Functional characterization and analgesic effects of mixed cannabinoid receptor/T-type channel ligands

Background

Increased neuronal excitability and spontaneous firing are hallmark characteristics of injured sensory neurons. Changes in expression of various voltage-gated Na⁺ channels (VGSCs) have been observed under neuropathic conditions and there is evidence for the involvement of protein kinase C (PKC) in sensory hyperexcitability. Here we demonstrate the contribution of PKC to P2X-evoked VGSC activation in dorsal root ganglion (DRG) neurons in neuropathic conditions.

Results

Using the spinal nerve ligation (SNL) model of neuropathic pain and whole-cell patch clamp recordings of dissociated DRG neurons, we examined changes in excitability of sensory neurons after nerve injury and observed that P2X3 purinoceptor-mediated currents induced by α,β-meATP triggered activation of TTX-sensitive VGSCs in neuropathic nociceptors only. Treatment of neuropathic DRGs with the PKC blocker staurosporine or calphostin C decreased the α,β-meATP-induced Na⁺ channels activity and reversed neuronal hypersensitivity. In current clamp mode, α,β-meATP was able to evoke action-potentials more frequently in neuropathic neurons than in controls. Pretreatment with calphostin C significantly decreased the proportion of sensitized neurons that generated action potentials in response to α,β-meATP. Recordings measuring VGSC activity in neuropathic neurons show significant change in amplitude and voltage dependence of sodium currents. In situ hybridization data indicate a dramatic increase in expression of embryonic Na_v1.3 channels in neuropathic DRG neurons. In a CHO cell line stably expressing the Na_v1.3 subunit, PKC inhibition caused both a significant shift in voltage-dependence of the channel in the depolarizing direction and a decrease in current amplitude.

Conclusion

Neuropathic injury causes primary sensory neurons to become hyperexcitable to ATP-evoked P2X receptor-mediated depolarization, a phenotypic switch sensitive to PKC modulation and mediated by increased activity of TTX-sensitive VGSCs. Upregulation in VGSC activity after injury is likely mediated by increased expression of the Na_v1.3 subunit, and the function of the Na_v1.3 channel is regulated by PKC.     

The cytochrome P450 inhibitor, ketoconazole, inhibits oxidized linoleic acid metabolite-mediated peripheral inflammatory pain

Background

Painful neuropathy is a common complication of diabetes. Previous studies have identified significant increases in the amount of voltage gated sodium channel isoforms Na_V1.7 and Na_V1.3 protein in the dorsal root ganglia (DRG) of rats with streptozotocin (STZ)-induced diabetes. We found that gene transfer-mediated release of the inhibitory neurotransmitters enkephalin or gamma amino butyric acid (GABA) from DRG neurons in diabetic animals reduced pain-related behaviors coincident with a reduction in Na_V1.7 protein levels in DRG in vivo. To further evaluate the role of Na_V?? subunit levels in DRG in the pathogenesis of pain in diabetic neuropathy, we constructed a non-replicating herpes simplex virus (HSV)-based vector expressing a microRNA (miRNA) against Na_V?? subunits.

Results

Subcutaneous inoculation of the miRNA-expressing HSV vector into the feet of diabetic rats to transduce DRG resulted in a reduction in Na_V?? subunit levels in DRG neurons, coincident with a reduction in cold allodynia, thermal hyperalgesia and mechanical hyperalgesia.

Conclusions

These data support the role of increased Na_V?? protein in DRG in the pathogenesis of pain in diabetic neuropathy, and provide a proof-of-principle demonstration for the development of a novel therapy that could be used to treat intractable pain in patients with diabetic neuropathy.     

Prostaglandin E2 Promotes Nav1.8 Trafficking via Its Intracellular RRR Motif Through the Protein Kinase A Pathway

Voltage‐gated sodium channels (Na_v) are essential for the initiation and propagation of action potentials in neurons. Na_v1.8 activity is regulated by prostaglandin E₂ (PGE₂). There is, however, no direct evidence showing the regulated trafficking of Na_v1.8, and the molecular and cellular mechanism of PGE₂‐induced sodium channel trafficking is not clear. Here, we report that PGE₂ regulates the trafficking of Na_v1.8 through the protein kinase A (PKA) signaling pathway, and an RRR motif in the first intracellular loop of Na_v1.8 mediates this effect. In rat dorsal root ganglion (DRG) neurons, prolonged PGE₂ treatment enhanced Na_v1.8 currents by increasing the channel density on the cell surface. Activation of PKA by forskolin had the same effect on DRG neurons and human embryonic kidney 293T cells expressing Na_v1.8. Inhibition of PKA completely blocked the PGE₂‐promoted effect on Na_v1.8. Mutation of five PKA phosphorylation sites or the RRR motif in the first intracellular loop of Na_v1.8 abolished the PKA‐promoted Na_v1.8 surface expression. Furthermore, a membrane‐tethered peptide containing the intracellular RRR motif disrupted the PGE₂‐induced promotion of the Na_v1.8 current in DRG neurons. Our data indicate that PGE₂ promotes the surface expression of Na_v1.8 via an intracellular RRR motif, and provide a novel mechanism for functional modulation of Na_v1.8 by hyperalgesic agents.     

Trigeminal-Rostral Ventromedial Medulla circuitry is involved in orofacial hyperalgesia contralateral to tissue injury

Background

Members of the degenerin/epithelial (DEG/ENaC) sodium channel family are mechanosensors in C elegans, and Nav1.7 and Nav1.8 voltage-gated sodium channel knockout mice have major deficits in mechanosensation. ?? and ??ENaC sodium channel subunits are present with acid sensing ion channels (ASICs) in mammalian sensory neurons of the dorsal root ganglia (DRG). The extent to which epithelial or voltage-gated sodium channels are involved in transduction of mechanical stimuli is unclear.

Results

Here we show that deleting ?? and ??ENaC sodium channels in sensory neurons does not result in mechanosensory behavioural deficits. We had shown previously that Nav1.7/Nav1.8 double knockout mice have major deficits in behavioural responses to noxious mechanical pressure. However, all classes of mechanically activated currents in DRG neurons are unaffected by deletion of the two sodium channels. In contrast, the ability of Nav1.7/Nav1.8 knockout DRG neurons to generate action potentials is compromised with 50% of the small diameter sensory neurons unable to respond to electrical stimulation in vitro.

Conclusion

Behavioural deficits in Nav1.7/Nav1.8 knockout mice reflects a failure of action potential propagation in a mechanosensitive set of sensory neurons rather than a loss of primary transduction currents. DEG/ENaC sodium channels are not mechanosensors in mouse sensory neurons.     

Discovery and biological evaluation of potent,selective, orally bioavailable,pyrazine-based blockers of the Nav1.8 sodium channel with efficacy in a model of neuropathic pain

Na_v1.8 (also known as PN3) is a tetrodotoxin-resistant (TTx-r) voltage-gated sodium channel (VGSC) that is highly expressed on small diameter sensory neurons. It has been implicated in the pathophysiology of inflammatory and neuropathic pain, and we envisioned that selective blockade of Na_v1.8 would be analgesic, while reducing adverse events typically associated with non-selective VGSC blocking therapeutic agents. Herein, we describe the preparation and characterization of a series of 6-aryl-2-pyrazinecarboxamides, which are potent blockers of the human Na_v1.8 channel and also block TTx-r sodium currents in rat dorsal root ganglia (DRG) neurons. Selected derivatives display selectivity versus human Na_v1.2. We further demonstrate that an example from this series is orally bioavailable and produces antinociceptive activity in vivo in a rodent model of neuropathic pain following oral administration.     

Network dynamics in nociceptive pathways assessed by the neuronal avalanche model

Background

Imiquimod (IQ) is known as an agonist of Toll-like receptor 7 (TLR7) and is widely used to treat various infectious skin diseases. However, it causes severe itching sensation as its side effect. The precise mechanism of how IQ causes itching sensation is unknown. A recent report suggested a molecular target of IQ as TLR7 expressed in dorsal root ganglion (DRG) neurons. However, we recently proposed a TLR7-independent mechanism, in which the activation of TLR7 is not required for the action of IQ in DRG neurons. To resolve this controversy regarding the involvement of TLR7 and to address the exact molecular identity of itching sensation by IQ, we investigated the possible molecular target of IQ in DRG neurons.

Findings

When IQ was applied to DRG neurons, we observed an increase in action potential (AP) duration and membrane resistance both in wild type and TLR7-deficient mice. Based on these results, we tested whether the treatment of IQ has an effect on the activity of K⁺ channels, K_v1.1 and K_v1.2 (voltage-gated K⁺ channels) and TREK1 and TRAAK (K_2P channels). IQ effectively reduced the currents mediated by both K⁺ channels in a dose-dependent manner, acting as an antagonist at TREK1 and TRAAK and as a partial antagonist at K_v1.1 and K_v1.2.

Conclusions

Our results demonstrate that IQ blocks the voltage-gated K⁺ channels to increase AP duration and K_2P channels to increase membrane resistance, which are critical for the membrane excitability of DRG neurons. Therefore, we propose that IQ enhances the excitability of DRG neurons by blocking multiple potassium channels and causing pruritus.     

PKC–NF-κB are involved in CCL2-induced Nav1.8 expression and channel function in dorsal root ganglion neurons

CCL2 [chemokine (C–C motif) ligand 2] contributes to the inflammation-induced neuropathic pain through activating VGSC (voltage-gated sodium channel)-mediated nerve impulse conduction, but the underlying mechanism is currently unknown. Our study aimed to investigate whether PKC (protein kinase C)–NF-κB (nuclear factor κB) is involved in CCL2-induced regulation of voltage-gated sodium Nav1.8 currents and expression. DRG (dorsal root ganglion) neurons were prepared from adult male Sprague–Dawley rats and incubated with various concentration of CCL2 for 24 h. Whole-cell patch-clamps were performed to record the Nav1.8 currents in response to the induction by CCL2. After being pretreated with 5 and10 nM CCL2 for 16 h, CCR2 [chemokine (C–C motif) receptor 2] and Nav1.8 expression significantly increased and the peak currents of Na_v1.8 elevated from the baseline 46.53±4.53 pA/pF to 64.28±3.12 pA/pF following 10 nM CCL2 (P<0.05). Compared with the control, significant change in Na_v1.8 current density was observed when the CCR2 inhibitor INCB3344 (10 nM) was applied. Furthermore, inhibition of PKC by AEB071 significantly eliminated CCL2-induced elevated Nav1.8 currents. In vitro PKC kinase assays and autoradiograms suggested that Nav1.8 within DRG neurons was a substrate of PKC and direct phosphorylation of the Na_v1.8 channel by PKC regulates its function in these neurons. Moreover, p65 expression was significantly higher in CCL2-induced neurons (P<0.05), and was reversed by treatment with INCB3344 and AEB071. PKC–NF-κB are involved in CCL2-induced elevation of Na_v1.8 current density by promoting the phosphorylation of Na_v1.8 and its expression.     

Aquaporin-1 Tunes Pain Perception by Interaction with Nav1.8 Na+ Channels in Dorsal Root Ganglion Neurons

Aquaporin-1 (AQP1) water channels are expressed in the plasma membrane of dorsal root ganglion (DRG) neurons. We found reduced osmotic water permeability in freshly isolated DRG neurons from AQP1^−/− versus AQP1^+/+ mice. Behavioral studies showed greatly reduced thermal inflammatory pain perception in AQP1^−/− mice evoked by bradykinin, prostaglandin E₂, and capsaicin as well as reduced cold pain perception. Patch clamp of freshly isolated DRG neurons showed reduced action potential firing in response to current injections. Single action potentials after pulse current injections showed reduced maximum inward current, suggesting impaired Na_v1.8 Na⁺ function. Whole-cell Na_v1.8 Na⁺ currents in Na_v1.8-expressing ND7-23 cells showed slowed frequency-dependent inactivation after AQP1 transfection. Immunoprecipitation studies showed AQP1- Na_v1.8 Na⁺ interaction, which was verified in live cells by single-particle tracking of quantum dot-labeled AQP1. Our results implicate the involvement of AQP1 in DRG neurons for the perception of inflammatory thermal pain and cold pain, whose molecular basis is accounted for, in part, by reduced Na_v1.8-dependent membrane Na⁺ current. AQP1 is, thus, a novel target for pain management.     

Upregulation of Cystathionine β-Synthetase Expression Contributes to Visceral Hyperalgesia Induced by Heterotypic Intermittent Stress in Rats

Background

Hydrogen sulfide (H₂S) functions as a neuromodulator, but whether it modulates visceral pain is not well known. This study was designed to determine the role for the endogenous H₂S producing enzyme cystathionine β-synthetase (CBS) and cystathionine γ-lyase (CSE) in a validated rat model of visceral hyperalgesia (VH).

Methods

VH was induced by nine-day heterotypic intermittent stress (HIS). Abdominal withdrawal reflex (AWR) scores were determined by measuring the visceromoter responses to colorectal distension (CRD). Dorsal root ganglia (DRG) neurons innervating the colon were labeled by injection of DiI (1,1''-dioleyl-3,3,3'',3-tetramethylindocarbocyanine methanesulfonate) into the colon wall. Patch clamp recording techniques were employed to examine excitability and sodium channel currents of colon specific DRG neurons. Tissues from colon related thoracolumbar DRGs were analyzed for CBS, CSE and sodium channel expression.

Results

HIS significantly increased the visceromotor responses to CRD in association with an upregulated expression of CBS not CSE proteins in colon related DRGs. Administration of O-(Carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor of CBS, attenuated the AWR scores in HIS-treated rats, in a dose dependent fashion. In contrast, AOAA did not produce any effect on AWR scores in healthy control rats. AOAA reversed the potentiation of sodium channel current densities of colon specific DRG neurons of HIS rats. To further confirm the role for CBS-H₂S signaling, NaHS was used to mimic the production of H₂S by CBS. Application of NaHS significantly enhanced neuronal excitability and potentiated sodium channel current densities of colon DRG neurons from healthy control rats. Furthermore, AOAA reversed the upregulation of Na_V1.7 and Na_V1.8 in colon related DRGs of HIS rats.

Conclusion

Our results suggest that upregulation of CBS expression might play an important role in developing VH via sensitization of sodium channels in peripheral nociceptors, thus identifying a specific neurobiological target for the treatment of VH in functional bowel syndromes.     

Tetrodotoxin‐resistant voltage‐gated sodium channel Nav1.8 constitutively interacts with ankyrin G

The tetrodotoxin‐resistant (TTX‐R) voltage‐gated sodium channel Na_v1.8 is predominantly expressed in peripheral afferent neurons, but in case of neuronal injury an ectopic and detrimental expression of Na_v1.8 occurs in neurons of the CNS. In CNS neurons, Na_v1.2 and Na_v1.6 channels accumulate at the axon initial segment, the site of the generation of the action potential, through a direct interaction with the scaffolding protein ankyrin G (ankG). This interaction is regulated by protein kinase CK2 phosphorylation. In this study, we quantitatively analyzed the interaction between Na_v1.8 and ankG. GST pull‐down assay and surface plasmon resonance technology revealed that Na_v1.8 strongly and constitutively interacts with ankG, in comparison to what observed for Na_v1.2. An ion channel bearing the ankyrin‐binding motif of Na_v1.8 displaced the endogenous Na_v1 accumulation at the axon initial segment of hippocampal neurons. Finally, Na_v1.8 and ankG co‐localized in skin nerves fibers. Altogether, these results indicate that Na_v1.8 carries all the information required for its localization at ankG micro‐domains. The constitutive binding of Na_v1.8 with ankG could contribute to the pathological aspects of illnesses where Na_v1.8 is ectopically expressed in CNS neurons.

     

Specific subunits of voltage-gated sodium channel underlying the subthreshold membrane potential oscillations: Potential therapeutic targets for neuropathic pain

The treatment of neuropathic pain remains a major challenge to pain clinicians. Certain nociceptive and non-nociceptive dorsal root ganglion (DRG) neurons may develop abnormal spontaneous activities following peripheral nerve injury, which is believed to be a major contributor to chronic pain. Subthreshold membrane potential oscillation (SMPO) observed in injured DRG neurons was reported to be involved in the generation of abnormal spontaneous activity. Tetrodotoxin-sensitive sodium (Na⁺) channels were testified to be involved in the generation of SMPO, but their specific subunits have not been clarified. We hypothesize that the subunits of voltage-gated sodium channel, Na_v1.3 and Na_v1.6, are involved in the generation of SMPO. An attempt to test this hypothesis may lead to a new therapeutic strategy for neuropathic pain.     

Enhancement of presynaptic glutamate release and persistent inflammatory pain by increasing neuronal cAMP in the anterior cingulate cortex

Background

Primary erythromelalgia is an autosomal dominant pain disorder characterized by burning pain and skin redness in the extremities, with onset of symptoms during the first decade in the families whose mutations have been physiologically studied to date. Several mutations of voltage-gated Na⁺ channel Na_V1.7 have been linked with primary erythromelalgia. Recently, a new substitution Na_V1.7/I136V has been reported in a Taiwanese family, in which pain appeared at later ages (9–22 years, with onset at 17 years of age or later in 5 of 7 family members), with relatively slow progression (8–10 years) to involvement of the hands. The proband reported onset of symptoms first in his feet at the age of 11, which then progressed to his hands at the age of 19. The new mutation is located in transmembrane segment 1 (S1) of domain I (DI) in contrast to all Na_V1.7 mutations reported to date, which have been localized in the voltage sensor S4, the linker joining segments S4 and S5 or pore-lining segments S5 and S6 in DI, II and III.

Results

In this study, we characterized the gating and kinetic properties of I136V mutant channels in HEK293 cells using whole-cell patch clamp. I136V shifts the voltage-dependence of activation by -5.7 mV, a smaller shift in activation than the other erythromelalgia mutations that have been characterized. I136V also decreases the deactivation rate, and generates larger ramp currents.

Conclusion

The I136V substitution in Na_V1.7 alters channel gating and kinetic properties. Each of these changes may contribute to increased excitability of nociceptive dorsal root ganglion neurons, which underlies pain in erythromelalgia. The smaller shift in voltage-dependence of activation of Na_V1.7, compared to the other reported cases of inherited erythromelalgia, may contribute to the later age of onset and slower progression of the symptoms reported in association with this mutation.     

Direct activation of Transient Receptor Potential Vanilloid 1(TRPV1) by Diacylglycerol (DAG)

Voltage-gated sodium channels play important roles in modulating dorsal root ganglion (DRG) neuron hyperexcitability and hyperalgesia after peripheral nerve injury or inflammation. We report that chronic compression of DRG (CCD) produces profound effect on tetrodotoxin-resistant (TTX-R) and tetrodotoxin-sensitive (TTX-S) sodium currents, which are different from that by chronic constriction injury (CCI) of the sciatic nerve in small DRG neurons. Whole cell patch-clamp recordings were obtained in vitro from L₄ and/or L₅ dissociated, small DRG neurons following in vivo DRG compression or nerve injury. The small DRG neurons were classified into slow and fast subtype neurons based on expression of the slow-inactivating TTX-R and fast-inactivating TTX-S Na⁺ currents. CCD treatment significantly reduced TTX-R and TTX-S current densities in the slow and fast neurons, but CCI selectively reduced the TTX-R and TTX-S current densities in the slow neurons. Changes in half-maximal potential (V_1/2) and curve slope (k) of steady-state inactivation of Na⁺ currents were different in the slow and fast neurons after CCD and CCI treatment. The window current of TTX-R and TTX-S currents in fast neurons were enlarged by CCD and CCI, while only that of TTX-S currents in slow neurons was increased by CCI. The decay rate of TTX-S and both TTX-R and TTX-S currents in fast neurons were reduced by CCD and CCI, respectively. These findings provide a possible sodium channel mechanism underlying CCD-induced DRG neuron hyperexcitability and hyperalgesia and demonstrate a differential effect in the Na⁺ currents of small DRG neurons after somata compression and peripheral nerve injury. This study also points to a complexity of hyperexcitability mechanisms contributing to CCD and CCI hyperexcitability in small DRG neurons.     

Transmembrane Segments Prevent Surface Expression of Sodium Channel Nav1.8 and Promote Calnexin-dependent Channel Degradation

The voltage-gated sodium channel (Na_v) 1.8 contributes substantially to the rising phase of action potential in small dorsal root ganglion neurons. Na_v1.8 is majorly localized intracellularly and its expression on the plasma membrane is regulated by exit from the endoplasmic reticulum (ER). Previous work has identified an ER-retention/retrieval motif in the first intracellular loop of Na_v1.8, which prevents its surface expression. Here we report that the transmembrane segments of Na_v1.8 also cause this channel retained in the ER. Using transferrin receptor and CD8α as model molecules, immunocytochemistry showed that the first, second, and third transmembrane segments in each domain of Na_v1.8 reduced their surface expression. Alanine-scanning analysis revealed acidic amino acids as critical factors in the odd transmembrane segments. Furthermore, co-immunoprecipitation experiments showed that calnexin interacted with acidic amino acid-containing sequences through its transmembrane segment. Overexpression of calnexin resulted in increased degradation of those proteins through the ER-associated degradation pathway, whereas down-regulation of calnexin reversed the phenotype. Thus our results reveal a critical role and mechanism of transmembrane segments in surface expression and degradation of Na_v1.8.     

Characterization of na(+) and ca(2+) channels in zebrafish dorsal root ganglion neurons

Background

Dorsal root ganglia (DRG) somata from rodents have provided an excellent model system to study ion channel properties and modulation using electrophysiological investigation. As in other vertebrates, zebrafish (Danio rerio) DRG are organized segmentally and possess peripheral axons that bifurcate into each body segment. However, the electrical properties of zebrafish DRG sensory neurons, as compared with their mammalian counterparts, are relatively unexplored because a preparation suitable for electrophysiological studies has not been available.

Methodology/Principal Findings

We show enzymatically dissociated DRG neurons from juvenile zebrafish expressing Isl2b-promoter driven EGFP were easily identified with fluorescence microscopy and amenable to conventional whole-cell patch-clamp studies. Two kinetically distinct TTX-sensitive Na⁺ currents (rapidly- and slowly-inactivating) were discovered. Rapidly-inactivating I_Na were preferentially expressed in relatively large neurons, while slowly-inactivating I_Na was more prevalent in smaller DRG neurons. RT-PCR analysis suggests zscn1aa/ab, zscn8aa/ab, zscn4ab and zscn5Laa are possible candidates for these I_Na components. Voltage-gated Ca²⁺ currents (I_Ca) were primarily (87%) comprised of a high-voltage activated component arising from ω-conotoxin GVIA-sensitive Ca_V2.2 (N-type) Ca²⁺ channels. A few DRG neurons (8%) displayed a miniscule low-voltage-activated component. I_Ca in zebrafish DRG neurons were modulated by neurotransmitters via either voltage-dependent or -independent G-protein signaling pathway with large cell-to-cell response variability.

Conclusions/Significance

Our present results indicate that, as in higher vertebrates, zebrafish DRG neurons are heterogeneous being composed of functionally distinct subpopulations that may correlate with different sensory modalities. These findings provide the first comparison of zebrafish and rodent DRG neuron electrical properties and thus provide a basis for future studies.     

Kinetics of sodium tail current during repolarization of axon membrane normally and in the presence of scorpion toxin

The kinetics of the sodium tail current (tail I_Na) in myelinated frog nerve fibers in the region of repolarization potentials (V_r) from –40 to –70 mV has two exponential components: fast and slow. The component composition of the tail I_Na depends on V_r: with an increase in negative values of V_r the contribution of the slow component of the tail (_s) decreases, and at V_r values higher than –80 mV, the tail I_Na follows virtually one fast exponential curve. The component composition of the tail I_Na at a fixed level of V_r depends on the initial conditions: with an increase in the duration of the test pulse (V_t) the amplitude of the fast component of the tail falls much faster than the amplitude of the slow component. In that case the kinetics of the fall in amplitude of the fast component corresponds to the kinetics of inactivation of I_Na. Scorpion toxin causes slowing of the kinetics of the tail I_Na at all values of V_r, mainly on account of an increase in _s. For qualitative interpretation of the results a kinetic scheme assuming the presence of two open states of the sodium channel of the axon membrane is suggested. The hypothesis is put forward that scorpion toxin interacts with the same site of the gating mechanism of the channel as DDT and trinitrophenol.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 5, pp. 541–549, September–October, 1980.     

Localization of receptor site on insect sodium channel for depressant β-toxin BmK IT2

Background

BmK IT2 is regarded as a receptor site-4 modulator of sodium channels with depressant insect toxicity. It also displays anti-nociceptive and anti-convulsant activities in rat models. In this study, the potency and efficacy of BmK IT2 were for the first time assessed and compared among four sodium channel isoforms expressed in Xenopus oocytes. Combined with molecular approach, the receptor site of BmK IT2 was further localized.

Principal Findings

2 µM BmK IT2 strongly shifted the activation of DmNa_v1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNa_v1.2, rNa_v1.3 and mNa_v1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu₈₉₆, Leu₈₉₉, Gly₉₀₄ in extracellular loop Domain II S3–S4 of DmNa_v1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNa_v1 could be conferred by Domain III. Analysis of subsequent DmNa_v1 mutants highlighted the residues in Domain III pore loop, esp. Ile₁₅₂₉ was critical for recognition and binding of BmK IT2.

Conclusions/Significance

In this study, BmK IT2 displayed total insect-selectivity. Two binding regions, comprising domains II and III of DmNa_v1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Na_v1.2, Na_v1.3 and Na_v1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.