Solution Structure and Functional Characterization of SGTx1, a Modifier of Kv2.1 Channel Gating<sup>,</sup>

Lee, Chul Won; Kim, Sunghwan; Roh, Sook Young; Endoh, Hiroshi; Kodera, Yasuhiro; Maeda, Tadakazu; Kohno, Toshiyuki; Wang, Julia M; Swartz, Kenton J.; Kim, Jae Il

doi:10.1021/bi0353373

Cited by 101 publications

(103 citation statements)

References 53 publications

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“…ProTx-II and a related tarantula venom peptide, ProTx-I, inhibit sodium channels by a mechanism similar to that of the potassium channel gating modifiers hanatoxin and SGTx1 (Swartz and MacKinnon, 1997;Lee et al, 2004). Indeed, ProTx-I shares significant sequence homology with hanatoxin.…”

Section: Discussionmentioning

confidence: 99%

ProTx-II, a Selective Inhibitor of Na_V1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Schmalhofer

Calhoun²,

Burrows³

et al. 2008

Mol Pharmacol

298

344

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Voltage-gated sodium (Na V 1) channels play a critical role in modulating the excitability of sensory neurons, and human genetic evidence points to Na V 1.7 as an essential contributor to pain signaling. Human loss-of-function mutations in SCN9A, the gene encoding Na V 1.7, cause channelopathy-associated indifference to pain (CIP), whereas gain-of-function mutations are associated with two inherited painful neuropathies. Although the human genetic data make Na V 1.7 an attractive target for the development of analgesics, pharmacological proof-of-concept in experimental pain models requires Na V 1.7-selective channel blockers. Here, we show that the tarantula venom peptide ProTx-II selectively interacts with Na V 1.7 channels, inhibiting Na V 1.7 with an IC 50 value of 0.3 nM, compared with IC 50 values of 30 to 150 nM for other heterologously expressed Na V 1 subtypes. This subtype selectivity was abolished by a point mutation in DIIS3. It is interesting that application of ProTx-II to desheathed cutaneous nerves completely blocked the C-fiber compound action potential at concentrations that had little effect on A␤-fiber conduction. ProTx-II application had little effect on action potential propagation of the intact nerve, which may explain why ProTx-II was not efficacious in rodent models of acute and inflammatory pain. Mono-iodo-ProTx-II ( 125 I-ProTx-II) binds with high affinity (K d ϭ 0.3 nM) to recombinant hNa V 1.7 channels. Binding of 125 IProTx-II is insensitive to the presence of other well characterized Na V 1 channel modulators, suggesting that ProTx-II binds to a novel site, which may be more conducive to conferring subtype selectivity than the site occupied by traditional local anesthetics and anticonvulsants. Thus, the 125 I-ProTx-II binding assay, described here, offers a new tool in the search for novel Na V 1.7-selective blockers.Pain relief remains an important, currently unmet, medical need. Voltage-gated sodium channels play a critical role in modulating the excitability of most neurons, including nociceptive sensory neurons signaling pain. Despite the clinical use of systemically administered lidocaine to treat chronic pain since the 1950s (Kugelberg and Lindblom, 1959) and the approval of the weak sodium channel blocker carbamazepine for the treatment of trigeminal neuralgia (Campbell et al., 1966), several oral sodium channel blockers have failed to show efficacy in large clinical trials (Wallace et al., 2002;Vinik et al., 2007). The failure of drugs in the clinic may at least partially be attributed to the narrow therapeutic window of non-subtype-selective sodium channel blockers.Recently, three publications have shown that loss-of-function mutations in the sodium channel subtype Na V 1.7 are the cause for channelopathy-associated insensitivity to pain (CIP) (Cox et al., 2006;Ahmad et al., 2007;Goldberg et al., 2007). Furthermore, genetic linkage analysis has identified gain-of-function mutations in Na V 1.7 as the cause of inherited erythromelalgia (Yang et al., 2004;Han et al., ...

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Section: Discussionmentioning

confidence: 99%

ProTx-II, a Selective Inhibitor of Na_V1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Schmalhofer

Calhoun²,

Burrows³

et al. 2008

Mol Pharmacol

298

344

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“…Venom peptides of the same family have been difficult to crystallize, and the currently known structures of Huwenoxin-IV, Hanatoxin-I, Hainantoxin-IV, SGTx1, and GrTx (42,(45)(46)(47)(48)(49) were determined by NMR techniques. We utilized a different approach and generated a high affinity antibody with a low dissociation rate and obtained a high resolution x-ray crystal structure of 2670 in complex with the Fab fragment of this antibody (supplemental Table 1).…”

Section: Effects Of Post-translational Modification On Potency Andmentioning

confidence: 99%

Engineering Highly Potent and Selective Microproteins against Nav1.7 Sodium Channel for Treatment of Pain

Shcherbatko

Rossi

Foletti

et al. 2016

Journal of Biological Chemistry

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The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtypeselective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC 50 to Nav1.7 of 2.5 nM) and selective. We achieved 80-and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC 50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nM. The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.

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“…Wang et al (19) also found basic residues in SGTx that are critical for inhibition of K v 2.1, whereas mutation of acidic residues in fact increased the affinity of that toxin for its target. In the SGTx solution structure, the basic residues form a ring around the hydrophobic patch, presumably creating the active face (31). Because the solution structure for ProTx-II is unknown, we chose to construct a molecular model based upon the known solution structure of a similar ICK toxin that targets K v 4 channels, HpTx-2.…”

Section: Mutationmentioning

confidence: 99%

Molecular Interactions of the Gating Modifier Toxin ProTx-II with Nav1.5

Smith

Cummins

Alphy

et al. 2007

Journal of Biological Chemistry

104

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Voltage-gated Na؉ channels are critical components in the generation of action potentials in excitable cells, but despite numerous structure-function studies on these proteins, their gating mechanism remains unclear. Peptide toxins often modify channel gating, thereby providing a great deal of information about these channels. ProTx-II is a 30-amino acid peptide toxin from the venom of the tarantula, Thrixopelma pruriens, that conforms to the inhibitory cystine knot motif and which modifies 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 and, therefore, differs from the classic site 4 toxins that shift voltage dependence of activation in the opposite direction. Despite this difference in functional effects, ProTx-II has been proposed to bind to neurotoxin site 4 because it modifies activation. Here, we investigate the bioactive surface of ProTx-II by alanine-scanning the toxin and analyzing the interactions of each mutant with the cardiac isoform, Na v 1.5. The active face of the toxin is largely composed of hydrophobic and cationic residues, joining a growing group of predominantly K v channel gating modifier toxins that are thought to interact with the lipid environment. In addition, we performed extensive mutagenesis of Na v 1.5 to locate the receptor site with which ProTx-II interacts. Our data establish that, contrary to prior assumptions, ProTx-II does not bind to the previously characterized neurotoxin site 4, thus making it a novel probe of activation gating in Na v channels with potential to shed new light on this process.

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Solution Structure and Functional Characterization of SGTx1, a Modifier of Kv2.1 Channel Gating^,

Cited by 101 publications

References 53 publications

ProTx-II, a Selective Inhibitor of Na_V1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

ProTx-II, a Selective Inhibitor of Na_V1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Engineering Highly Potent and Selective Microproteins against Nav1.7 Sodium Channel for Treatment of Pain

Molecular Interactions of the Gating Modifier Toxin ProTx-II with Nav1.5

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Solution Structure and Functional Characterization of SGTx1, a Modifier of Kv2.1 Channel Gating,

Cited by 101 publications

References 53 publications

ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

ProTx-II, a Selective Inhibitor of NaV1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

Engineering Highly Potent and Selective Microproteins against Nav1.7 Sodium Channel for Treatment of Pain

Molecular Interactions of the Gating Modifier Toxin ProTx-II with Nav1.5

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About

Solution Structure and Functional Characterization of SGTx1, a Modifier of Kv2.1 Channel Gating^,

ProTx-II, a Selective Inhibitor of Na_V1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors

ProTx-II, a Selective Inhibitor of Na_V1.7 Sodium Channels, Blocks Action Potential Propagation in Nociceptors