The East Asian scorpion Buthus martensii Karsch (BmK) is one of the classical traditional Chinese medicines for treating epilepsy for over a thousand years. Neurotoxins purified from BmK venom are considered as the main active ingredients, acting on membrane ion channels. Voltage-gated sodium channels (VGSCs) play a crucial role in the occurrence of epilepsy, which make them become important drug targets for epilepsy. Long chain toxins of BmK, composed of 60-70 amino acid residues, could specifically recognize VGSCs. Among them, α-like neurotoxins, binding to the receptor site-3 of VGSC, induce epilepsy in rodents and can be used to establish seizure models. The β or β-like neurotoxins, binding to the receptor site-4 of VGSC, have significant anticonvulsant effects in epileptic models. This review aims to illuminate the anticonvulsant/convulsant effects of BmK polypeptides by acting on VGSCs, and provide potential frameworks for the anti-epileptic drug-design.
Animals ; Anticonvulsants/therapeutic use* ; Neurotoxins/pharmacology* ; Scorpion Venoms/pharmacology* ; Scorpions/chemistry* ; Voltage-Gated Sodium Channels

Voltage-gated sodium channels (VGSCs), which are widely distributed in the excitable cells, are the primary mediators of electrical signal amplification and propagation. They play important roles in the excitative conduction of the neurons and cardiac muscle cells. The abnormalities of the structures and functions of VGSCs can change the excitability of the cells, resulting in a variety of diseases such as neuropathic pain, epilepsy and arrhythmia. At present, some voltage-gated sodium channel blockers are used for treating those diseases. In the recent years, several neurotoxins have been purified from the venom of the animals, which could inhibit the current of the voltage-gated sodium channels. Usually, these neurotoxins are compounds or small peptides that have been further designed and modified for targeted drugs of sodium channelopathies in the clinical treatment. In addition, a novel cysteine-rich secretory protein (CRBGP) has been isolated and purified from the buccal gland of the lampreys (Lampetra japonica), and it could inhibit the Na+ current of the hippocampus and dorsal root neurons for the first time. In the present study, the progress of the sodium channelopathies and the biological functions of voltage-gated sodium channel blockers are analyzed and summarized.
Animals ; Channelopathies ; physiopathology ; Hippocampus ; drug effects ; Neurons ; drug effects ; Neurotoxins ; pharmacology ; Venoms ; chemistry ; Voltage-Gated Sodium Channel Blockers ; pharmacology

PURPOSE: To examine whether citicoline has a neuroprotective effect on kainic acid (KA) -induced retinal damage. METHODS: KA (6 nmol) was injected into the vitreous of rat eyes. Citicoline (500mg/kg, i.p.) was administered to the rats once before and twice a day after KA-injection for 3- and 7-day intervals. The neuroprotective effects of citicoline were estimated by measuring the thickness of the various retinal layers using hematoxylin-eosin (H and E) staining. In addition, immunohistochemistry was conducted to elucidate the expression of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS). RESULTS: Morphometric analysis of retinal damage in KA-injected eyes showed significant cell loss in the inner nuclear layer (INL) and inner plexiform layer (IPL) of the retinas at 3 and 7 days after KA injection, but not in the outer nuclear layer (ONL). At 3 days after citicoline treatment, no significant changes were detected in the retinal thickness and immunoreactivities of eNOS and nNOS. The immunoreactivities of eNOS and nNOS increased in the retina at 7 days after the KA injection. However, prolonged treatment for 7 days significantly attenuated the immunoreactivities and the reduction of thickness. CONCLUSIONS: The results indicate that citicoline has a neuroprotective effect on KA-induced neurotoxicity in the retina.
Retina/*drug effects/*pathology ; Rats, Sprague-Dawley ; Rats ; Neurotoxins/*pharmacology ; Neuroprotective Agents/*pharmacology ; Male ; Kainic Acid/*pharmacology ; Cytidine Diphosphate Choline/*pharmacology ; Animals

Animals ; Bacterial Toxins ; chemistry ; pharmacology ; therapeutic use ; Humans ; Neoplasms ; therapy ; Neurotoxins ; chemistry ; pharmacology ; therapeutic use ; Proteins ; chemistry ; pharmacology ; therapeutic use ; Toxins, Biological ; chemistry ; pharmacology ; therapeutic use

OBJECTIVETo investigate the absorption enhancen effect of borneol/mentholum eutectic mixture (BO/ME) on nasal-brain delivery of neurotoxin loaded nanoparticles.

METHODUsing microdialysis sampling technique in awake freely-moving rats, the counter per minute (cpm) of dialysates in right PAG of NT-loaded nanoparticles with the BO/ME (BO/ME-NT-NP), radiolabeled with sodium 125I-Iodide, were measured in a gamma-counter for radioactivity. After converting cpm into corresponding concentrations of NT byin vivorecovery of microdialysis probes, the pharmacokinetic parameters were calculated.

RESULTThe BO/ME-NT-NP could be absorbed into the brain, much better to NT-NP and the nanoparticles with borneol or menthdlum only, and the pharmacokinetics accorded with the two-compartment model. The parameters tmax, cmax, AUC, t 1/2(beta) were 0.68 h, 27.32 ng x mL(-1), 132.68 ng x h x mL(-1), 3.1076 h.

CONCLUSIONWith adding BO/ME as absorption enhancer, NT could be significantly increased in the brain with the help of nanopartilces as carriers, and the time to maximal concentration was short, the elimination process was prolonged.

Absorption ; drug effects ; Animals ; Bornanes ; chemistry ; pharmacology ; Brain ; metabolism ; Drug Carriers ; pharmacokinetics ; Male ; Menthol ; chemistry ; pharmacology ; Microdialysis ; Nanoparticles ; Nasal Cavity ; metabolism ; Neurotoxins ; administration & dosage ; pharmacokinetics ; Rats

OBJECTIVETo investigate the neurotoxicity and its mechanism of quinolinic acid (QA) to spiral ganglion cells (SGC) and observe the protectable potential of MgCl(2) on SGC.

METHODSSGC were cultured in vitro for 72 h, and then were divided into 4 groups: control group, QA group (1 mmol/L QA), MK-801 group (1 mmol/L QA + 20 µmol/L MK-801)and MgCl(2) protected group (1 mmol/L QA + 1 mmol/L MgCl(2)). SGC apoptosis rate was analyzed by Annexin V staining and PI staining measurements after 24 h exposure to different medium. SGC cultured as methods above were divided into 4 groups as following: 100 µmol/L QA, 1 mmol/L QA, 20 µmol/L MK-801+1 mmol/L QA and 1 mmol/L MgCl(2) + 1 mmol/L QA. The intracellular calcium concentration was measured by laser scanning confocal microscope finally.

RESULTSApoptosis rate in QA group was higher than that in both of control group (59.1% ± 7.5% vs 9.2% ± 0.9%, x ± s, q = 11.9, P < 0.05) and MgCl(2) group (59.1% ± 7.5% vs 27.5% ± 8.3%, q = 7.5, P < 0.05). There was no significant difference between apoptosis rate of control and MK-801 group (12.8% ± 5.7% vs 9.2% ± 0.9%, q = 0.9, P > 0.05). It was shown that there was a significant increase of Ca(2+) in SGC in the presence of QA by laser scanning confocal microscope. MK-801 may completely block the increase of Ca(2+), and the increase of Ca(2+) can be reduce by the application of MgCl(2).

CONCLUSIONSQA might injure SGC by excessive activating NMDA receptors on the cell membrane. Mg(2+) may have the function to reduce the neurotoxicity of QA.

Animals ; Calcium ; analysis ; Cells, Cultured ; Magnesium Chloride ; pharmacology ; Neurotoxins ; toxicity ; Quinolinic Acid ; toxicity ; Rats ; Rats, Sprague-Dawley ; Spiral Ganglion ; cytology ; drug effects ; metabolism

Voltage-gated sodium channels (VGSCs) are transmembrane proteins responsible for generation and conduction of action potentials in excitable cells. Physiological and pharmacological studies have demonstrated that VGSCs play a critical role in chronic pain associated with tissue or nerve injury. Many long-chain peptide toxins (60-76 amino acid residues) purified from the venom of Asian scorpion Buthus martensii Karsch (BmK) are investigated to be sodium channel-specific modulators. The alpha-like neurotoxins that can bind to receptor site 3 of sodium channels, named as BmK I and BmK abT, could induce nociceptive effects in rats. On the contrast, the beta-like neurotoxins that can bind to receptor site 4 of sodium channels, named as BmK AS, BmK AS-1 and BmK IT2, could produce potent anti-nociceptive effects in animal pain models. BmK I could strongly prolong the fast inactivation of tetrodotoxin (TTX)-sensitive Na(+) currents on the rat dorsal root ganglia (DRG) neurons together with the augmentation of peak current amplitude. However, BmK IT2 and BmK ASs, potently suppressed both the peak TTX-resistant and TTX-sensitive Na(+) currents on rat small DRG neurons. Moreover, BmK ASs could decrease the excitability of small DRG neurons. Thus, the nociception/anti-nociception induced by scorpion neurotoxins may attribute to their distinct modulation on sodium channels in primary afferent sensory neurons. Therefore, the sodium channel-specific modulators from BmK venom could be used as not only pharmacological tools for better understanding the roles of VGSCs in pain signal conduction, but also lead molecules in the development of ideal analgesics targeting VGSCs.
Action Potentials ; Animals ; Ganglia, Spinal ; drug effects ; Neurons, Afferent ; drug effects ; Neurotoxins ; pharmacology ; Pain ; drug therapy ; Peptides ; pharmacology ; Rats ; Scorpion Venoms ; pharmacology ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; metabolism

Kv2.1 channel currents in pancreatic beta-cells are thought to contribute to action potential repolarization and thereby modulate insulin secretion. Because of its central role in this important physiological process, Kv2.1 channel is a promising target for the treatment of type 2 diabetes. Jingzhaotoxin-XI (JZTX-XI) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Two-microelectrode voltage clamp experiments had showed that the toxin inhibited Kv2.1 potassium currents expressed in Xenopus Laevis oocytes. In order to investigate the structure-function relationship of JZTX-XI, the natural toxin and a mutant of JZTX-XI in which Arg3 was replaced by Ala, were synthesized by solid-phase chemistry method with Fmoc-protected amino acids on the PS3 automated peptide synthesizer. Reverse-phase high performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) were used to monitor the oxidative refolding process of synthetic linear peptides to find the optimal renaturation conditions of these toxins. The experiments also proved that the relative molecular masses of refolded peptides were in accordance with their theoretical molecular masses. RP-HPLC chromatogram of co-injected native and refolded JZTX-XI was a single peak. Under the whole-cell patch-clamp mode, JZTX-XI could completely inhibit hKv2.1 and hNav1.5 channels currents expressed in HEK293T cells with IC50 values of 95.8 nmol/L and 437.1 nmol/L respectively. The mutant R3A-JZTX-XI could also inhibit hKv2.1 and hNav1.5 channel currents expressed in HEK293T cells with IC50 values of 1.22 micromol/L and 1.96 micromol/L respectively. However, the prohibitive levels of R3A-JZTX-XI on hKv2.1 and hNav1.5 channels were reduced by about 12.7 times and 4.5 times respectively, indicating that Arg3 was a key amino acid residue relative to the hKv2.1 channel activity of JZTX-XI, but it is also an amino acid residue correlated with the binding activity of JZTX-XI to hNav1.5 channel. Our findings should be helpful to develop JZTX-XI into a molecular probe and drug candidate targeting to Kv2.1 potassium channel in the pancreas.
Animals ; HEK293 Cells ; Humans ; Insulin-Secreting Cells ; metabolism ; Mutant Proteins ; genetics ; pharmacology ; NAV1.5 Voltage-Gated Sodium Channel ; metabolism ; Neurotoxins ; chemical synthesis ; genetics ; pharmacology ; Protein Refolding ; Shab Potassium Channels ; antagonists & inhibitors ; metabolism ; Sodium Channel Blockers ; pharmacology ; Spider Venoms ; genetics ; pharmacology ; Transfection

Kv4.3 channel is present in many mammalian tissues, predominantly in the heart and central nervous system. Its currents are transient, characterized by rapid activation and inactivation. In the hearts of most mammals, it is responsible for repolarization of the action potential of ventricular myocytes and is important in the regulation of the heart rate. Because of its central role in this important physiological process, Kv4.3 channel is a promising target for anti-arrhythmic drug development. Jingzhaotoxin-V (JZTX-V) is a novel peptide neurotoxin isolated from the venom of the spider Chilobrachys jingzhao. Whole-cell patch clamp recording showed that it partly blocked the transient outward potassium channels in dorsal root ganglion neurons of adult rats with an IC(50) value of 52.3 nmol/L. To investigate the effect of JZTX-V on Kv4.3 channel, JZTX-V was synthesized using the solid-phase chemical synthesis and separated by reverse phase high performance liquid chromatography (HPLC). The purity was tested by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MOLDI-TOF mass spectrometry). Two-electrode voltage-clamp technique was used to characterize the action of JZTX-V on Kv4.3 channels expressed in Xenopus laevis oocytes. As a result, JZTX-V displayed fast kinetics of inhibition and recovery from inactivation. Furthermore, it could inhibit Kv4.3 channel current in a time- and concentration-dependent manner with an IC(50) value of 425.1 nmol/L. The application of JZTX-V affected the activation and inactivation characteristics of Kv4.3 channel and caused a shift of the current-voltage relationship curve and the steady-state inactivation curve to depolarizing direction by approximately 29 mV and 10 mV, respectively. So we deduced that JZTX-V is a gating modifier toxin of Kv4.3 channel. Present findings should be helpful to develop JZTX-V into a molecular probe and drug candidate targeting to Kv4.3 channel in the myocardium.
Animals ; Ganglia, Spinal ; cytology ; Neurons ; drug effects ; Neurotoxins ; pharmacology ; Oocytes ; Patch-Clamp Techniques ; Peptides ; pharmacology ; Potassium Channel Blockers ; pharmacology ; Rats ; Shal Potassium Channels ; metabolism ; Spider Venoms ; pharmacology ; Xenopus laevis

In this study, cardiotonic and cardiotoxic effects of Buthus martensi Karsch (BmK) I, a modulator of voltage-gated sodium channels, were investigated on the isolated rat hearts. The results showed that BmK I evoked complex effects characterized by a change in both cardiac mechanical and electrical activity. Langendorff perfusion showed that: (1) maximal left ventricular developed pressure (LVDP(max)) and dp/dt(max) were markedly increased by BmK I (0.5-10 micromol/L) in a dose-dependent manner (n=6, P<0.05), positive chronotropic effects were also induced by BmK I (n=6, P<0.05); (2) negative inotropic action and bradycardia could be elicited at a larger dose of BmK I (20 micromol/L); (3) the coronary flow varied inversely with the positive inotropic effects, coronary flow reduced during positive inotropic effects from 14.5 to 8.6 ml/min after administration of 500 nmol/L BmK I (n=6, P<0.05). In addition, tachycardia and complex cardiac arrhythmias were induced by BmK I (0.5-10 micromol/L). The modulating of BmK I on the heart mechanical, electrical activity could be partially recovered after washing. As propranolol was applied to block the release of catecholamines before administration of BmK I, suggesting that the changes in cardiac mechanical and electrical activity induced by BmK I might not due to catecholamine release from the nerve terminal and subsequent stimulation of the beta-adrenoceptor but attributable to the modulation of BmK I on cardiac voltage-gated sodium channels.
Action Potentials ; drug effects ; Animals ; Electrophysiology ; In Vitro Techniques ; Insect Proteins ; Male ; Myocardial Contraction ; drug effects ; NAV1.5 Voltage-Gated Sodium Channel ; Neurotoxins ; pharmacology ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Scorpion Venoms ; pharmacology ; Sodium Channel Blockers ; pharmacology ; Sodium Channels ; drug effects