Mapping of scorpion toxin receptor sites at voltage-gated sodium channels

Scorpion alpha and beta toxins interact with voltage-gated sodium channels (Na_vs) at two pharmacologically distinct sites. Alpha toxins bind at receptor site-3 and inhibit channel inactivation, whereas beta toxins bind at receptor site-4 and shift the voltage-dependent activation toward more hyperpolarizing potentials. The two toxin classes are subdivided to distinct pharmacological groups according to their binding preferences and ability to compete for the receptor sites at Na_v subtypes. To elucidate the toxin-channel surface of interaction at both receptor sites and clarify the molecular basis of varying toxin preferences, an efficient bacterial system for their expression in recombinant form was established. Mutagenesis accompanied by toxicity, binding and electrophysiological assays, in parallel to determination of the three-dimensional structure using NMR and X-ray crystallography uncovered a bipartite bioactive surface in toxin representatives of all pharmacological groups. Exchange of external loops between the mammalian brain channel rNa_v1.2a and the insect channel DmNa_v1 highlighted channel regions involved in the varying sensitivity to assorted toxins. In parallel, thorough mutagenesis of channel external loops illuminated points of putative interaction with the toxins. Amino acid substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain II of rNa_v1.2a had prominent impact on the activity of the beta-toxin Css4 (from Centruroides suffusus suffusus), and substitutions at external loops S1-S2 and S3-S4 of the voltage sensor module in domain IV affected the activity of the alpha-toxin Lqh2 (from Leiurus quinquestriatus hebraeus). Rosetta modeling of toxin-Na_v interaction using the voltage sensor module of the potassium channel as template raises commonalities in the way alpha and beta toxins interact with the channel. Css4 interacts with rNa_v1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain II, while Lqh2 interacts with rNa_v1.2a at a crevice between S1-S2 and S3-S4 transmembrane segments in domain IV. Double-mutant cycle analysis and dissociation assays employing a battery of Lqh2 mutants against rNa_v1.2a mutants identified the docking orientation of alpha toxins at the channel external surface of the Gating-module in domain IV. The other point of interaction between the toxin and the channel has not yet been defined and may involve channel residues of either the Pore-module or the Gating-module.     

Pharmacological modulation of voltage-gated potassium channels as a therapeutic strategy

Importance of the field: The human genome encodes at least 40 distinct voltage-gated potassium channel subtypes, which vary in regional expression, pharmacological and biophysical properties. Voltage-dependent potassium (Kv) channels help orchestrate many of the physiological and pathophysiological processes that promote and sometimes hinder the healthy functioning of our bodies.

Areas covered in this review: This review summarizes patent and scientific literature reports from the past decade highlighting the opportunities that Kv channels offer for the development of new therapeutic interventions for a wide variety of disorders.

What the reader will gain: The reader will gain an insight from an analysis of the associations of different Kv family members with disease processes, summary and evaluation of the development of therapeutically relevant pharmacological modulators of these channels, particularly focusing on proprietary agents being developed.

Take home message: Development of new drugs that target Kv channels continue to be of great interest but is proving to be challenging. Nevertheless, opportunities for Kv channel modulators to have an impact on a wide range of disorders in the future remain an exciting prospect.     

Jingzhaotoxin-IX, a novel gating modifier of both sodium and potassium channels from Chinese tarantula Chilobrachys jingzhao

Tarantula Chilobrachys jingzhao is one of the most venomous species distributed in China. In this study, we have isolated and characterized a novel neurotoxin named Jingzhaotoxin-IX (JZTX-IX) from the venom of the tarantula. JZTX-IX is a C-terminally amidated peptide composed of 35 amino acid residues. The toxin shows 74% sequence identity with CcoTx3 from southeastern Africa tarantula Ceratogyrus cornuatus. JZTX-IX was found to interact with multiple types of ion channels including voltage-gated sodium channels (both tetrodotoxin-resistant and tetrodotoxin-sensitive isoforms) and Kv2.1 channel. The toxin had no effect on delayed rectifier potassium channel Kv1.1, 1.2 and 1.3. JZTX-IX shifted the voltage dependence of channel activation to more positive voltages, but binding of toxin to ion channels was not reversible by extreme depolarization. In addition, JZTX-IX could bias the activities of ion channels towards closed state because the time constant for decay (channel deactivation) of tail currents became faster in the presence of toxin. Taken together with the finding that 10 μM JZTX-IX completely blocked ion channels at resting potential without pulsing, we propose that JZTX-IX is a gating modifier showing low selectivity for ion channel types and trapping voltage sensor at closed state.     

Heteropoda toxin 2 is a gating modifier toxin specific for voltage-gated K+ channels of the Kv4 family.

Kv4 voltage-gated K(+) channels are responsible for transient K(+) currents in the central nervous system and in the heart. HpTx2 is a peptide toxin that selectively inhibits these currents; making it a useful probe for understanding Kv4 channel structure and drug binding. Therefore, we developed a method to produce large amounts of recombinant HpTx2. Recombinant toxin inhibits all three Kv4 isoforms to the same degree; however, the voltage-dependence of inhibition is less apparent for Kv4.1 than for Kv4.3. Similarly, recombinant HpTx2(GS) effects gating characteristics of both channels, but Kv4.1 to a much lesser degree. The toxin lacks affinity for Kv1.4, Kv2.1, and Kv3.4. To locate the binding site, the amino acids linking the third and forth membrane spanning segments of Kv4.3 were replaced with analogous amino acids of Kv1.4. The chimeric K(+) channel was completely insensitive to block by rHpTx2, suggesting that its binding site is near the channel's voltage sensor. These data show that rHpTx2(GS) is a gating modifier toxin that binds to a site remote from the pore.     

CSTX-1, a toxin from the venom of the hunting spider Cupiennius salei, is a selective blocker of L-type calcium channels in mammalian neurons

The inhibitor cystine-knot motif identified in the structure of CSTX-1 from Cupiennius salei venom suggests that this toxin may act as a blocker of ion channels. Whole-cell patch-clamp experiments performed on cockroach neurons revealed that CSTX-1 produced a slow voltage-independent block of both mid/low- (M-LVA) and high-voltage-activated (HVA) insect Ca_v channels. Since C. salei venom affects both insect as well as rodent species, we investigated whether Ca_v channel currents of rat neurons are also inhibited by CSTX-1. CSTX-1 blocked rat neuronal L-type, but no other types of HVA Ca_v channels, and failed to modulate LVA Ca_v channel currents. Using neuroendocrine GH3 and GH4 cells, CSTX-1 produced a rapid voltage-independent block of L-type Ca_v channel currents. The concentration–response curve was biphasic in GH4 neurons and the subnanomolar IC₅₀ values were at least 1000-fold lower than in GH3 cells. L-type Ca_v channel currents of skeletal muscle myoballs and other voltage-gated ion currents of rat neurons, such as I_Na(v) or I_K(v) were not affected by CSTX-1. The high potency and selectivity of CSTX-1 for a subset of L-type channels in mammalian neurons may enable the toxin to be used as a molecular tool for the investigation of this family of Ca_v channels.     

Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators.

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. Similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators.     

Carvacrol inhibits the neuronal voltage-gated sodium channels Nav1.2, Nav1.6, Nav1.3, Nav1.7, and Nav1.8 expressed in Xenopus oocytes with different potencies

Carvacrol is the predominant monoterpene in essential oils from many aromatic plants. Several animal studies showing analgesic effects of carvacrol indicate potential of carvacrol as a new medication for patients with refractory pain. Voltage-gated sodium channels (Na_v) are thought to have crucial roles in the development of inflammatory and neuropathic pain, but there is limited information about whether the analgesic mechanism of carvacrol involves Na_v. We used whole-cell, two-electrode, voltage-clamp techniques to examine the effects of carvacrol on sodium currents in Xenopus oocytes expressing α subunits of Na_v1.2, Na_v1.3, Na_v1.6, Na_v1.7, and Na_v1.8. Carvacrol dose-dependently suppressed sodium currents at a holding potential that induced half-maximal current. The half-maximal inhibitory concentration values for Na_v1.2, Na_v1.3, Na_v1.6, Na_v1.7, and Na_v1.8 were 233, 526, 215, 367, and 824 μmol/L, respectively, indicating that carvacrol had more potent inhibitory effects towards Na_v1.2 and Na_v1.6 than Na_v1.3, Na_v1.7, and Na_v1.8. Gating analysis showed a depolarizing shift of the activation curve and a hyperpolarizing shift of the inactivation curve in all five α subunits following carvacrol treatment. Furthermore, carvacrol exhibits a use-dependent block for all five α Na_v subunits. These findings provide a better understanding of the mechanisms associated with the analgesic effect of carvacrol.     

U-shaped dose-dependent effects of BmK AS,a unique scorpion polypeptide toxin,on voltage-gated sodium channels

Background and purpose:

Buthus martensi Karsch (BmK) AS is a scorpion polypeptide toxin, said to target the voltage-gated sodium channels (VGSCs). However, the mechanism of action of BmK AS on the VGSCs has yet to be defined.

Experimental approach:

We examined the electrophysiological effects of BmK AS in a wide dose range on the rat brain-type VGSC α-subunit, rNav1.2a, heterologously expressed in Xenopus oocytes and on the VGSCs endogenously expressed in the dorsal root ganglion neuroblastoma ND7-23 cell line.

Key results:

In the oocytes, BmK AS depolarized the voltage dependence of activation and inactivation of rNav1.2a at 0.1 and 500 nM whereas these parameters were hyperpolarized at 1 nM. In ND7-23 cells, BmK AS hyperpolarized the voltage dependence of activation and inactivation at 0.1, 1 and 100 nM but not 10 nM. BmK AS also hyperpolarized the voltage dependence of recovery from inactivation at 0.1 and 100 nM and slowed the recovery kinetics at all concentrations, but the effects of 1 and 10 nM were relatively smaller than those at 0.1 and 100 nM. Moreover, the inactivation of VGSCs was potentiated by 10 nM BmK AS in both systems, whereas it was inhibited by 0.1 or 100 nM BmK AS in the oocytes or ND7-23 cells respectively.

Conclusions and implications:

BmK AS modulated the VGSCs in a unique U-shaped dose-dependent manner, which could be due to the opposing effects of binding to two distinct receptor sites on the VGSCs.     

TREK-1双孔钾通道的功能研究进展

双孔钾通道是一类有多个亚型、可在基线水平上调节细胞膜兴奋性的通道超家族。其中一类TREK高表达于人的中枢神经系统，并受到一些物理和化学因素的调节。最近，有关基因敲除动物的实验研究表明，TREK-1可能参与了一些麻醉剂的全麻过程。     

The transient receptor potential channel antagonist SKF96365 is a potent blocker of low-voltage-activated T-type calcium channels

Background and purpose:

{"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365 (SKF), originally identified as a blocker of receptor-mediated calcium entry, is widely used diagnostically, as a blocker of transient receptor potential canonical type (TRPC) channels. While SKF has been used as a tool to define the functional roles of TRPC channels in various cell and tissue types, there are notable overlapping physiological and pathophysiological associations between TRPC channels and low-voltage-activated (LVA) T-type calcium channels. The activity of SKF against T-type Ca channels has not been previously explored, and here we systematically investigated the effects of SKF on recombinant and native voltage-gated Ca channel-mediated currents.

Experimental approach:

Effects of SKF on recombinant Ca channels were studied under whole-cell patch clamp conditions after expression in HEK293 cells. The effect of SKF on cerebellar Purkinje cells (PCs) expressing native T-type Ca channels was also assessed.

Key results:

SKF blocked recombinant Ca channels, representative of each of the three main molecular genetic classes (Ca_V1, Ca_V2 and Ca_V3) at concentrations typically utilized to assay TRPC function (10 µM). Particularly, human Ca_V3.1 T-type Ca channels were more potently inhibited by SKF (IC₅₀∼560 nM) in our experiments than previously reported for similarly expressed TRPC channels. SKF also inhibited native Ca_V3.1 T-type currents in a rat cerebellar PC slice preparation.

Conclusions and implications:

SKF was a potent blocker of LVA T-type Ca channels. We suggest caution in the interpretation of results using SKF alone as a diagnostic agent for TRPC activity in native tissues.     

Warifteine,a bisbenzylisoquinoline alkaloid,induces relaxation by activating potassium channels in vascular myocytes

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V102862 (Co 102862): a potent, broad-spectrum state-dependent blocker of mammalian voltage-gated sodium channels

1. 4-(4-Fluorophenoxy)benzaldehyde semicarbazone (V102862) was initially described as an orally active anticonvulsant with robust activity in a variety of rodent models of epilepsy. The mechanism of action was not known. We used whole-cell patch-clamp techniques to study the effects of V102862 on native and recombinant mammalian voltage-gated Na+ channels. 2. V102862 blocked Na+ currents (I(Na)) in acutely dissociated cultured rat hippocampal neurons. Potency increased with membrane depolarization, suggesting a state-dependent mechanism of inhibition. There was no significant effect on the voltage dependence of activation of I(Na). 3. The dissociation constant for the inactivated state (K(I)) was approximately 0.6 microM, whereas the dissociation constant for the resting state (K(R)) was >15 microM. 4. The binding to inactivated channels was slow, requiring a few seconds to reach steady state at -80 mV. 5. The mechanism of inhibition was characterized in more detail using human embryonic kidney-293 cells stably expressing rat brain type IIA Na+ (rNa(v)1.2) channels, a major Na+ channel alpha subunit in rat hippocampal neurons. Similar to hippocampal neurons, V102862 was a potent state-dependent blocker of rNa(v)1.2 channels with a K(I) of approximately 0.4 microM and K(R) approximately 30 microM. V102862 binding to inactivated channels was relatively slow (k(+) approximately = 1.7 microM(-1) s(-1)). V102862 shifted the steady-state availability curve in the hyperpolarizing direction and significantly retarded recovery of Na+ channels from inactivation. 6. These results suggest that inhibition of voltage-gated Na+ channels is a major mechanism underlying the anticonvulsant properties of V102862. Moreover, understanding the biophysics of the interaction may prove to be useful in designing a new generation of potent Na+ channel blocker therapeutics.     

OdK2, a Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae

The first Kv1.3 channel-selective toxin from the venom of the Iranian scorpion Odonthobuthus doriae (OdK2) was purified, sequenced and characterized physiologically. OdK2 consists of 38 amino acids, including six conserved cysteine and a C-terminal lysine residue, as revealed by the unique use of a quadrupole ion cyclotron resonance Fourier-transform mass spectrometer. Based on multiple sequence alignments, OdK2 was classified as alpha-KTX3.11. The pharmacological effects of OdK2 were studied on a panel of eight different cloned K(+) channels (vertebrate Kv1.1-Kv1.6, Shaker IR and hERG) expressed in Xenopus laevis oocytes. Interestingly, OdK2 selectively inhibits the currents through Kv1.3 channels with an IC50 value of 7.2+/-2.7nM.     

Are big potassium-type Ca2+-activated potassium channels a viable target for the treatment of epilepsy?

Introduction: BK (big potassium) channels are Ca²⁺-activated K⁺ channels widely expressed in mammalian cells. They are extensively distributed in the CNS, the most abundant level being found in brain areas largely involved in epilepsy, namely cortex, hippocampus, piriform cortex, and other limbic structures. BK channels control action potential shape/duration, thereby regulating membrane excitability and Ca²⁺ signaling.

Areas covered: The potassium channel superfamily represents a rich source of potential targets for therapeutic intervention in epilepsy. Some studies have identified alterations in BK channel function, therefore, supporting the development of drugs acting on these channels for epilepsy treatment.

Expert opinion: The actual sketch is intriguing and controversial, since mechanisms altering the physiological role of BK channels leading to either a loss- or gain-of-function have both been linked to seizure onset. Not many studies have been performed to unravel the efficacy of drugs acting on these channels as potential antiepileptics; however, paradoxically, efficacy has been demonstrated for both BK channel openers and blockers. Furthermore, their potential usefulness in preventing epileptogenesis has not been investigated at all. Substantial data on risks and benefits of modulating these channels are urgently needed to draw a definitive conclusion on whether BK channels are a viable future target for the treatment of epilepsy.     

Expression and isotopic labelling of the potassium channel blocker ShK toxin as a thioredoxin fusion protein in bacteria

The polypeptide toxin ShK is a potent blocker of Kv1.3 potassium channels, which play a crucial role in the activation of human effector memory T-cells (T_EM). Selective blockers constitute valuable therapeutic leads for the treatment of autoimmune diseases mediated by T_EM cells, such as multiple sclerosis, rheumatoid arthritis, and type-1 diabetes. We have established a recombinant peptide expression system in order to generate isotopically-labelled ShK and various ShK analogues for in-depth biophysical and pharmacological studies. ShK was expressed as a thioredoxin fusion protein in Escherichia coli BL21 (DE3) cells and purified initially by Ni²⁺ iminodiacetic acid affinity chromatography. The fusion protein was cleaved with enterokinase and purified to homogeneity by reverse-phase HPLC. NMR spectra of ¹⁵N-labelled ShK were similar to those reported previously for the unlabelled synthetic peptide, confirming that recombinant ShK was correctly folded. Recombinant ShK blocked Kv1.3 channels with a K_d of 25 pM and inhibited the proliferation of human and rat T lymphocytes with a preference for T_EM cells, with similar potency to synthetic ShK in all assays. This expression system also enables the efficient production of ¹⁵N-labelled ShK for NMR studies of peptide dynamics and of the interaction of ShK with Kv1.3 channels.     

线粒体ATP敏感性钾通道不参与异丙酚预处理的心肌保护

目的　观察异丙酚预处理对心肌缺血再灌注损伤的保护机制是否通过开放线粒体ATP敏感性K通道 (KATP)。方法　非循环式Langendorff离体心脏灌注模型 ,灌注 1h ,常温下行全心缺血 2 5min ,恢复再灌注 30min。通过Maclab仪记录左室舒张末压 (LVEDP)、左室发展压 (LVDP)、左室压上升和下降最大速率 (±dp/dtmax)。测恢复再灌注末心肌组织MDA含量。结果　恢复再灌注 30min末 ,对照组(Con)、异丙酚预处理组 (PP)、5 HD +PP和 5 HD组心肌组织的MDA含量分别为 (113 7± 2 0 9)、(89 4± 13 7)、(91 9± 14 4 )和 (114 8± 19 7)nmol·10 0mg-1。PP组和 5 HD+PP组的心肌MDA含量都明显低于Con组和 5 HD组 (P<0 0 5 ) ;PP组和 5 HD +PP组两组间的MDA差异无显著性 (P >0 0 5 )。恢复再灌注 30min末 ,Con组、PP组、5 HD+PP组和 5 HD组的LVEDP值分别为基础值的 5 1、3 2、3 6和 5 3倍。PP组和 5 HD +PP组LVEDP值的上升幅度均明显低于Con组和 5 HD组 (P <0 0 5 ) ,而 5 HD +PP组和PP组之间差异无显著性 (P >0 0 5 )。结论　异丙酚预处理的心肌保护不是通过开放线粒体ATP敏感性K通道 ,其心肌保护作用和线粒体KATP无关     

Characterization of the potassium channels involved in EDHF-mediated relaxation in cerebral arteries

1. In the presence of N^G-nitro-L-arginine (L-NOARG, 0.3 mM) and indomethacin (10 μM), the relaxations induced by acetylcholine and the calcium (Ca) ionophore {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 are considered to be mediated by endothelium-derived hyperpolarizing factor (EDHF) in the guinea-pig basilar artery.
2. Inhibitors of adenosine 5′-triphosphate (ATP)-sensitive potassium (K)-channels (K_ATP; glibenclamide, 10 μM), voltage-sensitive K-channels (K_V; dendrotoxin-I, 0.1 μM or 4-aminopyridine, 1 mM), small (SK_Ca; apamin, 0.1 μM) and large (BK_Ca; iberiotoxin, 0.1 μM) conductance Ca-sensitive K-channels did not affect the L-NOARG/indomethacin-resistant relaxation induced by acetylcholine.
3. Synthetic charybdotoxin (0.1 μM), an inhibitor of BK_Ca and K_V, caused a rightward shift of the concentration-response curve for acetylcholine and reduced the maximal relaxation in the presence of L-NOARG and indomethacin, whereas the relaxation induced by {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187 was not significantly inhibited.
4. A combination of charybdotoxin (0.1 μM) and apamin (0.1 μM) abolished the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187. However, the acetylcholine-induced relaxation was not affected by a combination of iberiotoxin (0.1 μM) and apamin (0.1 μM).
5. Ciclazindol (10 μM), an inhibitor of K_V in rat portal vein smooth muscle, inhibited the L-NOARG/indomethacin-resistant relaxations induced by acetylcholine and {"type":"entrez-nucleotide","attrs":{"text":"A23187","term_id":"833253","term_text":"A23187"}}A23187, and the relaxations were abolished when ciclazindol (10 μM) was combined with apamin (0.1 μM).
6. Human pial arteries from two out of four patients displayed an L-NOARG/indomethacin-resistant relaxation in response to substance P. This relaxation was abolished in both cases by pretreatment with the combination of charybdotoxin (0.1 μM) and apamin (0.1 μM), whereas each toxin had little effect alone.
7. The results suggest that K_V, but not K_ATP and BK_Ca, is involved in the EDHF-mediated relaxation in the guinea-pig basilar artery. The synergistic action of apamin and charybdotoxin (or ciclazindol) could indicate that both K_V and SK_Ca are activated by EDHF. However, a single type of K-channel, which may be structurally related to K_V and allosterically regulated by apamin, could also be the target for EDHF.

     

Effects of Resibufogenin and Cinobufagin on voltage-gated potassium channels in primary cultures of rat hippocampal neurons

Outward delayed rectifier potassium channel and outward transient potassium channel have multiple important roles in maintaining the excitability of hippocampal neurons. The present study investigated the effects of two bufadienolides, Resibufogenin (RBG) and Cinobufagin (CBG), on the outward delayed rectifier potassium current (I_K) and outward transient potassium current (I_A) in rat hippocampal neurons. RBG and CBG have similar structures and both were isolated from the venom gland of toad skin. RBG inhibited both I_K and I_A, whereas CBG inhibited I_K without noticeable effect on I_A. Moreover, at 1 μM concentration both RBG and CBG could alter some channel kinetics and gating properties of I_K, such as steady-state activation and inactivation curves, open probability and time constants. These findings suggested that I_K is probably a target of bufadienolides, which may explain the mechanisms of bufadienolides’ pathological effects on central nervous system.     

A-887826 is a structurally novel, potent and voltage-dependent Nav1.8 sodium channel blocker that attenuates neuropathic tactile allodynia in rats

Activation of sodium channels is essential to action potential generation and propagation. Recent genetic and pharmacological evidence indicates that activation of Na_v1.8 channels contributes to chronic pain. Herein, we describe the identification of a novel series of structurally related pyridine derivatives as potent Na_v1.8 channel blockers. A-887826 exemplifies this series and potently (IC₅₀ = 11nM) blocked recombinant human Na_v1.8 channels. A-887826 was ∼3 fold less potent to block Na_v1.2, ∼10 fold less potent to block tetrodotoxin-sensitive sodium (TTX-S Na⁺) currents and was >30 fold less potent to block Na_V1.5 channels. A-887826 potently blocked tetrodotoxin-resistant sodium (TTX-R Na⁺) currents (IC₅₀ = 8nM) from small diameter rat dorsal root ganglion (DRG) neurons in a voltage-dependent fashion. A-887826 effectively suppressed evoked action potential firing when DRG neurons were held at depolarized potentials and reversibly suppressed spontaneous firing in small diameter DRG neurons from complete Freund’s adjuvant inflamed rats. Following oral administration, A-887826 significantly attenuated tactile allodynia in a rat neuropathic pain model. Further characterization of TTX-R current block in rat DRG neurons demonstrated that A-887826 (100 nM) shifted the mid-point of voltage-dependent inactivation of TTX-R currents by ∼4 mV without affecting voltage-dependent activation and did not exhibit frequency-dependent inhibition. The present data demonstrate that A-887826 is a structurally novel and potent Na_v1.8 blocker that inhibits rat DRG TTX-R currents in a voltage-, but not frequency-dependent fashion. The ability of this structurally novel Na_v1.8 blocker to effectively reduce tactile allodynia in neuropathic rats further supports the role of Na_v1.8 sodium channels in pathological pain states.     

Molecular determinants of state-dependent block of voltage-gated sodium channels by pilsicainide

Background and purpose:

Pilsicainide, an anti-arrhythmic drug used in Japan, is described as a pure sodium channel blocker. We examined the mechanisms by which it is able to block open channels, because these properties may be especially useful to reduce hyperexcitability in pathologies characterized by abnormal sodium channel opening.

Experimental approach:

The effects of pilsicainide on various heterologously expressed human sodium channel subtypes and mutants were investigated using the patch clamp technique.

Key results:

Pilsicainide exhibited tonic and use-dependent effects comparable to those of mexiletine and flecainide on hNav1.4 channels. These use-dependent effects were abolished in the mutations F1586C and Y1593C within segment 6 of domain IV, suggesting that the interaction of pilsicainide with these residues is critical for its local anaesthetic action. Its affinity constants for closed channels (K_R) and channels inactivated from the closed state (K_I) were high, suggesting that its use-dependent block (UDB) requires the channel to be open for it to reach a high-affinity blocking site. Accordingly, basic pH, which slightly increased the proportion of neutral drug, dramatically decreased K_R and K_I values. Effects of pilsicainide were similar on skeletal muscle hNav1.4, brain hNav1.1 and heart hNav1.5 channels. The myotonic R1448C and G1306E hNav1.4 mutants were more and less sensitive to pilsicainide, respectively, due to mutation-induced gating modifications.

Conclusions and implications:

Although therapeutic concentrations of pilsicainide may have little effect on resting and closed-state inactivated channels, it induces a strong UDB due to channel opening, rendering the drug ideally suited for inhibition of high-frequency action potential firing.