8.B.19 The Sea Anemone K⁺ Channel Blocker Toxin, BcsTx3 (BcsTx3) Family Sea anemone venoms are a rich source of peptide toxins which provide tools for studying the structures and functions of ion channels. BcsTx3, a toxin found in the venom of a Bunodosoma caissarum (from Brazil) has been purified and biochemically and pharmacologically characterized (Orts et al. 2013). The pharmacological effects were studied on 12 different subtypes of voltage-gated potassium channels (K(V)1.1-K(V)1.6; K(V)2.1; K(V)3.1; K(V)4.2; K(V)4.3; hERG and Shaker IR) and three cloned voltage-gated sodium channel isoforms (Na(V)1.2, Na(V)1.4 and BgNa(V)1.1), all expressed in Xenopus laevis oocytes. BcsTx3 showed a high affinity for Drosophila Shaker IR channels over rKv1.2, hKv1.3 and rKv1.6, and was not active on Na_Vchannels. Biochemical characterization revealed that BcsTx3 is a 50 amino acid peptide crosslinked by four disulfide bridges, and sequence comparison allowed BcsTx3 to be classified as a novel type of sea anemone toxin acting on K_V channels. Putative toxins homologous to BcsTx3 from two additional actiniarian species were identified (Orts et al. 2013). These sea anemone toxins (subfamily 1) are clearly related to spider toxins (subfamily 2), and families 8.B.6 and 8.B.19 seem to be related to each other as well as possibly to 1.C.52, 8.B.3 and 8.B.5. These tentative possibilities need to be confirmed. However 8.B.3 and 8.B.5 are for sure related.
This family belongs to the Huwentoxin Superfamily.
References:
Agwa, A.J., L.V. Blomster, D.J. Craik, G.F. King, and C.I. Schroeder. (2018). Efficient Enzymatic Ligation of Inhibitor Cystine Knot Spider Venom Peptides: Using Sortase A To Form Double-Knottins That Probe Voltage-Gated Sodium Channel Na1.7. Bioconjug Chem. [Epub: Ahead of Print]
Cassola, A.C., H. Jaffe, H.M. Fales, S.C. Afeche, F. Magnoli, and J. Cipolla-Neto. (1998). ω-Phonetoxin-IIA: a calcium channel blocker from the spider Phoneutria nigriventer. Pflugers Arch 436: 545-552.
Clémençon, B., L. Kuhn-Nentwig, N. Langenegger, L. Kopp, S. Peigneur, J. Tytgat, W. Nentwig, and B.P. Lüscher. (2020). Neurotoxin Merging: A Strategy Deployed by the Venom of the Spider to Potentiate Toxicity on Insects. Toxins (Basel) 12:.
Kubista, H., R.A. Mafra, Y. Chong, G.M. Nicholson, P.S. Beirão, J.S. Cruz, S. Boehm, W. Nentwig, and L. Kuhn-Nentwig. (2007). CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons. Neuropharmacology 52: 1650-1662.
Kuhn-Nentwig, L., I.M. Fedorova, B.P. Lüscher, L.S. Kopp, C. Trachsel, J. Schaller, X.L. Vu, T. Seebeck, K. Streitberger, W. Nentwig, E. Sigel, and L.G. Magazanik. (2012). A venom-derived neurotoxin, CsTx-1, from the spider Cupiennius salei exhibits cytolytic activities. J. Biol. Chem. 287: 25640-25649.
Orts, D.J., Y. Moran, C.T. Cologna, S. Peigneur, B. Madio, D. Praher, L. Quinton, E. De Pauw, J.E. Bicudo, J. Tytgat, and J.C. de Freitas. (2013). BcsTx3 is a founder of a novel sea anemone toxin family of potassium channel blocker. FEBS J. 280: 4839-4852.
Pinheiro, A.C., A.J. da Silva, M.A. Prado, M.d.o.N. Cordeiro, M. Richardson, M.C. Batista, C.J. de Castro Junior, A.R. Massensini, C. Guatimosim, M.A. Romano-Silva, C. Kushmerick, and M.V. Gomez. (2009). Phoneutria spider toxins block ischemia-induced glutamate release, neuronal death, and loss of neurotransmission in hippocampus. Hippocampus 19: 1123-1129.
Reis, H.J., M.A. Prado, E. Kalapothakis, M.N. Cordeiro, C.R. Diniz, L.A. De Marco, M.V. Gomez, and M.A. Romano-Silva. (1999). Inhibition of glutamate uptake by a polypeptide toxin (phoneutriatoxin 3-4) from the spider Phoneutria nigriventer. Biochem. J. 343Pt2: 413-418.
Sachkova, M.Y., A.A. Slavokhotova, E.V. Grishin, and A.A. Vassilevski. (2014). Structure of the yellow sac spider Cheiracanthium punctorium genes provides clues to evolution of insecticidal two-domain knottin toxins. Insect Mol Biol 23: 527-538.
Schalle, J., U. Kämpfer, S. Schürch, L. Kuhn-Nentwig, S. Haeberli, and W. Nentwig. (2001). CSTX-9, a toxic peptide from the spider Cupiennius salei: amino acid sequence, disulphide bridge pattern and comparison with other spider toxins containing the cystine knot structure. Cell Mol Life Sci 58: 1538-1545.
Vassilevski, A.A., I.M. Fedorova, E.E. Maleeva, Y.V. Korolkova, S.S. Efimova, O.V. Samsonova, L.V. Schagina, A.V. Feofanov, L.G. Magazanik, and E.V. Grishin. (2010). Novel class of spider toxin: active principle from the yellow sac spider Cheiracanthium punctorium venom is a unique two-domain polypeptide. J. Biol. Chem. 285: 32293-32302.
Wullschleger, B., W. Nentwig, and L. Kuhn-Nentwig. (2005). Spider venom: enhancement of venom efficacy mediated by different synergistic strategies in Cupiennius salei. J Exp Biol 208: 2115-2121.
Examples:
TC#	Name	Organismal Type	Example
8.B.19.1.1	*The mature K⁺ channel blocking toxin, BcsTx3 of 50 aas (Orts et al.* 2013).**	Animals (Sea anemones)	BcsTx3 of Bunodosoma caissarum

8.B.19.1.2	Putative pro-toxin of 190 aas	Animals (see anemones)	Toxin precursor of Nematostella vectensis

Examples:
TC#	Name	Organismal Type	Example
8.B.19.2.1	U1-ctenitoxin Cs1a (CstX-7). Blocks mammalian neuronal L-type (Cav1/CACNA1) high-voltage-activated calcium channels. Causes paralysis in Drosophila. Its insecticidal activity is synergistically increased by potassium ions, M-ctenitoxin-Cs1a (P83619), omega-ctenitoxin-Cs1a (P81694), and U1-ctenitoxin-Cs2a (P83919). Because of the disulfide knot strcuture, this neurotoxin is called a knottin (Schalle et al. 2001).	Animals (spiders)	CstX-7 of Cupiennius salei

8.B.19.2.2	Peptide toxin, Cs1a omega ctenitoxin, of 122 aas from a spider multicomponent venom. It contains a cystine knot structure and is therefore called a knottin (Kuhn-Nentwig et al. 2012). Double-knottin peptides from spider venom have been used to reveal aspects of the pharmacology of transmembrane channels (Agwa et al. 2018). It shows calcium channel blocking activity and exhibits cytolytic activity by affecting the outer leaflet curvature and/or pore formation across the membrane (Kuhn-Nentwig et al. 2012, Clémençon et al. 2020). It blocks L-type calcium channels (Cav1/CACNA1) (TC# 1.A.1.11.4) in mammalian neurons at nanomolar concentrations. Furthermore, it produces a slow voltage-independent block of mid/low and high voltage-activated calcium channels in cockroach neurons (Kubista et al. 2007). Potassium ions, histamine, M-ctenitoxin-Cs1a (P83619), CSTX-9 (P58604), and CSTX-13 (P83919) synergistically increase the insecticidal activity of this toxin (Wullschleger et al. 2005, Clémençon et al. 2020). In vivo, it causes paralysis in blow flies and provokes death in Drosophila (Clémençon et al. 2020).	Animals (wandering spiders)	Cs1a toxin of Cupiennius salei

8.B.19.2.3	UT-Lycotoxin, Ls1t (LsTx-A35) of 107 aas. The disulfide knot defines it as a knottin. It is a member of the CsTx superfamily and the U1-lycotoxin family. This protein is processed to the mature form.	Animals (spiders)	Ks1t of Lycosa (Aranea) singoriensis

8.B.19.2.4	Ω Lsp-IA (47aas) Ca²⁺ channel inhibitor	Spiders	*Ω Lsp-IA of Geolycosa* sp. (P85079)**

8.B.19.2.5	Latartoxin 1b of 86 aas. It is further processed to the mature form. Belongs to the CsTx superfamily. The presence of disulfide through disulfide knot structually defines it as a 'knottin'.	Spiders	Latartoxin 1b of Lachesana tarabaevi

8.B.19.2.6	U2-Lycotoxin Ls1c of 105 aas; calcium channel impairing toxin. Also called LsTxM3.	Animals	U2 Lycotoxin Ls1c of Lycosa singoriensis

8.B.19.2.7	DELTA-miturgitoxin-Cp1b of 183 aas and 1 N-terminal TMS. It is a spider venom toxin that exhibits cytolytic activity by forming an α-helix across the membrane and is lethal to insect larvae (Vassilevski et al. 2010, Sachkova et al. 2014). It causes instant paralysis and death to larvae of the flesh fly (S. carnaria) at doses of 20 µg/g, and at doses of less than 10 µg/g, it causes reversible paralysis by causing stable and irreversible depolarization of fly muscle fibers while destabilizing the membrane (Vassilevski et al. 2010).		*Toxin of Cheiracanthium punctorium* (Yellow sac spider)**

8.B.19.2.8	ω-ctenitoxin-Pn3a of 116 aas and 1 N-terminal TMS. This toxin is a potent and practically irreversible antagonist of both Cav2.1/CACNA1A and Cav2.2/CACNA1B calcium channels, while it displays a partial and rapidly reversible block of Cav2.3/CACNA1E calcium channels and no effect on Cav3/CACNA1 calcium channels (Cassola et al. 1998). It also inhibits glutamate uptake from rat brain synaptosomes by an interaction between cysteines from both glutamate transporter and toxin (Reis et al. 1999). It blocks potassium-induced exocytosis of synaptic vesicles in brain cortical synaptosomes with an IC₅₀ of 1.1 nM. In mice, induces rapid general flaccid paralysis followed by death in 10-30 minutes at dose levels of 5 µg per mouse. In rat brain, it inhibits glutamate release, neuronal death and loss of neurotransmission in the hippocampus resulting from ischemia (Pinheiro et al. 2009).		Ctenitoxin of Phoneutria nigriventer (Brazilian armed spider) (Ctenus nigriventer)