Yonghua Ji

Exploring the obscure profiles of pharmacological binding sites on voltage-gated sodium channels by BmK neurotoxins

Diverse subtypes of voltage-gated sodium channels (VGSCs) have been found throughout tissues of the brain, muscles and the heart. Neurotoxins extracted from the venom of the Asian scorpion Buthus martensi Karsch (BmK) act as sodium channel-specific modulators and have therefore been widely used to study VGSCs. α-type neurotoxins, named BmK I, BmK αIV and BmK abT, bind to receptor site-3 on VGSCs and can strongly prolong the inactivation phase of VGSCs. In contrast, β-type neurotoxins, named BmK AS, BmK AS-1, BmK IT and BmK IT2, occupy receptor site-4 on VGSCs and can suppress peak currents and hyperpolarize the activation kinetics of sodium channels. Accumulating evidence from binding assays of scorpion neurotoxins on VGSCs, however, indicate that pharmacological sensitivity of VGSC subtypes to different modulators is much more complex than that suggested by the simple α-type and β-type neurotoxin distinction. Exploring the mechanisms of possible dynamic interactions between site 3-/4-specific modulators and region- and/or species-specific subtypes of VGSCs would therefore greatly expand our understanding of the physiological and pharmacological properties of diverse VGSCs. In this review, we discuss the pharmacological and structural diversity of VGSCs as revealed by studies exploring the binding properties and cross-competitive binding of site 3- or site 4-specific modulators in VGSC subtypes in synaptosomes from distinct tissues of diverse species.

Molecular determination of selectivity of the site 3 modulator (BmK I) to sodium channels in the CNS: a clue to the importance of Nav1.6 in BmK I-induced neuronal hyperexcitability.

BmK I, a site-3-specific modulator of VGSCs (voltage-gated sodium channels) from the Chinese scorpion Buthus martensi Karsch, can induce spontaneous nociception and hyperalgesia and generate epileptiform responses in rats, which is attributed to the modulation of VGSCs in the neural system. However, which VGSC subtype is targeted by BmK I remains to be identified. Using two-electrode voltage-clamp recording, we studied the efficacy and selectivity of BmK I to three neuronal VGSCs co-expressed with the auxiliary β1 subunit in Xenopus oocytes. Results revealed that BmK I induced a large increase in both transient and persistent currents in mNav1.6α/β1 (where m indicates mouse), which correlated with a prominent reduction in the fast component of inactivating current. In comparison, BmK I-increased currents of rNav1.2α/β1 (where r indicates rat) and rNav1.3α/β1 were much smaller. The EC50 values of BmK I for rNav1.2α/β1 (252±60xa0nM) and mNav1.6α/β1 (214±30xa0nM) were similar and roughly half of that for rNav1.3α/β1 (565±16xa0nM). Moreover, BmK I only accelerated the slow inactivation development and delay recovery of mNav1.6α/β1 through binding to the channel in the open state. Residue-swap analysis verified that an acidic residue (e.g. Asp1602 in mNav1.6) within the domain IV S3-S4 extracellular loop of VGSCs was crucial for the selectivity and modulation pattern of BmK I. Our findings thus provide the molecular determinant explaining the divergent and intriguing behaviour of neuronal VGSCs in response to site-3-specific modulators, indicating that these subtypes play different roles in BmK I-induced hyperexcitablity in rat models.

Epigallocatechin-3-gallate induces apoptosis, inhibits proliferation and decreases invasion of glioma cell.

Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, has been considered a potential therapeutic and chemopreventive agent for cancer. Glioma is a malignant tumor with high mortality but effective therapy has not yet been developed. In this study, we found that EGCG induced apoptosis in U251 glioma cells via the laminin receptor (molecular weight 67kDa) in a time- and dose-dependent manner, decreased their invasiveness and inhibited their proliferation. The mitogen-activated protein kinase pathway was shown to be involved in glioma cell apoptosis and proliferation. Furthermore, the mRNA levels of matrix metalloproteinase (MMP)-2 and MMP-9 were reduced after EGCG treatment. These results suggest that EGCG has important therapeutic effects with low toxicity and side-effects, and could be used in cancer chemoprevention.

Deciphering the Venomic Transcriptome of Killer-Wasp Vespa velutina

Wasp stings have been arising to be a severe public health problem in China in recent years. However, molecular information about lethal or toxic factors in wasp venom is extremely lacking. In this study, we used two pyrosequencing platforms to analyze the transcriptome of Vespa velutina, the most common wasp species native in China. Besides the substantial amount of transcripts encoding for allergens usually regarded as the major lethal factor of wasp sting, a greater abundance of hemostasis-impairing toxins and neurotoxins in the venom of V. velutina were identified, implying that toxic reactions and allergic effects are envenoming strategy for the dangerous outcomes. The pattern of differentially expressed genes before and after venom extraction clearly indicates that the manifestation of V. velutina stings depends on subtle regulations in the metabolic pathway required for toxin recruitment. This comparative analysis offers timely clues for developing clinical treatments for wasp envenoming in China and around the world.

Comparative pharmacology and cloning of two novel arachnid sodium channels: Exploring the adaptive insensitivity of scorpion to its toxins.

Scorpion toxins have been found lacking effect on Na+ current of its own sodium channel, whereas the molecular mechanism remains mystery. In this study, the binding affinity of pharmacologically distinct scorpion toxins was found much weaker to scorpion (Buthus martensii) nerve synaptosomes than to spider (Ornithoctonus huwena) ones. The sodium channel cDNA from these two species were further cloned. The deduced proteins contain 1871 and 1987 amino acids respectively. Several key amino acid substitutions, i.e., A1610V, I1611L and S1617K, are found in IVS3–S4 constituting receptor site‐3, and for receptor site‐4, two residues (Leu‐Pro) are inserted near IIS4 of scorpion sodium channel.

Activation of mammalian target of rapamycin mediates rat pain-related responses induced by BmK I, a sodium channel-specific modulator

The mammalian target of rapamycin (mTOR) is known to regulate cell proliferation and growth by controlling protein translation. Recently, it has been shown that mTOR signaling pathway is involved in long-term synaptic plasticity. However, the role of mTOR under different pain conditions is less clear. In this study, the spatiotemporal activation of mTOR that contributes to pain-related behaviors was investigated using a novel animal inflammatory pain model induced by BmK I, a sodium channel-specific modulator purified from scorpion venom. In this study, intraplantar injections of BmK I were found to induce the activation of mTOR, p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) in rat L5-L6 spinal neurons. In the spinal cord, mTOR, p70 S6K and 4E-BP1 were observed to be activated in the ipsilateral and contralateral regions, peaking at 1–2 h and recovery at 24 h post-intraplantar (i.pl.) BmK I administration. In addition, intrathecal (i.t.) injection of rapamycin – a specific inhibitor of mTOR – was observed to result in the reduction of spontaneous pain responses and the attenuation of unilateral thermal and bilateral mechanical hypersensitivity elicited by BmK I. Thus, these results indicate that the mTOR signaling pathway is mobilized in the induction and maintenance of pain-activated hypersensitivity.

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 DmNav1, the sodium channel from Drosophila, to more hyperpolarized potentials; whereas it hardly affected the gating properties of rNav1.2, rNav1.3 and mNav1.6, three mammalian central neuronal sodium channel subtypes. (1) Mutations of Glu896, Leu899, Gly904 in extracellular loop Domain II S3–S4 of DmNav1 abolished the functional action of BmK IT2. (2) BmK IT2-preference for DmNav1 could be conferred by Domain III. Analysis of subsequent DmNav1 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 DmNav1, play separated but indispensable roles in the interaction with BmK IT2. The insensitivity of Nav1.2, Nav1.3 and Nav1.6 to BmK IT2 suggests other isoforms or mechanism might be involved in the suppressive activity of BmK IT2 in rat pathological models.

Activation of mammalian target of rapamycin contributes to pain nociception induced in rats by BmK I, a sodium channel-specific modulator.

The mammalian target of rapamycin (mTOR) pathway is essential for maintenance of the sensitivity of certain adult sensory neurons. Here, we investigated whether the mTOR cascade is involved in scorpion envenomation-induced pain hypersensitivity in rats. The results showed that intraplantar injection of a neurotoxin from Buthus martensii Karsch, BmK I (10 μg), induced the activation of mTOR, as well as its downstream molecules p70 ribosomal S6 protein kinase (p70 S6K) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), in lumbar 5–6 dorsal root ganglia neurons on both sides in rats. The activation peaked at 2 h and recovered 1 day after injection. Compared with the control group, the ratios of p-mTOR/p-p70 S6K/p-4EBP1 in three types of neurons changed significantly. The cell typology of p-mTOR/p-p70 S6K/p-4E-BP1 immuno-reactive neurons also changed. Intrathecal administration of deforolimus, a specific inhibitor of mTOR, attenuated BmK I-induced pain responses (spontaneous flinching, paroxysmal pain-like behavior, and mechanical hypersensitivity). Together, these results imply that the mTOR signaling pathway is mobilized by and contributes to experimental scorpion sting-induced pain.

Knockdown of PKCε expression inhibits growth, induces apoptosis and decreases invasiveness of human glioma cells partially through Stat3.

Glioma is the most common primary central nervous system tumor. Despite considerable research effort, little progress has been made in the therapeutic treatment of this disease. Protein kinase Cε (PKCε), an important intracellular signaling molecule, modulates diverse cellular functions, including cell proliferation, apoptosis, invasion and differentiation. The aim of the study is to investigate whether knockdown of PKCε expression by RNA interference (RNAi) could affect the growth, apoptosis and invasion of human glioma cells, and the involvement of the signal transducer and activator of transcription 3 (Stat3) signaling pathway in these effects. Our data showed that knockdown of PKCε expression inhibited proliferation, induced apoptosis and decreased invasiveness of human glioma cell lines U251 and U87, as well as suppressed the growth of U87 cell-derived tumors in nude mice. Moreover, PKCε physically interacts with Stat3, and knockdown of PKCε expression attenuated Stat3Ser727 phosphorylation and B-cell lymphoma-extra large (Bcl-xL) expression in the two human glioma cell lines. These results support an important role for PKCε in glioma cell growth, apoptosis and invasion, and PKCε exerting its above effects at least in part through Stat3. Thus, PKCε has the potential to be an attractive therapeutic target for glioma therapy.

Visual deprivation induce cross-modal enhancement of olfactory perception

The underlying mechanisms responsible for enhanced olfactory perception of congenital blind humans remain elusive so far. Here, animal behavioral test showed that congenital visual deprivation (from postnatal day 0-28) or one-week visual deprivation during juvenile stage (from postnatal day 21-28) could reduce the latency time of food-seeking but increase the odor discrimination performance of rodents. The enhanced olfactory perception induced by one-week visual deprivation could be returned to base level when visual input was recovered. Accordingly, local field potential (LFP) oscillation recording inxa0vivo showed that the power of high-frequency β and γ oscillations were increased in olfactory bulb (OB) and anterior piriform cortex (aPC) of vision deprived animals. This research discovered the enhancement of olfactory perception and adaptive plasticity of oscillations in olfactory system of rodents induced by visual deprivation, which may facilitate better understanding of mechanisms underlying cross-modal plasticity.