Yucheng Xiao

Tarantula huwentoxin-IV inhibits neuronal sodium channels by binding to receptor site 4 and trapping the domain ii voltage sensor in the closed configuration.

Peptide toxins with high affinity, divergent pharmacological functions, and isoform-specific selectivity are powerful tools for investigating the structure-function relationships of voltage-gated sodium channels (VGSCs). Although a number of interesting inhibitors have been reported from tarantula venoms, little is known about the mechanism for their interaction with VGSCs. We show that huwentoxin-IV (HWTX-IV), a 35-residue peptide from tarantula Ornithoctonus huwena venom, preferentially inhibits neuronal VGSC subtypes rNav1.2, rNav1.3, and hNav1.7 compared with muscle subtypes rNav1.4 and hNav1.5. Of the five VGSCs examined, hNav1.7 was most sensitive to HWTX-IV (IC50 ∼ 26 nm). Following application of 1 μm HWTX-IV, hNav1.7 currents could only be elicited with extreme depolarizations (>+100 mV). Recovery of hNav1.7 channels from HWTX-IV inhibition could be induced by extreme depolarizations or moderate depolarizations lasting several minutes. Site-directed mutagenesis analysis indicated that the toxin docked at neurotoxin receptor site 4 located at the extracellular S3-S4 linker of domain II. Mutations E818Q and D816N in hNav1.7 decreased toxin affinity for hNav1.7 by ∼300-fold, whereas the reverse mutations in rNav1.4 (N655D/Q657E) and the corresponding mutations in hNav1.5 (R812D/S814E) greatly increased the sensitivity of the muscle VGSCs to HWTX-IV. Our data identify a novel mechanism for sodium channel inhibition by tarantula toxins involving binding to neurotoxin receptor site 4. In contrast to scorpion β-toxins that trap the IIS4 voltage sensor in an outward configuration, we propose that HWTX-IV traps the voltage sensor of domain II in the inward, closed configuration.

Voltage-gated sodium channel in grasshopper mice defends against bark scorpion toxin.

Bite Me! As unpleasant as it is, pain serves a purpose, to alert the body to potential damage. This protective function may explain why few predators have evolved resistance to the painful venoms used as a defense by their prey. The grasshopper mouse, however, is insensitive to one of the most painful stings in the animal kingdom—that of the bark scorpion. Rowe et al. (p. 441; see the Perspective by Lewin) now show that grasshopper mice use the toxins present in the scorpion venom to block voltage-gated pain transmission, temporarily reducing their sensitivity to nonvenom-induced pain. Thus, grasshopper mice use the scorpions painful defense to their advantage and have evolved a mechanism that allows for reduction of pain sensitivity only when it is needed. Grasshopper mice use the scorpion’s strength against it—instead of inducing pain, scorpion venom blocks pain. [Also see Perspective by Lewin] Painful venoms are used to deter predators. Pain itself, however, can signal damage and thus serves an important adaptive function. Evolution to reduce general pain responses, although valuable for preying on venomous species, is rare, likely because it comes with the risk of reduced response to tissue damage. Bark scorpions capitalize on the protective pain pathway of predators by inflicting intensely painful stings. However, grasshopper mice regularly attack and consume bark scorpions, grooming only briefly when stung. Bark scorpion venom induces pain in many mammals (house mice, rats, humans) by activating the voltage-gated Na+ channel Nav1.7, but has no effect on Nav1.8. Grasshopper mice Nav1.8 has amino acid variants that bind bark scorpion toxins and inhibit Na+ currents, blocking action potential propagation and inducing analgesia. Thus, grasshopper mice have solved the predator-pain problem by using a toxin bound to a nontarget channel to block transmission of the pain signals the venom itself is initiating.

CRMP2 Protein SUMOylation Modulates NaV1.7 Channel Trafficking

Background: Post-translational modifications of CRMP2 protein direct its regulation of effector proteins. Results: Destruction of a CRMP2 SUMOylation site reduces surface expression and current density of sodium channel NaV1.7. Conclusion: CRMP2 SUMOylation choreographs NaV1.7, but not NaV1.1 or NaV1.3, trafficking. Significance: Learning how neuronal NaV1.7 trafficking is modulated by CRMP2 is important for understanding the mechanism of action of NaV-targeted anti-epileptic and anti-nociceptive drugs. Voltage-gated sodium channel (NaV) trafficking is incompletely understood. Post-translational modifications of NaVs and/or auxiliary subunits and protein-protein interactions have been posited as NaV-trafficking mechanisms. Here, we tested if modification of the axonal collapsin response mediator protein 2 (CRMP2) by a small ubiquitin-like modifier (SUMO) could affect NaV trafficking; CRMP2 alters the extent of NaV slow inactivation conferred by the anti-epileptic (R)-lacosamide, implying NaV-CRMP2 functional coupling. Expression of a CRMP2 SUMOylation-incompetent mutant (CRMP2-K374A) in neuronal model catecholamine A differentiated (CAD) cells did not alter lacosamide-induced NaV slow inactivation compared with CAD cells expressing wild type CRMP2. Like wild type CRMP2, CRMP2-K374A expressed robustly in CAD cells. Neurite outgrowth, a canonical CRMP2 function, was moderately reduced by the mutation but was still significantly higher than enhanced GFP-transfected cortical neurons. Notably, huwentoxin-IV-sensitive NaV1.7 currents, which predominate in CAD cells, were significantly reduced in CAD cells expressing CRMP2-K374A. Increasing deSUMOylation with sentrin/SUMO-specific protease SENP1 or SENP2 in wild type CRMP2-expressing CAD cells decreased NaV1.7 currents. Consistent with a reduction in current density, biotinylation revealed a significant reduction in surface NaV1.7 levels in CAD cells expressing CRMP2-K374A; surface NaV1.7 expression was also decreased by SENP1 + SENP2 overexpression. Currents in HEK293 cells stably expressing NaV1.7 were reduced by CRMP2-K374A in a manner dependent on the E2-conjugating enzyme Ubc9. No decrement in current density was observed in HEK293 cells co-expressing CRMP2-K374A and NaV1.1 or NaV1.3. Diminution of sodium currents, largely NaV1.7, was recapitulated in sensory neurons expressing CRMP2-K374A. Our study elucidates a novel regulatory mechanism that utilizes CRMP2 SUMOylation to choreograph NaV1.7 trafficking.

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

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 α-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 voltage-sensor. 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 voltage-sensor in the resting configuration selectively impairs open-channel 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 voltage-sensor 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.

Isolation and Characterization of CvIV4: A Pain Inducing α- Scorpion Toxin

Background Among scorpion species, the Buthidae produce the most deadly and painful venoms. However, little is known regarding the venom components that cause pain and their mechanism of action. Using a paw-licking assay (Mus musculus), this study compared the pain-inducing capabilities of venoms from two species of New World scorpion (Centruroides vittatus, C. exilicauda) belonging to the neurotoxin-producing family Buthidae with one species of non-neurotoxin producing scorpion (Vaejovis spinigerus) in the family Vaejovidae. A pain-inducing α-toxin (CvIV4) was isolated from the venom of C. vittatus and tested on five Na+ channel isoforms. Principal Findings C. vittatus and C. exilicauda venoms produced significantly more paw licking in Mus than V. spinigerus venom. CvIV4 produced paw licking in Mus equivalent to the effects of whole venom. CvIV4 slowed the fast inactivation of Nav1.7, a Na+ channel expressed in peripheral pain-pathway neurons (nociceptors), but did not affect the Nav1.8-based sodium currents of these neurons. CvIV4 also slowed the fast inactivation of Nav1.2, Nav1.3 and Nav1.4. The effects of CvIV4 are similar to Old World α-toxins that target Nav1.7 (AahII, BmK MI, LqhIII, OD1), however the primary structure of CvIV4 is not similar to these toxins. Mutant Nav1.7 channels (D1586A and E1589Q, DIV S3–S4 linker) reduced but did not abolish the effects of CvIV4. Conclusions This study: 1) agrees with anecdotal evidence suggesting that buthid venom is significantly more painful than non-neurotoxic venom; 2) demonstrates that New World buthids inflict painful stings via toxins that modulate Na+ channels expressed in nociceptors; 3) reveals that Old and New World buthids employ similar mechanisms to produce pain. Old and New World α-toxins that target Nav1.7 have diverged in sequence, but the activity of these toxins is similar. Pain-inducing toxins may have evolved in a common ancestor. Alternatively, these toxins may be the product of convergent evolution.

Alternative splicing of Na V 1.7 exon 5 increases the impact of the painful pepD mutant channel I1461T

Alternative splicing is known to alter pharmacological sensitivities, kinetics, channel distribution under pathological conditions, and developmental regulation of VGSCs. Mutations that alter channel properties in NaV1.7 have been genetically implicated in patients with bouts of extreme pain classified as inherited erythromelalgia (IEM) or paroxysmal extreme pain disorder (PEPD). Furthermore, patients with IEM or PEPD report differential age onsets. A recent study reported that alternative splicing of NaV1.7 exon 5 affects ramp current properties. Since IEM and PEPD mutations also alter NaV1.7 ramp current properties we speculated that alternative splicing might impact the functional consequences of IEM or PEPD mutations. We compared the effects alternative splicing has on the biophysical properties of NaV1.7 wild-type, IEM (I136V), and PEPD (I1461T) channels. Our major findings demonstrate that although the 5A splice variant of the IEM channel had no functional impact, the 5A splice variant of the PEPD channel significantly hyperpolarized the activation curve, slowed deactivation and closed-state inactivation, shifted the ramp current activation to more hyperpolarized potentials, and increased ramp current amplitude. We hypothesize a D1/S3-S4 charged residue difference between the 5N (Asn) and the 5A (Asp) variants within the coding region of exon 5 may contribute to shifts in channel activation and deactivation. Taken together, the additive effects observed on ramp currents from exon 5 splicing and the PEPD mutation (I1461T) are likely to impact the disease phenotype and may offer insight into how alternative splicing may affect specific intramolecular interactions critical for voltage-dependent gating.

The human Nav1.5 F1486 deletion associated with long QT syndrome leads to impaired sodium channel inactivation and reduced lidocaine sensitivity.

•  We investigated how the F1486 deletion LQT3 mutation impairs the functional properties of the human cardiac voltage‐gated sodium channel (hNav1.5) and alters action potential firing. •  Voltage‐clamp recordings from HEK 293 cells and cardiomyocytes expressing recombinant channels demonstrated that the F1486del mutation reduces peak current density but also impairs inactivation and increases late current density. •  Current‐clamp recordings from cardiomyocytes indicated that the increase in late current density would result in prolonged action potential duration and this was confirmed using computer simulations. •  The deletion of F1486 abolished the ability of lidocaine to stabilize the inactivated state and eliminated the high‐affinity binding of lidocaine to inactivated channels. •  Our data show that the hNav1.5‐F1486del mutation has complex functional consequences and indicate that knowledge of the specific molecular defect is critical when developing potential treatments for individuals with prolonged QT intervals.

Substituted N-(biphenyl-4'-yl)methyl (R)-2-acetamido-3-methoxypropionamides: potent anticonvulsants that affect frequency (use) dependence and slow inactivation of sodium channels.

We prepared 13 derivatives of N-(biphenyl-4′-yl)methyl (R)-2-acetamido-3-methoxypropionamide that differed in type and placement of a R-substituent in the terminal aryl unit. We demonstrated that the R-substituent impacted the compound’s whole animal and cellular pharmacological activities. In rodents, select compounds exhibited excellent anticonvulsant activities and protective indices (PI = TD50/ED50) that compared favorably with clinical antiseizure drugs. Compounds with a polar, aprotic R-substituent potently promoted Na+ channel slow inactivation and displayed frequency (use) inhibition of Na+ currents at low micromolar concentrations. The possible advantage of affecting these two pathways to decrease neurological hyperexcitability is discussed.