Evidence for multiple effects of ProTxII on activation gating in NaV1.5

Edgerton, Gabrielle B.; Blumenthal, Kenneth M.; Hanck, Dorothy A.

doi:10.1016/j.toxicon.2008.06.023

Cited by 18 publications

(22 citation statements)

References 27 publications

(35 reference statements)

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“…ProTx-II has complex actions on VGSC currents (Edgerton et al, 2008) and the molecular determinants of these actions have been elusive and controversial (Bosmans et al, 2006(Bosmans et al, , 2008Smith et al, 2007;Schmalhofer et al, 2008;Sokolov et al, 2008a;Xiao et al, 2010). Our data show that ProTx-II substantially modulates the conformation of both the DI and DII voltage-sensors in Na v 1.5.…”

Section: Discussionmentioning

confidence: 57%

Gating-Pore Currents Demonstrate Selective and Specific Modulation of Individual Sodium Channel Voltage-Sensors by Biological Toxins

2014

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Voltage-gated sodium channels are critical determinants of nerve and muscle excitability. Although numerous toxins and small molecules target sodium channels, identifying the mechanisms of action is challenging. Here we used gating-pore currents selectively generated in each of the voltage-sensors from the four a-subunit domains (DI-DIV) to monitor the activity of individual voltage-sensors and to investigate the molecular determinants of sodium channel pharmacology. The tarantula toxin huwentoxin-IV (HWTX-IV), which inhibits sodium channel current, exclusively enhanced inward gating-pore currents through the DII voltagesensor. By contrast, the tarantula toxin ProTx-II, which also inhibits sodium channel currents, altered the gating-pore currents in multiple voltage-sensors in a complex manner. Thus, whereas HWTX-IV inhibits central-pore currents by selectively trapping the DII voltage-sensor in the resting configuration, ProTx-II seems to inhibit central-pore currents by differentially altering the configuration of multiple voltage-sensors. The sea anemone toxin anthopleurin B, which impairs open-channel inactivation, exclusively enhanced inward gating-pore currents through the DIV voltage-sensor. This indicates that trapping the DIV voltagesensor in the resting configuration selectively impairs openchannel inactivation. Furthermore, these data indicate that although activation of all four voltage-sensors is not required for central-pore current generation, activation of the DII voltagesensor is crucial. Overall, our data demonstrate that gating-pore currents can determine the mechanism of action for sodium channel gating modifiers with high precision. We propose this approach could be adapted to identify the molecular mechanisms of action for gating modifiers of various voltage-gated ion channels.

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Section: Discussionmentioning

confidence: 57%

Gating-Pore Currents Demonstrate Selective and Specific Modulation of Individual Sodium Channel Voltage-Sensors by Biological Toxins

2014

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“…45) that are thought to act through the membrane (25,46) and two scorpion toxins [AaHII (47) and TsVII (48)] for which the membrane interaction is unclear (12,46,49). Each of these toxins target one or more of the voltage sensors in rNav1.2a, and collectively they interact with all four (12,28,(50)(51)(52)(53)(54) (Fig. S2A).…”

Section: Resultsmentioning

confidence: 99%

Palmitoylation influences the function and pharmacology of sodium channels

Bosmans

Milescu

Swartz

2011

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

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Palmitoylation is a common lipid modification known to regulate the functional properties of various proteins and is a vital step in the biosynthesis of voltage-activated sodium (Nav) channels. We discovered a mutation in an intracellular loop of rNav1.2a (G1079C), which results in a higher apparent affinity for externally applied PaurTx3 and ProTx-II, two voltage sensor toxins isolated from tarantula venom. To explore whether palmitoylation of the introduced cysteine underlies this observation, we compared channel susceptibility to a range of animal toxins in the absence and presence of 2-Br-palmitate, a palmitate analog that prevents palmitate incorporation into proteins, and found that palmitoylation contributes to the increased affinity of PaurTx3 and ProTx-II for G1079C. Further investigations with 2-Br-palmitate revealed that palmitoylation can regulate the gating and pharmacology of wild-type (wt) rNav1.2a. To identify rNav1.2a palmitoylation sites contributing to these phenomena, we substituted three endogenous cysteines predicted to be palmitoylated and found that the gating behavior of this triple cysteine mutant is similar to wt rNav1.2a treated with 2-Br-palmitate. As with chemically depalmitoylated rNav1.2a channels, this mutant also exhibits an increased susceptibility for PaurTx3. Additional mutagenesis experiments showed that palmitoylation of one cysteine in particular (C1182) primarily influences PaurTx3 sensitivity and may enhance the inactivation process of wt rNav1.2a. Overall, our results demonstrate that lipid modifications are capable of altering the gating and pharmacological properties of rNav1.2a.ecause of their essential role in generating and propagating action potentials in excitable tissues (1-3), voltage-activated sodium (Nav) channels are a primary target of drugs (4, 5) and toxins found in animal venoms (6-8). As a result, Nav channel activity can be influenced by a variety of naturally occurring molecules, with the voltage sensors in all four domains representing the foremost target of tarantula toxins (9). The conventional view is that animal toxins interact with ion channel voltage sensors through direct protein-protein interactions (10-13). However, crystallographic and functional studies on voltage-activated potassium (Kv) channels have revealed an important role for the lipid membrane in regulating how Kv channels gate in response to changes in voltage (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In addition, recent reports show that tarantula toxins can interact with Kv channel voltage sensors by partitioning into the membrane and binding to S3b-S4 paddle motifs at the protein-lipid interface (23-27). The discovery that several of these toxins also interact with paddle motifs found in Nav channels implies that partitioning can enable tarantula toxins to interact with voltage sensors in this family of ion channels as well (25,28). Therefore, an emerging concept in the ion channel field is that the toxin pharmacology of an ion channel is not only determined by ligand-p...

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“…Such complex modifications suggest a multimode of action that could be related to "multifaceted," i.e. involving several binding sites, toxin-channel interaction (58). Recently, the gating modification of K v channel chimeras by VSTx1 was proven to be a side effect of the membrane partitioning of this toxin, rather than dependent on direct toxin-channel interaction (59).…”

Section: Discussionmentioning

confidence: 99%

Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels

Redaelli

Cassulini

Silva

et al. 2010

Journal of Biological Chemistry

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Venom-derived peptide modulators of ion channel gating are regarded as essential tools for understanding the molecular motions that occur during the opening and closing of ion channels. In this study, we present the characterization of five spider toxins on 12 human voltage-gated ion channels, following observations about the target promiscuity of some spider toxins and the ongoing revision of their "canonical" gating-modifying mode of action. The peptides were purified de novo from the venom of Grammostola rosea tarantulas, and their sequences were confirmed by Edman degradation and mass spectrometry analysis. Their effects on seven tetrodotoxin-sensitive Na ؉ channels, the three human ether-à-go-go (hERG)-related K ؉ channels, and two human Shaker-related K ؉ channels were extensively characterized by electrophysiological techniques. All the peptides inhibited ion conduction through all the Na ؉ channels tested, although with distinctive patterns. The peptides also affected the three pharmaceutically relevant hERG isoforms differently. At higher concentrations, all peptides also modified the gating of the Na ؉ channels by shifting the activation to more positive potentials, whereas more complex effects were recorded on hERG channels. No effects were evident on the two Shaker-related K ؉ channels at concentrations well above the IC 50 value for the affected channels. Given the sequence diversity of the tested peptides, we propose that tarantula toxins should be considered both as multimode and target-promiscuous ion channel modulators; both features should not be ignored when extracting mechanistic interpretations about ion channel gating. Our observations could also aid in future structure-function studies and might help the development of novel ion channel-specific drugs.

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Evidence for multiple effects of ProTxII on activation gating in NaV1.5

Cited by 18 publications

References 27 publications

Gating-Pore Currents Demonstrate Selective and Specific Modulation of Individual Sodium Channel Voltage-Sensors by Biological Toxins

Gating-Pore Currents Demonstrate Selective and Specific Modulation of Individual Sodium Channel Voltage-Sensors by Biological Toxins

Palmitoylation influences the function and pharmacology of sodium channels

Target Promiscuity and Heterogeneous Effects of Tarantula Venom Peptides Affecting Na+ and K+ Ion Channels

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