M. Rees M. Neuron. 2006; 52: 767-774Abstract Full Text Full Text PDF PubMed Scopus (573) Google Scholar, 10Cox J.J. Reimann F. Nicholas A.K. Thornton G. Roberts E. Springell K. Karbani G. Jafri H. Mannan J. Raashid Y. Al-Gazali L. Hamamy H. Valente E.M. Gorman S. Williams R. McHale D.P. Wood J.N. Gribble F.M. Woods C.G. Nature. 2006; 444: 894-898Crossref PubMed Scopus (1145) Google Scholar), and the TTX-S Nav1.3 is up-regulated in inflammatory pain (5Wood J.N. Boorman J.P. Okuse K. Baker M.D. J. Neurobiol. 2004; 61: 55-71Crossref PubMed Scopus (306) Google Scholar). To learn more about the different roles played by each of the VGSC subtypes in normal and disease states requires new inhibitors with improved VGSC subtype selectivity. Inhibitors of TTX-S VGSCs include tetrodotoxin (TTX) from puffer fish, the saxitoxins (STXs) from marine dinoflagellates, and the μ-conotoxins from the venom of cone snails. These toxins act competitively at Site 1 in the P-loop region of the ion-conducting pore of the α-subunit of the VGSC (3Catterall W.A. Goldin A.L. Waxman S.G. Pharmacol. Rev. 2005; 57: 397-409Crossref PubMed Scopus (1054) Google Scholar, 11Cestele S. Catterall W.A. Biochimie (Paris). 2000; 82: 883-892Crossref PubMed Scopus (609) Google Scholar). While TTX and STX act with nanomolar potency across the six TTX-S VGSCs (Nav1.1–1.4, 1.6, and 1.7), μ-conotoxins are more discriminating. Currently 12 μ-conotoxins have been identified from nine species of fish hunting cone snails (Table 1). μ-Conotoxins GIIIA, GIIIB, and GIIIC from Conus geographus selectively target the skeletal muscle Nav1.4, PIIIA from Conus purpurasence (12Shon K.J. Stocker M. Terlau H. Stuhmer W. Jacobsen R. Walker C. Grilley M. Watkins M. Hillyard D.R. Gray W.R. Olivera B.M. J. Biol. Chem. 1998; 273: 33-38Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 13Safo P. Rosenbaum T. Shcherbatko A. Choi D.Y. Han E. Toledo-Aral J.J. Olivera B.M. Brehm P. Mandel G. J. Neurosci. 2000; 20: 76-80Crossref PubMed Google Scholar, 14Nielsen K.J. Watson M. Adams D.J. Hammarstrom A.K. Gage P.W. Hill J.M. Craik D.J. Thomas L. Adams D. Alewood P.F. Lewis R.J. J. Biol. Chem. 2002; 277: 27247-27255Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and TIIIA from Conus tulipa (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar) target Nav1.2 and 1.4, and μ-conotoxins SmIIIA from Conus stercusmuscsarum (16West P.J. Bulaj G. Garrett J.E. Olivera B.M. Yoshikami D. Biochemistry. 2002; 41: 15388-15393Crossref PubMed Scopus (86) Google Scholar, 17Keizer, D. W., West, P. J., Lee, E. F., Yoshikami, D., Olivera, B. M., Bulaj, G., and Norton, R. S. (2003)Google Scholar), SIIIA from Conus striatus (18Bulaj G. West P.J. Garrett J.E. Marsh M. Zhang M.-M. Norton R.S. Smith B.J. Yoshikami D. Olivera B.M. Biochemistry. 2005; 44: 7259-7265Crossref PubMed Scopus (107) Google Scholar, 19Wang C.Z. Zhang H. Jiang H. Lu W. Zhao Z.Q. Chi C.W. Toxicon. 2006; 47: 122-132Crossref PubMed Scopus (44) Google Scholar), and KIIIA from Conus kinoshitai (18Bulaj G. West P.J. Garrett J.E. Marsh M. Zhang M.-M. Norton R.S. Smith B.J. Yoshikami D. Olivera B.M. Biochemistry. 2005; 44: 7259-7265Crossref PubMed Scopus (107) Google Scholar) target amphibian TTX-R sodium channels. More recently, KIIIA has also been shown to inhibit TTX-S sodium channels in mouse DRG and rat TTX-S sodium channels expressed in Xenopus oocytes (20Zhang M.M. Green B.R. Catlin P. Fiedler B. Azam L. Chadwick A. Terlau H. McArthur J.R. French R.J. Gulyas J. Rivier J.E. Smith B.J. Norton R.S. Oliviera B.M. Yoshikami D. Bulaj G. J. Biol. Chem. 2007; 282: 30699-30706Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar). Lastly, μ-conotoxins CnIIIA, CnIIIB from Conus consors, CIIIA from Conus catus, and MIIIA from Conus magus were found to inhibit TTX-R and TTX-S sodium channels in frog DRG but not rat or mouse DRG neurons (21Zhang M.-M. Fiedler B. Green B.R. Catlin P. Watkins M. Garrett J.E. Smith B.J. Yoshikami D. Olivera B.M. Bulaj G. Biochemistry. 2006; 45: 3731-3840Google Scholar). Most reported μ-conotoxins have a conserved arginine in loop 2, which is essential for high affinity interactions at TTX-S sodium channels (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar, 22Chahine M. Chen L.-Q. Fotouhi N. Walsky R. Fry D. Santarelli V. Horn R. Kallen R.G. Receptors Channels. 1995; 3: 161-174PubMed Google Scholar, 23Chang G. Guida W.C. Still W.C. J. Am. Chem. Soc. 1989; 111: 4379-4386Crossref Scopus (1207) Google Scholar, 24Shon K.J. Olivera B.M. Watkins M. Jacobsen R.B. Gray W.R. Floersca C.Z. Cruz L.J. Hillyard D.R. Brink A. Terlau H. Yoshikami D. J. Neurosci. 1998; 18: 4473-4481Crossref PubMed Google Scholar, 25Wakamatsu K. Kohda D. Hatanaka H. Lancelin J.-M. Ishida Y. Oya M. Nakamura H. Inagaki F. Sato K. Biochemistry. 1992; 31: 12577-12584Crossref PubMed Scopus (90) Google Scholar). In contrast, SIIIA and KIIIA have a much shorter loop 2 and a lysine instead of an arginine in the corresponding position. Recent structure-activity studies on TIIIA (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar) and KIIIA (20Zhang M.M. Green B.R. Catlin P. Fiedler B. Azam L. Chadwick A. Terlau H. McArthur J.R. French R.J. Gulyas J. Rivier J.E. Smith B.J. Norton R.S. Oliviera B.M. Yoshikami D. Bulaj G. J. Biol. Chem. 2007; 282: 30699-30706Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) have confirmed that the arginine in loop 2 is crucial for TIIIA affinity at both neuronal and skeletal muscle sodium channels, whereas the structurally equivalent lysine was only important for affinity at the skeletal muscle VGSC. Three-dimensional structures of μ-conotoxins GIIIA, GIIIB, PIIIA, TIIIA, and SmIIIA have been determined by NMR (14Nielsen K.J. Watson M. Adams D.J. Hammarstrom A.K. Gage P.W. Hill J.M. Craik D.J. Thomas L. Adams D. Alewood P.F. Lewis R.J. J. Biol. Chem. 2002; 277: 27247-27255Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar, 17Keizer, D. W., West, P. J., Lee, E. F., Yoshikami, D., Olivera, B. M., Bulaj, G., and Norton, R. S. (2003)Google Scholar, 26Lancelin J.-M. Kohda D. Tate T. Yanagawa Y. Abe T. Satake M. Inagaki F. Biochemistry. 1991; 30: 6908-6916Crossref PubMed Scopus (89) Google Scholar, 27Hill J.M. Alewood P.F. Craik D.J. Biochemistry. 1996; 35: 8824-8835Crossref PubMed Scopus (96) Google Scholar). These structures reveal a similar fold for the μ-conotoxins despite high sequence divergence. At present, structures of μ-conotoxins SIIIA and KIIIA are modeled from SmIIIA (18Bulaj G. West P.J. Garrett J.E. Marsh M. Zhang M.-M. Norton R.S. Smith B.J. Yoshikami D. Olivera B.M. Biochemistry. 2005; 44: 7259-7265Crossref PubMed Scopus (107) Google Scholar, 20Zhang M.M. Green B.R. Catlin P. Fiedler B. Azam L. Chadwick A. Terlau H. McArthur J.R. French R.J. Gulyas J. Rivier J.E. Smith B.J. Norton R.S. Oliviera B.M. Yoshikami D. Bulaj G. J. Biol. Chem. 2007; 282: 30699-30706Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 21Zhang M.-M. Fiedler B. Green B.R. Catlin P. Watkins M. Garrett J.E. Smith B.J. Yoshikami D. Olivera B.M. Bulaj G. Biochemistry. 2006; 45: 3731-3840Google Scholar).TABLE 1μ-Conotoxin sequences and VGSC selectivity Z, Pyroglutamate; O, hydroxyproline.PeptideaSIIIA, SIIIB, and KIIIA are smaller μ-conotoxins, whereas the others are defined as larger μ-conotoxinsSequenceSelectivityRef.SIIIBZN–CCNG––GCSSKWCKGHARCCbC-terminal amidation1.4 ≈ 1.2This workSIIIAZN–CCNG––GCSSKWCRDHARCCbC-terminal amidation1.2 ≈ 1.4This work (18Bulaj G. West P.J. Garrett J.E. Marsh M. Zhang M.-M. Norton R.S. Smith B.J. Yoshikami D. Olivera B.M. Biochemistry. 2005; 44: 7259-7265Crossref PubMed Scopus (107) Google Scholar, 19Wang C.Z. Zhang H. Jiang H. Lu W. Zhao Z.Q. Chi C.W. Toxicon. 2006; 47: 122-132Crossref PubMed Scopus (44) Google Scholar)KIIIA–––CCN––––CSSKWCRDHSRCCbC-terminal amidation1.2 ≈ 1.4(18Bulaj G. West P.J. Garrett J.E. Marsh M. Zhang M.-M. Norton R.S. Smith B.J. Yoshikami D. Olivera B.M. Biochemistry. 2005; 44: 7259-7265Crossref PubMed Scopus (107) Google Scholar, 20Zhang M.M. Green B.R. Catlin P. Fiedler B. Azam L. Chadwick A. Terlau H. McArthur J.R. French R.J. Gulyas J. Rivier J.E. Smith B.J. Norton R.S. Oliviera B.M. Yoshikami D. Bulaj G. J. Biol. Chem. 2007; 282: 30699-30706Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar)SmIIIAZR–CCNGRRGCSSRWCRDHSRCCbC-terminal amidationAmphibian(16West P.J. Bulaj G. Garrett J.E. Olivera B.M. Yoshikami D. Biochemistry. 2002; 41: 15388-15393Crossref PubMed Scopus (86) Google Scholar)MIIIAZ–GCCNVPNGCSGRWCRDHAQCCbC-terminal amidationAmphibian(21Zhang M.-M. Fiedler B. Green B.R. Catlin P. Watkins M. Garrett J.E. Smith B.J. Yoshikami D. Olivera B.M. Bulaj G. Biochemistry. 2006; 45: 3731-3840Google Scholar)CIIIA–GRCCEGPNGCSSRWCKDHARCCbC-terminal amidationAmphibian(21Zhang M.-M. Fiedler B. Green B.R. Catlin P. Watkins M. Garrett J.E. Smith B.J. Yoshikami D. Olivera B.M. Bulaj G. Biochemistry. 2006; 45: 3731-3840Google Scholar)CnIIIA–GRCCDVPNACS–RWCRDHAQCCbC-terminal amidationAmphibian(21Zhang M.-M. Fiedler B. Green B.R. Catlin P. Watkins M. Garrett J.E. Smith B.J. Yoshikami D. Olivera B.M. Bulaj G. Biochemistry. 2006; 45: 3731-3840Google Scholar)CnIIIBZ–GCCGEPNLCFTRWCRNNARCCRQQcFree acid C-terminal. Cysteines 1–4, 2–5, 3–6 are pairedAmphibian(21Zhang M.-M. Fiedler B. Green B.R. Catlin P. Watkins M. Garrett J.E. Smith B.J. Yoshikami D. Olivera B.M. Bulaj G. Biochemistry. 2006; 45: 3731-3840Google Scholar)TIIIARHGCCKGOKGCSSRECRO–QHCCbC-terminal amidation1.4 ≈ 1.2(15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar)PIIIAZRLCCGFOKSCRSRQCKO–HRCCbC-terminal amidation1.4 ≈ 1.2(24Shon K.J. Olivera B.M. Watkins M. Jacobsen R.B. Gray W.R. Floersca C.Z. Cruz L.J. Hillyard D.R. Brink A. Terlau H. Yoshikami D. J. Neurosci. 1998; 18: 4473-4481Crossref PubMed Google Scholar)GIIIARD–CCTOOKKCKDRQCKO–QRCCAbC-terminal amidation1.4 >> 1.2(38Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar)GIIIBRD–CCTOORKCKDRRCKO–MKCCAbC-terminal amidation1.4 > 1.2(38Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar)GIIICRD–CCTOOKKCKDRRCKO–LKCCAbC-terminal amidation1.4 >> 1.2(38Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar)a SIIIA, SIIIB, and KIIIA are smaller μ-conotoxins, whereas the others are defined as larger μ-conotoxinsb C-terminal amidationc Free acid C-terminal. Cysteines 1–4, 2–5, 3–6 are paired Open table in a new tab This study reports the isolation and structure-activity of μ-conotoxins SIIIA and SIIIB from the milked venom of C. striatus. To define aspects of their structure that contribute to binding to mammalian VGSCs, we determined the NMR structure of SIIIA. Distinct from other μ-conotoxins, SIIIA adopts a single conformation in solution that includes an α-helical motif not previously seen in other members of this class. This structural change was associated with a significant pharmacophore shift from the dominant arginine residue in loop 2 of the larger μ-conotoxins to a series of five similarly important residues, including four associated with the α-helical region, in the smaller SIIIA. Interestingly, a number of SIIIA analogues had significantly enhanced neuronal selectivity, opening the way to the development of subtype-selective neuronal VGSC inhibitors. Isolation of μ-Conotoxins SIIIA and SIIIB—Crude C. striatus milked venom was separated into 80 1-min fractions by semipreparative RP-HPLC using a Vydac C18 column (250 × 10 mm, 5 μ) eluted at 3 ml/min with a 1% gradient from 100% A to 80% B over 80 min (solvent A 0.05% trifluoroacetic acid, solvent B 90% ACN + 0.045% trifluoroacetic acid). The eluant was monitored at 230 nm and 125I-TIIIA-guided fractionation (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar) of the crude milked venom RP-HPLC fractions of C. striatus used to isolate SIIIA and SIIIB to homogeneity. The sequences of SIIIA and SIIIB (Table 1) were determined by Edman sequencing only after pyroglutamate aminopeptidase digestion, indicating that the N-terminal residue of both these conotoxins was a pyroglutamate. The observed masses for native SIIIA and SIIIB (2206.8 and 2120.7 Da, respectively) were consistent with the Edman sequencing results for peptides with an amidated C terminus. Co-injected synthetic and native SIIIA and SIIIB (2:1 ratio) co-eluted on a Vydac C18 reversed-phase HPLC column (250 × 4.6 mm, 5 μ) eluted with a 1% gradient from 100% A to 50% B over 50 min, confirming the identity of the native peptides. Peptide Synthesis—μ-Conotoxins SIIIA and SIIIB and analogues were prepared by Boc chemistry (28Schnölzer M. Alewood P.F. Jones A. Alewood D. Kent S.B.H. Int. J. Pept. Protein Res. 1992; 40: 180-193Crossref PubMed Scopus (938) Google Scholar) using methods described previously (14Nielsen K.J. Watson M. Adams D.J. Hammarstrom A.K. Gage P.W. Hill J.M. Craik D.J. Thomas L. Adams D. Alewood P.F. Lewis R.J. J. Biol. Chem. 2002; 277: 27247-27255Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Side-chain protecting groups chosen were Arg(Tos), Asp(OcHex), Lys(CIZ), Ser(Bzl), Asn(Xan), His(DNP), and Cys(p-MeBzl). The crude reduced peptides were purified by preparative RP-HPLC using a 1% gradient (100% A to 80% B over 80 min) and UV monitoring (230 nm), and oxidized at 0.02 mm in either aqueous 0.33 m NH4OAc/0.5 M GnHCl/2 M NH4OH, or 100 mm ammonium bicarbonate in the presence of oxidized and reduced glutathione (29Nielsen K.J. Adams D. Thomas L. Bond T. Alewood P.F. Craik D.J. Lewis R.J. J. Mol. Biol. 1999; 289: 1405-1421Crossref PubMed Scopus (68) Google Scholar). Oxidized peptides were purified by preparative RP-HPLC. Peptides were quantified initially by triplicate amino acid analysis to create an external reference standard for HPLC quantitation of each peptide (30Moffatt F. Senkans P. Ricketts D. J. Chromatogr. A. 2000; 891: 235-242Crossref PubMed Scopus (28) Google Scholar). Mass spectra were acquired on a PE-Sciex API III triple quadrupole electrospray mass spectrometer in positive ion mode (m/z 500–2000 at 0.1–0.2 Da steps, declustering potentials of 10–90 V, and dwell times of 0.4–1.0 s). Data were deconvoluted using MacSpec 3.2 (Sciex, Canada) to obtain the molecular weight from the multiply charged ion species. Mass spectrometry was used to confirm purity and to monitor peptide oxidation (data not shown). Chemicals and Reagents—Acetonitrile, methanol, ethanol, and glacial acetic acid were from BDH (Poole, UK). Phenyl isothiocyanate and amino acid standards were from Pierce. HBTU, dimethyl formamide, dichloromethane, trifluoroacetic acid, N,N-diisopropylethylamine, dicyclohexylcarbodiimide, and 1-hydroxybenzotriazole were all peptide synthesis grade from Auspep (Melbourne, Australia). p-Methylbenzhydrylamine 1% cross-linked divinylbenzene polystyrene resin was from the Peptide Institute (Osaka, Japan), and BocPAM-Phe resin was from Applied Biosystems (Fosters City, CA). Purified water was obtained from a tandem Milli-RO/Milli-Q system (Bedford, MA). p-Cresol, p-thiocresol, guanidine·HCl (99%+), reduced and oxidized glutathione, and triethylamine (freshly distilled) were from Sigma Aldrich. Anhydrous hydrogen fluoride (HF) was supplied by BOC Gases (Brisbane, Australia). Ammonium acetate (AR) and ammonium sulfate (AR) was from Ajax Chemicals (Australia). Boc-l-amino acids were purchased from Bachem and Novabiochem (La Jolla, CA). All other reagents and solvents were ACS analytical reagent grade. Radioligand Binding Studies—125I-TIIIA (prepared using IODOGEN) radioligand binding studies were performed using rat brain and rat skeletal muscle preparations to measure Nav1.2 and Nav1.4 affinity, as previously described (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar). Nonlinear regressions (Hillslope-1) were fitted to triplicate data obtained for each experiment using Prism software (GraphPad, San Diego, CA). Recording of Depolarization-activated Sodium Ion Currents in Rat DRG Neurons—Neonatal rat DRG neurons were prepared as described previously (31Daly N.L. Ekberg J.A. Thomas L. Adams D.J. Lewis R.J. Craik D.J. J. Biol. Chem. 2004; 279: 25774-25782Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Whole cell TTX-S and TTX-R sodium ion currents were recorded in using an Axopatch 200A patch clamp amplifier (Molecular Devices Corp.). Series resistance was routinely compensated by 70–80%. Capacitive and leakage currents were digitally subtracted using a -P/6 pulse protocol. Internal pipette solution contained (in mm): NaCl 10, CsF 130, CsCl 10, EGTA 10, HEPES-CsOH 10, pH 7.2, 290–300 mOsm (with sucrose). The bath solution contained (in mm): NaCl 50, KCl 5, MgCl2 1, CaCl2 1, glucose 10, tetraethylammonium (TEA)-Cl 90, HEPES-TEA-OH 10, pH 7.35. TTX-resistant sodium ion currents were recorded from small DRG neurons (≤25 μm diameter) in the presence of 300 nm TTX. Large neurons (≥40 μm diameter), in which the sodium ion current exhibited fast activation and inactivation kinetics and was mostly TTX-sensitive (>90%), were used to study toxin effects on TTX-S sodium ion current after complete washout of TTX block. Toxins were diluted in extracellular solution and added via a gravity-fed perfusion system. Xenopus Oocyte Experiments—Depolarization-activated sodium ion currents were recorded from Xenopus oocytes expressing various VGSC α-subunit subtypes using a two-electrode (virtual ground circuit) voltage clamp at room temperature (20–23 °C) with a GeneClamp 500B amplifier and pCLAMP 8 software (Axon Instruments Inc, Union City, CA). Bath solution contained (in mm) 100 NaCl, 2 KCl, 1 MgCl2, 0.3 CaCl2, and 20 HEPES-NaOH, pH 7.5. Data were low pass-filtered at 2 kHz, digitized at 10 kHz, and leak-subtracted on-line using a -P/6 protocol and analyzed off-line. Data were analyzed using Clampfit 8 software (Axon Instruments Inc., Union City, CA). Rat Nav1.2 and rat Nav1.3 was a gift from A. Goldin (University of California), human Nav1.5 a gift from R. Kass (Columbia University), and human Nav1.7 a gift from N. Klugbauer (Teknischen Universität, Munich). Toxins were added by direct bath application (static bath) followed by extensive mixing to reach the final concentrations stated. The steady-state block was reached after ∼8 min of toxin exposure, and washout of SIIIA was followed for 20–30 min. 1H Nuclear Magnetic Resonance (NMR) Spectroscopy—All NMR experiments were recorded on a Bruker ARX 500 spectrometer equipped with a z-gradient unit, a Bruker DMX 750, or a Bruker 600 Avance spectrometer equipped with a x,y,z-gradient unit. Peptide concentrations were ∼2 mm. SIIIA and SIIIB were examined in 95% H2O/5% D2O (pH 3.0, 275–298 K) and in 100% D2O (260–293 K). SIIIA analogues were examined in 90% H2O/10% D2O, pH 3.0. For SIIIA, 1H NMR experiments recorded included NOESY, (mixing times 120, 300, and 400 ms), TOCSY (mixing time 80 ms), double-quantum filtered (DQF)-COSY, and E-COSY in 100% D2O. Slowly exchanging amide protons were detected by acquiring one-dimensional NMR spectra immediately after dissolving the peptide in 100% D2O at pH 3.5. For all other analogues, TOCSY experiments with a mixing time of 80 ms were acquired. Additional NOESY experiments were only run if ambiguities arose during assignment. All spectra were run over 6024 Hz (500 MHz), 7184 Hz (600 MHz), or 8192 Hz (750 MHz) with 4K data points, 400–512 FIDs, 8–64 scans, and a recycle delay of 1–2 s. The solvent was suppressed using the WATERGATE sequence (32Piotto M. Saudek V. Sklenar V. J. Biomol. NMR. 1992; 2: 661-665Crossref PubMed Scopus (3522) Google Scholar). Spectra were processed using UXNMR as described previously (29Nielsen K.J. Adams D. Thomas L. Bond T. Alewood P.F. Craik D.J. Lewis R.J. J. Mol. Biol. 1999; 289: 1405-1421Crossref PubMed Scopus (68) Google Scholar) and using Aurelia, subtraction of background was used to minimize T1-noise. Chemical shift values of SIIIA and analogues were obtained in 90% H2O, 10% D2O and referenced internally to DSS at 0.00 ppm. Secondary Hα shifts were measured and compared with values for random coil shifts (33Wishart D.S. Bigam C.G. Holm A. Hodges R.S. Sykes B.D. J. Biomol. NMR. 1995; 5: 67-81Crossref PubMed Scopus (1416) Google Scholar). SIIIA was assigned in sparky (34Goddard T.D. Kneller D.G. SPARKY 3. University of California, San Francisco, CA2004Google Scholar), and distance information obtained by integration of a 300 ms mixing time NOESY spectrum. Backbone dihedral restraints were derived from 3JHN-Hαcoupling constants from a DQF-COSY spectrum or a one-dimensional 1H NMR spectrum. The ϕ dihedral angle restraints included were restrained to 120 ± 30° for 3JHN-Hα >8 Hz and -60 ± 30° for 3JHN-Hα <5.8 Hz. χ1 dihedral angels were determined from intraresidue nuclear Overhauser effect (NOE) and 3JHα-Hβ coupling patterns for an E-COSY spectrum. Initial structures were generated using dyana (35Guntert P. Mumenthaler C. Wüthrich K. J. Mol. Biol. 1997; 273: 283-298Crossref PubMed Scopus (2553) Google Scholar), and final structures were calculated in explicit water with CNS (36Brünger A.T. Adams P.D. Rice L.M. Structure. 1997; 5: 325-336Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar) as previously described (37Rosengren K.J. Daly N.L. Plan M.R. Waine C. Craik D.J. J. Biol. Chem. 2003; 278: 8606-8616Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). An initial 50 structures were calculated, and the 17 lowest overall energy structures selected to represent the solution structure of SIIIA. Isolation of μ-Conotoxins SIIIA and SIIIB—To identify μ-conotoxins selective for neuronal VGSCs, fractionated milked C. striatus venom was assayed for the ability to displace 125I-TIIIA from rat brain sodium channels (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar). Three active fractions with retention times of 20, 21, and 24 min (Fig. 1) were purified to homogeneity, digested using pyroglutaminase, and Edman-sequenced to reveal three peptides with a CC-C-C-CC motif characteristic of μ-conotoxins (38Cruz L.J. Gray W.R. Olivera B.M. Zeikus R.D. Kerr L. Yoshikami D. Moczydlowski E. J. Biol. Chem. 1985; 260: 9280-9288Abstract Full Text PDF PubMed Google Scholar). Fraction 24 contained the sequence of TIIIA, previously identified in C. tulipa by PCR (15Lewis R.J. Schroeder C.I. Ekberg J. Nielsen K.J. Loughnan M. Thomas L. Adams D.A. Drinkwater R. Adams D.J. Alewood P.F. Mol. Pharmacol. 2007; 71: 676-685Crossref PubMed Scopus (58) Google Scholar), fraction 20 the sequence for SIIIA, while fraction 21 contained a sequence for a new μ-conotoxin we named SIIIB (Table 1). SIIIA and SIIIB each comprised 20 residues with a net charge of +2 and +3, respectively. Mass spectrometry and co-elution studies of the native and synthetic peptides confirmed the sequences and C-terminal amidation of both peptides (data not shown). Radioligand Binding Studies—Synthetic μ-conotoxin SIIIA and SIIIB each potently displaced 125I-TIIIA from VGSCs in rat brain and rat skeletal muscle (Fig. 2). The pIC50values for SIIIA and SIIIB and selected additional μ-conotoxins are summarized in Table 2. Similar to TTX and STX, SIIIA had a small but significant preference for neuronal Nav1.2 over the skeletal muscle Nav1.4, whereas SIIIB had a preference for Nav1.4. Both SIIIA and SIIIB were full inhibitors of 125I-TIIIA binding to rat brain or skeletal muscle sodium channels. Interestingly, removing the pyro
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