8.B.5 The Na⁺/K⁺/Ca²⁺ Channel Targeting Tarantula Huwentoxin (THT) Family Protoxins inhibit voltage-gated calcium (Ca_v3.1/CACNA1G), potassium (K_v2.1/KCNB1) and sodium (Na_v1.5) channels and shift the voltage-dependence of channel activation to more positive potentials. They potently inhibit all sodium channel subtypes tested (Na_v1.2/SCN2A, Na_v1.5/SCN5A, Na_v1.7/SCN9A, and Na_v1.8/SCN10A). Protoxins 1 (35aas) and 2 (30aas) are peptide toxins from the venom of the tarantula, Thrixopelma pruriens, that conform to the inhibitory cystine knot motif and which modify activation kinetics of Na_v and Ca_v, but not K_v, channels. ProTx-II inhibits current by shifting the voltage dependence of activation to more depolarized potentials (Smith et al., 2007). Many plant and animal toxins cause aversive behaviors in animals due to their pungent or unpleasant taste or because they cause other unpleasant senstations like pain. Cromer and McIntyre (2007) have reviewed toxins that act at the TRPV1 ion channel expressed in primary sensory neurons. This channel is activated by multiple painful stimuli and is thought to be a key pain sensor and integrator. The painful peptide 'vanillotoxin' components of tarantula toxin activate the TRPV1 ion channel to cause pain. Toxins from plants, spiders and jellyfish that act on TRPV1 have been identified. Structural information about sites of interaction (toxin-binding sites on the K_v ion channel) have been evaluated. Toxin agonists such as resiniferatoxin and vanillotoxins were proposed to interact with a region of TRPV1 that is homologous to the 'voltage sensor' in the K_v1.2 ion channel, to open the channel and activate primary sensory nerves, causing pain (Cromer and McIntyre, 2007). The voltage-sensor paddle is a crucial structural motif in voltage-activated potassium (K(v)) channels that has been proposed to move at the protein-lipid interface in response to changes in membrane voltage. Tarantula toxins like hanatoxin and SGTx1 inhibit K(v) channels by interacting with paddle motifs within the membrane (Milescu et al., 2007). These toxins can partition into membranes under physiologically relevant conditions, but the toxin-membrane interaction is not sufficient to inhibit K(v) channels. These require specific binding to the paddle motif.
This family belongs to the Conotoxin Superfamily.
References:
Collaço, R.C., F. Van Petegem, and F. Bosmans. (2024). ω-Grammotoxin-SIA inhibits voltage-gated Na⁺ channel currents. J Gen Physiol 156:.
Cromer, B.A., and P. McIntyre. (2008). Painful toxins acting at TRPV1. Toxicon. 51: 163-73.
Diao, J., Y. Lin, J. Tang, and S. Liang. (2003). cDNA sequence analysis of seven peptide toxins from the spider Selenocosmia huwena. Toxicon. 42: 715-723.
Escoubas, P., S. Diochot, M.L. Célérier, T. Nakajima, and M. Lazdunski. (2002). Novel tarantula toxins for subtypes of voltage-dependent potassium channels in the K_v2 and K_v4 subfamilies. Mol. Pharmacol. 62: 48-57.
Henriques, S.T., E. Deplazes, N. Lawrence, O. Cheneval, S. Chaousis, M. Inserra, P. Thongyoo, G.F. King, A.E. Mark, I. Vetter, D.J. Craik, and C.I. Schroeder. (2016). Interaction of Tarantula Venom Peptide ProTx-II with Lipid Membranes is a Prerequisite for its Inhibition of Human Voltage-gated Sodium Channel NaV1.7. J. Biol. Chem. [Epub: Ahead of Print]
Lau, C.H., G.F. King, and M. Mobli. (2016). Molecular basis of the interaction between gating modifier spider toxins and the voltage sensor of voltage-gated ion channels. Sci Rep 6: 34333.
Liao, Z., C. Yuan, M. Deng, J. Li, J. Chen, Y. Yang, W. Hu, and S. Liang. (2006). Solution structure and functional characterization of jingzhaotoxin-XI: a novel gating modifier of both potassium and sodium channels. Biochemistry. 45: 15591-15600.
Middleton, R.E., V.A. Warren, R.L. Kraus, J.C. Hwang, C.J. Liu, G. Dai, R.M. Brochu, M.G. Kohler, Y.D. Gao, V.M. Garsky, M.J. Bogusky, J.T. Mehl, C.J. Cohen, and M.M. Smith. (2002). Two tarantula peptides inhibit activation of multiple sodium channels. Biochemistry 41: 14734-14747.
Milescu, M., J. Vobecky, S.H. Roh, S.H. Kim, H.J. Jung, J.I. Kim, and K.J. Swartz. (2007). Tarantula toxins interact with voltage sensors within lipid membranes. J. Gen. Physiol. 130: 497-511.
Ono, S., T. Kimura, and T. Kubo. (2011). Characterization of voltage-dependent calcium channel blocking peptides from the venom of the tarantula Grammostola rosea. Toxicon 58: 265-276.
Ozawa, S., T. Kimura, T. Nozaki, H. Harada, I. Shimada, and M. Osawa. (2015). Structural basis for the inhibition of voltage-dependent K⁺ channel by gating modifier toxin. Sci Rep 5: 14226.
Priest, B.T., K.M. Blumenthal, J.J. Smith, V.A. Warren, and M.M. Smith. (2007). ProTx-I and ProTx-II: gating modifiers of voltage-gated sodium channels. Toxicon 49: 194-201.
Ramracheya, R., C. Ward, M. Shigeto, J.N. Walker, S. Amisten, Q. Zhang, P.R. Johnson, P. Rorsman, and M. Braun. (2010). Membrane potential-dependent inactivation of voltage-gated ion channels in α-cells inhibits glucagon secretion from human islets. Diabetes 59: 2198-2208.
Smith, J.J., T.R. Cummins, S. Alphy, K.M. Blumenthal. (2007). Molecular interactions of the gating modifier toxin ProTx-II with Na_v 1.5. J. Biol. Chem. 282.17; 12687-12697.
Takahashi, H., J.I. Kim, H.J. Min, K. Sato, K.J. Swartz, and I. Shimada. (2000). Solution structure of hanatoxin1, a gating modifier of voltage-dependent K⁺ channels: common surface features of gating modifier toxins. J. Mol. Biol. 297: 771-780.
Zhang, P.F., P. Chen, W.J. Hu, and S.P. Liang. (2003). Huwentoxin-V, a novel insecticidal peptide toxin from the spider Selenocosmia huwena, and a natural mutant of the toxin: indicates the key amino acid residues related to the biological activity. Toxicon. 42: 15-20.
Examples:
TC#	Name	Organismal Type	Example
8.B.5.1.1	Processed protoxin-1 (ProTx1; ProTx-I) of 35 aas. Inhibits voltage-gated calcium channels Cav3.1/CACNA1G, voltage-gated potassium channels Kv2.1/KCNB1 and all sodium channels tested (Nav1.2/SCN2A, Nav1.5/SCN5A, Nav1.7/SCN9A, and Nav1.8/SCN10A). Shifts the voltage-dependence of channel activation to more positive potentials. Most potent against Nav1.8/SCN10A (Middleton et al. 2002; Priest et al. 2007). A hydrophobic patch on the ProTx-II surface anchors it at the cell surface in a position that optimizes interaction of the peptide with the binding site on the voltage sensor domain. Binding of ProTx-II to the lipid membrane is directly linked to its potency as a hNaV1.7 channel inhibitor (Henriques et al. 2016).	Tarantulas	*ProTx1 of Thrixopelma pruriens* (P83480)**

Examples:
TC#	Name	Organismal Type	Example
8.B.5.2.1	Protoxin-2 (ProTx2; ProTx-II)	Tarantulas	*ProTx2 of Thrixopelma pruriens* (P83476)**

8.B.5.2.2	κ-Sparatoxin-Hv1b or heteropodatoxin 2 of 30 aas; It is an inhibitor of voltage-gated potassium channels of the Kv4/KCND family (Ramracheya et al. 2010). Inhibition of Kv4.3/KCND3 and Kv4.2/KCND2 is strongly voltage-dependent, while inhibition of Kv4.1/KCND1 shows less voltage-dependence. Its binding site may be near the potassium channel voltage sensor. This toxin also blocks calcium channels.	Animals (spiders)	heteropda toxin 2 of Heteropoda venatoria (Brown huntsman spider) (Aranea venatoria)

Examples:
TC#	Name	Organismal Type	Example
8.B.5.3.1	Vanillotoxin-1 (VaTx1) of Trinidad chevron tarantula (35 aas) (activates TRPV1 causing pain) (Cramer and McIntyre, 2008)	Spiders	*VaTx1 of Psalmopoeus cambridgei* (P0C244)**

8.B.5.3.2	Vanillotoxin-3 (VaTx3) (34 aas; 50% identical to VATx1) (activates TRPV1 causing pain) (Cramer and McIntyre, 2008)	Spiders	*VaTx3 of Psalmopoeus cambridgei* (P0C246)**

8.B.5.3.3	Heteroscodratoxin-1 (HMTx1) of the tarantula (35 aas) (blocks the voltage-gated K⁺ channels: K_v2.1 (KCNB1), K_v2.2 (KCNB2), K_v4.1 (KCND1), K_v4.2 (KCND2) and K_v4.3 (KCND3) causing convulsions and death in mice (Escoubas et al, 2002))	Spiders	*HMTx1 of Heteroscodra maculata* (P60992)**

8.B.5.3.4	*Huwentoxin-5 (HwTx5) precursor (86 aas) (insecticidal neurotoxin, Knottin-type; Diao et al., 2003; Zhang et al., 2003)*	Spiders	*HwTx5 of Ornithoctonus huwena* (P61104)**

8.B.5.3.5	*Jingzhaotoxin-11 (JzTx11) precursor (86 aas) (Inhibits K⁺ and Na⁺ channels (e.g., K_v2.1, K_v4.1, K_v4.2, and K_v1.5)(Structure known: Liao et al., 2006))*	Spiders	*JzTx11 of Chilobrachys jingzhao* (P0C247)**

8.B.5.3.6	*Hantotoxin-1 (HATx1) (35 aas) (Inhibits K⁺ and Ca²⁺ channels, e.g., K_v2.1, K_v4.2 and Ca_v2.1); (Structure known: Takahashi et al., 2000)*	Spiders	*Hanatoxin-1 of Grammostola rosea* (tarantula) (P56852)**

8.B.5.3.7	ω-grammotoxin, SIA. Blocks P/Q type voltage-dependent calcium channels, Ca_v2.1 (Ono et al., 2011). ω-Grammotoxin-SIA (GrTX-SIA) was originally isolated from the venom of the Chilean rose tarantula and shown to function as a gating modifier of voltage-gated Ca2+ (CaV) channels. It also inhibits voltage-gated K⁺ (KV) channel currents via a similar mechanism that involved binding to a conserved S3-S4 region in the voltage-sensing domains (VSDs). Since voltage-gated Na⁺ (NaV) channels contain homologous structural motifs, It might also inhibit members of this ion channel family as well. Collaço et al. 2024 showed that GrTX-SIA can impede the gating process of multiple NaV channel subtypes with NaV1.6 being the most susceptible target. Molecular docking of GrTX-SIA onto NaV1.6, supported by a p.E1607K mutation, revealed the voltage sensor in domain IV (VSDIV) as a primary site of action. The biphasic manner in which current inhibition appeared to occur suggested a second, possibly lower-sensitivity binding locus, which was identified as VSDII by using KV2.1/NaV1.6 chimeric voltage-sensor constructs. Subsequently, the NaV1.6p.E782K/p.E838K (VSDII), NaV1.6p.E1607K (VSDIV), and particularly the combined VSDII/VSDIV mutant lost virtually all susceptibility to GrTX-SIA. Together with existing literature, these results suggest that GrTX-SIA recognizes modules in NaV channel VSDs that are conserved among ion channel families, thereby allowing it to act as a comprehensive ion channel gating modifier peptide (Collaço et al. 2024).	Tarantula	*SIA of Grammostola rosea* (P60590)**

8.B.5.3.8	K⁺ channel blocker, a gating modifier toxin that inhibits by binding to the voltage sensor domain (VSD) of various VIC superfamily members, VSTx1 of 62 aas and 1 TMS (Ozawa et al. 2015). Many spider-venom peptides function as gating modifiers by binding to the VSDs of voltage-gated channels and trapping them in a closed or open state (Lau et al. 2016). The toxin interacts with residues in an aqueous cleft formed between the extracellular S1-S2 and S3-S4 loops of the VSD whilst maintaining lipid interactions in the gaps formed between the S1-S4 and S2-S3 helices. The resulting network of interactions increases the energetic barrier to the conformational changes required for channel gating, and this is the mechanism by which gating modifier toxins inhibit voltage-gated ion channels (Lau et al. 2016).		VSTx1 of Grammostola rosea (Chilean rose tarantula) (Grammostola spatulata)