Tut Journal or Biologjcal Chkmistry Vol. 270, No. 28, 19-9UG of July 14, pp. 16796-16802. 1995 £> 1995 by The American Society for Biochemistry and Mnlccutar Biology, Inc. Printed in USA-
A N ew Fam ily of C onotoxins T hat Blocks V oltage-gated Sodium Channels*
(Received for publication, March 23, 1995, and in revised form, May 8 , 1995)
J. Michael McIntoshtSU, Arik Hasson, Micha E. Spiral, William R, GrayS, Wenqin Li§, Maren Marsh**, David R. HillyardS**, and Baldomero M. Olivera § From the Departments of ^Biology, **Pathology, and tPsychiatry, University of Utah, Salt Lake City, Utah., 84112, the IIDepartment of Neurabiology, Institu te of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904 Israel, and The Interuniversity Institute for Marine Science*, Eilat SSI03, Israel
Conus peptides, including o>-conatoxins a n d ct-cono- subtypes. For example, in the calcium channel field, the £Q toxins (targeting calcium channels and nicotinic acetyl
(iO-conotoxin MrVIB: acskkweycivpilgfvyccpglicgpfvcv In this paper, we describe the isolation, characterization, and www.jbc.org synthesis of novel peptides, jiO-conatoxins MrVIA and MrVIB, fiO-conotoxin MrVIA was chemically synthesized and from the venom of the snail-hunting species Conus marmoretis proved indistinguishable from the natural product. Sur e prisingly, the jxO-cono toxins sh o w no sequence similar (Fig, 1), Although these peptides produce a biological effect 3 ity to the pt-conotoxins. However, ananalysis of c D N A which is similar to that of the jx-eonotoxins, they are unrelated 2009 14, October UTAH, on UNIV OF at clones encoding the fxO-conotoxin MrVIB demonstrated structurally. Thus, the /xO-conotoxins define a new family of 1 striking sequence similarity to to- a n d 8-conotoxin pre peptides which block Na+ currents of voltage-sensitive sodium I cursors. Together, the w-, and /xO-conotoxins define channels. The discovery of this novel family provides a new the O-superfamily of Conus peptides. The probable bio opportunity to obtain a large armamentarium of subtype-spe o logical role an d evolutionary affinities of these peptides cific sodium channel-targeted Conus peptides, are discussed. c EXPERIMENTAL PROCEDURES Peptide Purification
Fig. 1. The m arble cone C. m ar moreus and other snail-hunting Co nus species. Top left, an etching of C. marmoreus by Rembrandt. Note that Rembrandt neglected to reverse the pic ture, so that when printed, the helical shell comes out sinistral, instead of dex- tral. He did, however, remember to render his signature as a mirror image so the correct signature impression is made. Top right, a photograph of Conus mar moreus. The photographer has signed as Rembrandt did. In a mirror, the photo graph has the handedness of the etching m and vice versa. Lower panels show mem bers of two major clades of snail-hunting CD Conus. In the C. marmoreus clade, left, the following species and forms are in 2 cluded: top row, left to right, Conus vidua and C. marmoreus (both Philippines). onodd fromDownloaded Middle row, a dwarf, melanistic C. mar moreus specimen (for nigrescens) (Samoa) and Conus bandanus (Hawaii). Bottom row, Conus nicobaricus (Sulu Sea), Conus nocturnus (Palawan Island), and Conus araneosus (India). In the lower right panel is the C. textile clade of snail-hunting spe
cies. Top row, left to right, Conus glo- www.jbc.org riamaris (Philippines), Conus retifer (Oki nawa), and Conus natalis (South Africa). Middle row, C. textile and Conus legatus (both Philippines). Bottom row, Conus eu- trios (Mozambique), Conus dalli (Mexico), 2009 14, October UTAH, on UNIV OF at and Conus bengalensis (Bay of Bengal). I Photographs by Kerry Matz. §
<3 e
chemistry was used, with single couplings using dicyclohexylcarbodi- and redissolved in distilled HaO (7 jd). Sequenase reaction buffer (2 p.1)
tween 20-24 °C. 20-100 jxl of toxin solution were applied to the experimental dish (0.25-0.75 ml) using a Gilson tip pipette placed a few millimeters away from the neuron under study. The indicated toxin concentrations cor respond to the final concentration in the experimental dish. Solutions—Control experiments were carried out in artificial sea water composed of: 460 mM NaCl, 10 mM KCl, 11 mM CaCl.,, 55 mM MgCl.j, and 10 m u HEPES. The pH was adjusted to 7.6, To minimize potassium currents we used an external potassium channel-blocking solution, in which KCI was substitute for by CsCl. In addition, the solution contained 50 mM tetraethyl ammonium chloride
and 0.1 mM 3,4-diaminopyridine. The osmolarity of the solution was Tim e (min) adjusted by reducing the NaCl concentration to 410 mM. To monitor sodium current, a calcium-free potassium channcl-block- D ing solution was used. In this solution, Ca2' was substituted for by MrVIA M f *, and the solution was supplemented by 8 mM Coa' to prevent any ACRKKWE’f CIVPIIGFIYCCPGUCGPFVCV C a '' influx. For these experiments the patch pipette contained 440 mM CsCl, 40 mM CsOH, 20 mM NaCl, 6.S mM MgCI.,, 10 mM EGTA, 100 mM MrVIB £Q HEPES, and 3 mM adenosine 5’-triphosphate (ATP). The pH was ad ACSKKWEYCIVPI LGFVYCCPGLICGPFVCV justed to 7.3, To monitor calcium currents the external sodium ions were replaced m by tetraethylammonium chloride. The patch pipette contained 440 mM co CsCl, 40 mM CsOH, 2 mM CaCl, 6,8 mM MgCl2, 100 mM HEPES, 10 mM < Cs4BAPTA' (Molecular Probes, Eugene, OR), and 5.6 mM glucose, [n order to prevent run-down of calcium channels (16, 17), the internal fromDownloaded solution was supplemented with 0.5 mM guanosine 5’-triphosphate Fig. 2. Isolation and characterization of /iO-conotoxins MrVIA (xO-conotdXin MrVIB: ACSKKWEYCIVPILGPV^CCPGLICGPFVCV natural material and was equipotent in biological assays. Precursor Sequence—In order to determine the precursor Liquid secondary ion mass spectrometry indicated that Cys sequence of these peptides, a cDNA library was prepared from residues are present as disulfides and that the COOH-terminal venom ducts of Conus marmoreus and clones related to the new ct-carboxyl group is the free acid for both peptides (monoiso peptides were identified. Two cDNA clones for ^O-conotoxin topic MH+: MrVIA calculated 3487.66, observed 3487.8; MrVIB MrVIB were characterized. The nucleotide sequence and pre calculated 3404.58, observed 3404.8). The sequence was fur dicted amino acid sequence of the encoded precursor is shown ther verified by chemical synthesis and cDNA cloning (see in Fig. 3. It is seen that the last 31 residues exactly match the below). experimentally determined sequence of /iO -conotoxin MrVIB. Surprisingly, the new conotoxins show no detectable amino The putative 82-amino acid precursor sequence is presumably acid sequence homology to the well characterized sodium chan- cleaved after Arg-51 to yield the mature 31-amino acid peptide. The cDNA sequence also verifies the biochemical assignment for a free carboxyl terminus for MrVIB. 1 The abbreviations used are: BAPTA, l,2-bis(2-aminophe- noxy)cthane'iV,W.(V‘ JV-tetraacetic acid; RPLC. reversed phase liquid Electrophysiology—Action potentials generated by intracel chromatography. lular stimulation of cultured A plysia neurons were rapidly fxO-conotoxins MrVIA/B, Novel Sodium Channel Probes 16799 Table I met lys leu thr cys me" net ile vzd ala val leu jhe 1«j thr ala Bioassay nO-conotaxin MrVIA ATG AAA CTO ACG 1GC ATS ATG H C GIT OCT GIG CTG TTC TIE ACA GOC Toxin was injected intracranially (i.e.) or intraperitoneally (i.p.) into young (6 -8 g) mice (29). ME. no effect; NT, not tested. trp thr leu val met ala asp asp ser asn esn gly lea ala asn his Dose i.e. ip TCG M J i CTC QIC ATG OCT GWT GAC TCC M C AKT 33A CTG 033 AAT CAT nmoi phe leu lys ser oig asp glu net glu asp pro glu ala ser lys leu 0.1 Ataxia/light coma NE IT T TIG AAA OTT GAC GAA ATG GAG rounds of colony hybridization of this library using a probe specific for recover even after prolonged washing (45 min) (Fig. SD), the fromDownloaded (o-conotoxin signal sequence. The 15 clones were then sequenced with blockade of the calcium current recovered to the control level this same oligonucleotide. Of these, four encoded jAO-conotoxin MrVIB. within a few seconds of wash (Fig. 5, C and D). The high MrVIA concentration necessary to induce the blockade of 1^,,*-, the (30 s) blocked by bath application of 350 nm conotoxin MrVIA slow blocking rate, and the rapid washout of the blockade (Fig. 4, A and B). The action potential blockade was not asso suggest that this blockade of calcium conductance by MrVIA is ciated with changes in the transmembrane potential or input unlikely to play a major role in prey immobilization by C. www.jbc.org resistance. An increase in the intracellular stimulus intensity- m arm oreus. Since 250 nM MrVIA is sufficient to completely failed to evoke a fall-blown action potential, but induced a block a concentration that does not affect 1 ^ -, /aO-cono e small local response (not shown). The local response was most toxin MrVIA can be used effectively as a sodium channel block likely generated by unblocked voltage-gated calcium channels, ing agent in neurobiological studies of the A plysia nervous 2009 14, October UTAH, on UNIV OF at 3 as it could be abolished by the addition of 8 mM cobalt ions to system . 1 the bath solution (see also Fig. 5A), l±0-conutoxin MrVIA Is Active in Vertebrate Systems—The To identify the mechanisms underlying the action potential results above demonstrate that jiO-conotoxins purified from I blockage by jj.0-con□ toxins MrVIA/B, we used experimental the venom of a mollusc-hunting Conus potently block voltage- o protocols that permitted us to examine the isolated macro sensitive sodium channels in molluscan systems. The activity c scopic currents of either K+, Ca2 1, or N a'. The toxins had no of juO-conotoxin MrVIA was also examined using injections into effects on the voltage-gated potassium currents at concentra mice; results are shown in Table I. The data reveal that the tions of Up to 10 JAM, peptide is extremely potent when injected into the rodent cen 2 0 m V 5 n A Fig. 4. Blockade of action potential and inward sodium current by |iO- B, V— m conotoxin MrVIA as revealed by cur T( rent and voltage clamp experiments. A . (control), the action potential was gen m erated by an intracellular rectangular depolarizing pulse. A 2, 10 s after toxin < application to reach a final bath concen tration of 350 nM, the action potential was fromDownloaded blocked. An increase in the stimulus in tensity after the blockade failed to elicit a 20 mV 20 mV regenerative response. The inward INa+ -50 mV I -SO niV J evoked by depolarizing the neuron from a holding potential of -50 to 20 mV (iJj) was completely blocked 30 s following the application of 250 nM MrVIA (B.j. In the record of Bs the control and two con www.jbc.org secutive traces are shown. C, partial blockade of IN„ by 40 If v. toxin revealed e that the block is not associated with a V(mV) $ D change in the current-voltage relation -40 -20 2D 40 60 80 100 2009 14, October UTAH, on UNIV OF at 3 ships. The .'.wv/ shows the control current I__ _I 1 m i 1 trace and the partially blocked INll-. □ □ i I □ o [] □ ♦ -4 c □ □ ° ♦ -6 ■10 f t f -12 channels in general. Thus, three distinctive pharmacological groups of Conus pep Superfamilies of Conus Peptides—Surprisingly, although the tides appear to belong to the same structural group, which we new conotoxins clearly block N a1 currents elicited through will define as the “O-superfamily” of Conus peptides: the voltage-sensitive sodium channels, they show no detectable ij-conotoxins which inhibit calcium channels, the S-conotoxins amino acid sequence identity to the (u-conotoxms from fish- which alow down the inactivation rate of voltage-sensitive so hunting Conus, the other group of Conus peptides that block dium channels, and the fiO-conotoxins which block voltage- sodium currents. Instead, the MrVIA/B peptides have a cys sensitive sodium currents. By contrast, the ^i-conotoxins from teine pattern similar to that of the ti>-eonot Fro. 5, Effect of Mr\TA On th* So* dium and calcium cvfrent For the ex periment the potassium currents were blocked, The neuron was depolarized from a holding potential of -50 to 20 mV. I nA In the control [A the depolarization in J duced an early rapidly inactivating so dium currcnt and a plateau of calcium current. Application of 150 hm MrVIA D I SO nM J^rVIA 3.5mMMjV|A wash in PCUS rapidly blocked the early sodium current 1.2—1 (second trace in A and see inset). The rate i A I at which INo+ was blocked is shown by the open diamond# in D. Note that the cal # cium current was not affected by 150 nM ■ 0-8 £Q MrVIA iA and black circles in D). Follow * ing the application of 3.5 jutM MrVIA, l<-„2* j 0,6 was gradually reduced I.R and black cir- m ctes in Dh Ipa2' rapidly recovered follow 0.4 co ing wash by artificial sea water, whereas O < the sodium current remained blocked. 0.2 fromDownloaded 0 i* o ^-o-o.o o fro-o,n » y 500 1000 1500 2000 2500 Time (s) gle superfamily to produce functionally distinct toxin families. In vivo results already suggest that the first peptides char Such an extreme interspecific diversification of venom peptides acterized from these two conotoxin families (p. and jxO) may www.jbc.org may account in part for the unusual proliferation of species in have distinctly different sodium channel subtype specificity in the genus Conus, which is presently believed to be the largest mammalian systems. Thus, relatively high doses of /u,0-cono- living ^enus of marine invertebrates (approximately 500 living toxin MrVIA (>10 nmol/10 g) are inactive peripherally in ro 3 species) (27). dents, whereas /x-conotoxin GIIIB is a potent paralytic when 2009 14, October UTAH, on UNIV OF at Biological Role—Our results suggest that snail-hunting Co injected intraperitoneal (28); this is consistent with the well 1 nus have evolved at least two solutions to the same biological established specificity of p,-conotoxins for voltage-sensitive so I problem of feeding on snails. When harpooned, the prey would dium channels from mammalian skeletal muscle. In contrast, o naturally retract into their shells to escape from a predatory low doses of /xO-conotoxin MrVIA cause ataxia and/or reversi Conus snail. Our observations of feeding in aquaria suggest ble coma in a few minutes when injected intracranially, c that two snail-hunting Cobus groups have generated very dif whereas no detectable symptoms are observed after several ferent ways to prevent prey from becoming paralyzed inside the hours of a similar central injection ofV-conotoxin GIIIB. These Conns textile, results suggest that MtVIA is a potent ligand in the mamma cone snail after immobilization of the victim, albeit by very nels expressed in X em ipus oocytes are, however, consistent different means. with the in vivo central effects of ^O conotoxins.3 Many more The Potential of Conus Peptides for Generating a Subtype- members of the /x- and /.tO-co no toxin families can be identified specific Sodium Channel Pharmacology—juO-conotoxins by molecular genetic techniques and the predicted peptides MrVIA and MrVIB are harbingers of a potentially much wider chemically synthesized. By screening for those peptides that range of peptides active against sodium channels. The avail induce different in vivo symptoms, an expanding set of prom ability of the cloned precursor sequence has allowed us to ising ligands for establishing a subtype-specific pharmacology identify potential homologs from venoms of other Conus spe for sodium channels should be established. cies.2 The sequences available suggest that there is consider General Perspectives—Different ion channel subtypes might able sequence hypervari ability in this family of Conns peptides, be expected to play distinctive functional roles in nervous sys making it likely that different members may have differing tems. For voltage-sensitive calcium channels, this functional sodium channel subtype specificity profiles. Similarly, al differentiation of subtypes has become increasingly well docu though the structurally unrelated /i-eono toxins have only been mented. This has been made possible in large part by the characterized from a single fish-hunting Ccmus species, Conus availability of a family of Contis peptides, the t(>-conotoxms: geographus, a molecular genetic analysis has revealed that a thus, «1(1- and a ^-containing calcium channel complexes are number of other fish-hunting Conus species also have jx-cono- differentially targeted by different tij-conotoxins (1). toxins; the predicted peptides are presently being synthesized The functional differentiation of sodium channel subtypes is and will be further characterized. much more poorly understood; the paucity of sodium channel 1 M. M arsh and D. Hillyard, unpublished results. ! H. Terlau, B. M. Olivera, and W. Stuhmer, unpublished results. 16802 H.0-conotoxins MrVIA / B, Novel Sodium Channel Probes A Comparison of O-supeifamily precursor sequences J lO -M r V lB 8 -T n V I A (0 -G V J A [a l a t r p t h r l e u v a l n e t a l a a s o a s p s e r a s n a s n g l y l e a a l a ld J.a t r p t h r p h e a la t h r a l a a s p p r o a r g a s n g l y le u g l y I a l a c y s g i n l e u i l e t h r a l a a s p a s p s e r a r g ------g l y t h r g i n a s n h i s p h e l e u l y s s e r a r g a s p g l u met. g l u a s p p r o g l u a l a a s n l e u p h e s e r a s n a l a h is h i s g l u n te t l y s a s n p r o g l u a la — — Pic. 6. Comparison of O-superfam- l y s h i s — a r g a l a l e u g l y s e r t h r t h r — g l u l e u TT.it ate toxin ily precursor sequences. The inferred ------— - - — i sequence of the fiO-conotoxin MrVIB pre r cursor sequence (see Fig. 3) is aligned s e r l y s l e u g l u l y s a r g a la c y s s e r l y s lys t r p glu t y r cyra CD with the prepropeptide sequences of s e r l y s l e u a*sn l y s a r g t r p c y s l y s g i n ser gly glu n e t c y s 8-conotoxin TxVIA (formerly called the — 03 s e r — l e u s e r t h r a r g c y B l y s s e r p r o gly ser s e r c y B < King-Kong peptide) and ui-conntojtin GVTA, The signal sequence and mature fromDownloaded toxin regions are indicated, with an inter vening proregion. The conserved amino i l e v a l p r o i l e l e u g i y pl"fi v a l t y r c y s aye p r o g l y le w acids are boxed. Note the almost total con — a s n le u — l e u a s p g i n a s n — c y s c y s a s p g l y t y r — servation of signal sequences versus the — s e r p r o — t h r s e r t y r a s n — c y s c y s a r g s e r striking divergence of the mature toxins. “ l I c y s g l y p r o p h e v a l c y s v a l www.jbc.org c y s i l e v a l l e u v a l — ------c y S tlnr c y s a s n p r o t y r t h r l y s a r g c y s t y r g l y B Summary of Identical Amino Acid Residues t NV F TH o Otbr 4 2009 14, October UTAH, on UNIV OF at S ign al "Fro" Mature Toxirt sequence region Cys residues Non-Cys Residues Total residues in uQ-MrVlB 22 29 6 25 # Identical to TkVIA 19 19 6 4 # Identical jn all three sequences 14 7 6 0 subtype'specific ligands is a major reason for this situation. M . ( 1994> Biochemistry 33, 11420-11425 10. Col ledge, C. J.h Hunsperger, J, P., Imperial, .1. S., and Hillyard, I). R. < 1992» This report describes a new family of Conus peptides, the Toxicon 30, ]Ill'll 16 jiO-conotoxins, which potently block voltage-sensitive sodium 11. Spira, M. E., Benbassat, D., and Dormann, A. < 1993) J. Neurobiol. 24,300-316 currents- The ^O-conotoxins are potentially as important to 12. Spira, M. E., Ha.sson, A., Fainztlber, M., Gordon, [>., and Zlotkin, E. (1993) Isr. J, Med. Sci- 29, 530-543 sodium channel subtype differentiation as the ci>-conotoxins are 13. Ziv, E. N., and Spira, M. E. {1993) Eur. J. Neurosci. 5, 657-668 e i to calcium channels. 14. Hamill, O. P., Marty, A., Nehcr, E., Sakmann. B., and Sigworth, F. J. (1981) P fluegers A rck. 391, 85-100 Acknowledgments—Mass spectrometry was performed by A. G. Craig 15- Benba&sat, D., and Spira, M. E. (1993)*/. Exp. Neurol. 122, 295-300 at the Clayton Foundation Laboratories of the Salk Institute. Construc 16. Fox, A. P., Nowycky, M. C., and Tsien, R. W (1987) J. Physiol. {Lond.) 394, tion of the peptide resin and sequence analysis were carricd out by R. 49-172 ^ Schackmann at the Utah Regional Cancer Center. Dr. K.-J. Shon syn 17. Rosenmund, C., and Westbrook, G. L. (1993) N euron 10, 805-814 18. O livera, B. M.j Cruz, L. J., de Santou, V., LeCheminant, G. W., Griffin, I>., thesized ufle of the synthetic MrVIA samples used in these studies. Zeikus, R.p McIntosh► J., Galyean, R.n Varga, J., Gray, W, R,. and Rivier, J. Biochemistry REFERENCES (1987) 26, 2086-2090 19- Woudward, S, R., Crui, L. J„ Olivera, B .M , and Hillyard, D R, 11990) E M HO 1. Olivers B, M n Miljanich, Ramachandran, j„ and Adams, M, E. (1994> J , 1, 1015-1020 Annie* Rev. Biochem. 63, 823-367 20. HoBRon, A., Kainzilher. M , Gordon. D.» Zlotkin, E-. and Spira, M, £. (1993i 2. Ohizumi, Y .t Minoshima, S., Takahashi, M .r Kajiwara, A., Nakamura, H., and Eur. J, Neurosci. 5, 5S-64 Kobayashi, J. (1986) J. Pharmacol. Exp. Ther. 239, 243-248 21. Hasson, A.„ Shont K.-J., Olivera, B. 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