The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Na<sub>v1.6</sub>voltage-dependent sodium channel

Rosker, Christian; Lohberger, Birgit; Hofer, Doris; Steinecker, Bibiane; Quasthoff, Stefan; Schultze-Seemann, Wolfgang

doi:10.1152/ajpcell.00070.2007

Cited by 114 publications

(113 citation statements)

References 32 publications

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“…In contrast, the residual current after CnTX blockade was insensitive to acute hypoxia, SUMO1 and SENP1 (Figure 1g), suggesting that other Na V channels did not contribute significantly to the hypoxic response. Supporting the notion that most of the residual current is passed by Na V 1.6, 50 nm 4,9 anhydrotetrodotoxin (AnTTX), a toxin that blocks Na V 1.6~150-fold more effectively than Na V 1.2 (Rosker et al, 2007), inhibited~10% of I Na under control conditions and >90% of the current remaining after CnTX application (Figure 1e,f). Moreover, with SENP1 in the pipette, the fraction of I Na blocked by CnTX was decreased and suppression by AnTTX was increased (Figure 1h), the expected response if deSUMOylation decreased the activity of Na V 1.2 channels so that Na V 1.6 channels passed a larger fraction of the current.…”

Section: Namentioning

confidence: 66%

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Plant

Marks

Goldstein

2017

Biophysical Journal

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The mechanism for the earliest response of central neurons to hypoxia-an increase in voltage-gated sodium current (I Na )-has been unknown. Here, we show that hypoxia activates the Small Ubiquitin-like Modifier (SUMO) pathway in rat cerebellar granule neurons (CGN) and that SUMOylation of Na V 1.2 channels increases I Na . The time-course for SUMOylation of single Na V 1.2 channels at the cell surface and changes in I Na coincide, and both are prevented by mutation of Na V 1.2-Lys38 or application of a deSUMOylating enzyme. Within 40 s, hypoxia-induced linkage of SUMO1 to the channels is complete, shifting the voltage-dependence of channel activation so that depolarizing steps evoke larger sodium currents. Given the recognized role of I Na in hypoxic brain damage, the SUMO pathway and Na V 1.2 are identified as potential targets for neuroprotective interventions.

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Section: Namentioning

confidence: 66%

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Plant

Marks

Goldstein

2017

Biophysical Journal

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“…We were unable to reproduce the subtype-specific action of 4,9-anhydroTTX (4) (Rosker et al, 2007). We verified the purity of synthetic 4,9-anhydroTTX (4) via NMR (Ohyabu et al, 2003;Nishikawa et al, 2004;Urabe et al, 2006) and used it to treat HEK293T cells expressing a single VSSC subtype; however, we observed that the working solution containing 100 nM 4,9-anhydroTTX (4) caused inhibition of Na v 1.6 (Supporting Information Figure S4) when it was stored for half a year, implying that the production of TTX (1) and 4-epiTTX occurred, probably attributable to an equilibrium between TTX (1), 4-epiTTX and 4,9-anhydroTTX (4), which may not support Rosker's results.…”

Section: Discussionmentioning

confidence: 99%

“…The IC 50 value of either of the two compounds for each subtype of VSSC was approximately three orders of magnitude larger than that of TTX (1). Rosker et al (2007) showed that 4,9-anhydroTTX (4) selectively inhibits Na v 1.6 with an IC 50 value of 7.8 ± 2.3 nM but does not affect other subtypes. Teramoto et al revealed the inhibition of a resurgent-like current through Na v 1.6 expressed in mouse vas deferens myocytes (Teramoto et al, 2012;Teramoto and Yotsu-Yamashita, 2015).…”

Section: Inhibition Of the Vssc Subtypes By Ttx And Its Analoguesmentioning

confidence: 99%

Differential binding of tetrodotoxin and its derivatives to voltage‐sensitive sodium channel subtypes (Na_v1.1 to Na_v1.7)

Tsukamoto

Chiba

Wakamori

et al. 2017

British J Pharmacology

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BACKGROUND AND PURPOSEThe development of subtype-selective ligands to inhibit voltage-sensitive sodium channels (VSSCs) has been attempted with the aim of developing therapeutic compounds. Tetrodotoxin (TTX) is a toxin from pufferfish that strongly inhibits VSSCs. Many TTX analogues have been identified from marine and terrestrial sources, although their specificity for particular VSSC subtypes has not been investigated. Herein, we describe the binding of 11 TTX analogues to human VSSC subtypes Na v 1.1-Na v 1.7. EXPERIMENTAL APPROACHEach VSSC subtype was transiently expressed in HEK293T cells. The inhibitory effects of TTX analogues on each subtype were assessed using whole-cell patch-clamp recordings.

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“…The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11)(12)(13)(14)(15)(16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5,6,(21)(22)(23). The lack of structural information motivates a comprehensive, systematic study of toxin-protein interactions.…”

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confidence: 99%

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7

Thomas‐Tran

Bois

2016

Proc. Natl. Acad. Sci. U.S.A.

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Improper function of voltage-gated sodium channels (Na V s), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na V s hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na V through a systematic structure-activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na V 1.7 (hNa V 1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na V inhibitors.sodium channel | guanidinium toxin | mutant cycle analysis M odulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na V s) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na V 1.1-Na V 1.9, Na x ) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na V s have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1-4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na V isoforms with nanomolar affinity (5-7). Guanidinium toxins inhibit Na + influx through Na V s by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8-10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na V has been solved to date (11-16). Na V homology models, constructed based on X-ray analyses of prokaryotic Na + and K + voltage-gated channels, do not sufficiently account for experimental structure-activity relationship (SAR) data (6,(17)(18)(19)(20), and the molecular details underlying distinct differences in toxin potencies toward individual Na V subtypes remain undefined (5, 6, 21-23)....

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The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Na_v1.6voltage-dependent sodium channel

Cited by 114 publications

References 32 publications

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Differential binding of tetrodotoxin and its derivatives to voltage‐sensitive sodium channel subtypes (Na_v1.1 to Na_v1.7)

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7

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The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Nav1.6voltage-dependent sodium channel

Cited by 114 publications

References 32 publications

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Sumoylation of Na V 1.2 Channels Mediates the Early Response to Acute Hypoxia in Central Neurons

Differential binding of tetrodotoxin and its derivatives to voltage‐sensitive sodium channel subtypes (Nav1.1 to Nav1.7)

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa V 1.7

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The TTX metabolite 4,9-anhydro-TTX is a highly specific blocker of the Na_v1.6voltage-dependent sodium channel

Differential binding of tetrodotoxin and its derivatives to voltage‐sensitive sodium channel subtypes (Na_v1.1 to Na_v1.7)

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNa _V 1.7