Effects of Intrathecally Administerd NaV1.8 Antisense Oligonucleotide on the Expression of Sodium Channel mRNA in Dorsal Root Ganglion

Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1.8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion （DRG） neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200--260 g were anesthetized with the intraperitoneal injection of 300 mg· kg^-1 choral hydrate. The CCI model was made by loose ligation of sciatic nerve trunk by 4--0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2--4. The animals were decapitated 14 days after the surgery. The L4--L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1.8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1.8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 μg as compared with that in CCI group （P〈0.01）, and it was even greater at the dosage of 90 μg. The intrathecally injected NaV1.8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.

Deglycosylation altered the gating properties of rNav1.3:glycosylation/deglycosylation homeostasis probably complicates the functional regulation of voltage-gated sodium channel

Objective To examine the effect of deglycosylation on gating properties of rNav1.3. Methods rNav1.3 was expressed in Xenopus oocyte, with glycosylation inhibition by using tunicamycin. Two-electrode voltage clamp was employed to record the whole-cell sodium current and data were analyzed by Origin software. Those of glycosylated rNav1.3 were kept as control. Results Compared with glycosylated ones, the steady-state activation curve of deglycosylated rNav1.3 was positively shifted by about 10 mV, while inactivation curve was negatively shifted by about 8 mV. Conclusion Glycosylation altered the gating properties of rNav 1.3 and contributed to the functional diversity.

Targeting voltage-gated sodium channels for treatment for chronic visceral pain

Voltage-gated sodium channels (VGSCs) play a fundamental role in controlling cellular excitability,and their abnormal activity is related to several pathological processes,including cardiac arrhythmias,epilepsy,neurodegenerative diseases,spasticity and chronic pain.In particular,chronic visceral pain,the central symptom of functional gastrointestinal disorders such as irritable bowel syndrome,is a serious clinical problem that affects a high percentage of the world population.In spite of intense research efforts and after the dedicated decade of pain control and research,there are not many options to treat chronic pain conditions.However,there is a wealth of evidence emerging to give hope that a more refined approach may be achievable.By using electronic databases,available data on structural and functional properties of VGSCs in chronic pain,particularly functional gastrointestinal hypersensitivity,were reviewed.We summarize the involvement and molecular bases of action of VGSCs in the pathophysiology of several organic and functionalgastrointestinal disorders.We also describe the efficacy of VGSC blockers in the treatment of these neurological diseases,and outline future developments that may extend the therapeutic use of compounds that target VGSCs.Overall,clinical and experimental data indicate that isoform-specific blockers of these channels or targeting of their modulators may provide effective and novel approaches for visceral pain therapy.

A novel mutation in the sodium channel α1 subunit gene in a child with Dravet syndrome in Turkey

Dravet syndrome is a rare epileptic encephalopathy characterized by frequent seizures beginning in the first year of life and behavioral disorders. Mutations in the sodium channel α1 subunit gene are the main cause of this disease. We report two patients with refractory seizures and psychomotor retardation in whom the final diagnosis was Dravet syndrome with confirmed mutations in the sodium channel α1 subunit gene. The mutation identified in the second patient was a novel frame shift mutation, which resulted from the deletion of five nucleotides in exon 24.

Effect of orphanin FQ and morphine on sodium channel current in somatosensory area of rat cerebral cortex

BACKGROUND： Some experiments have demonstrated that injecting orphanin FQ （OFQ） into lateral ventricle, which can obviously decrease the pain threshold. It is indicated that OFQ is an anti-opiate substance. However, whether OFQ has effects on sensory neuron ion channel in cerebral cortex needs to be further studied. OBJECTIVE： To investigate the effects of OFQ, morphine or their combination on sodium channel current of somatosensory neurons in rat cerebral cortex. DESIGN： Repeated measurement trial. SETTING： Department of Physiology, Harbin Medical University. MATERIALS： Fifty healthy Wistar rats, aged 12 - 16 days, of either gender, were provided by the Experimental Animal Center, Second Hospital Affiliated to Harbin Medical University. OFQ was purchased from Sigma-Aldrich Company, and morphine was provided by the Shenyang First Pharmaceutical Factory. PC2C patch clamp amplifier and LabmasterTLlwere purchased from Yibo Life Science Instrument Co.,Ltd. of Huazhong University of Science and Techgnology. METHODS： This experiment was carried out in the Department of Physiology （provincial laboratory）, Harbin Medical University between January 2005 and May 2006. Cortical neurons were acutely isolated from rats, and prepared into cell suspension following culture. ①Sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by whole-cell Patch clamp technique after 50 nmol/L OFQ being added to extracellular fluid. ②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was recorded before and after administration by the same method after 20 μmol/L morphine being added to extracellular fluid, and then the change of sodium channel current was recorded after 50 nmol/L OFQ being added. MAIN OUTCOME MEASURES： The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex following the administration of OFQ, morphine separately or their combination.. RESULTS： ①The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was significantly lower after administration of 50 nmol/L OFQ than before at the clampe of the voltage of -30 mV （P 〈 0.05）.②The amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex was significantly lower after administration of 20 μmol/L morphine than before at the clampe of the voltage of-30 mV （P 〈 0.05）. The sodium channel current recovered to - （2 345.24±174.18） pA after 50 nmol/L OFQ was administrated. There were significant differences in the amplitude of Na^＋ channel current between two interventions （P 〈 0.05）. CONCLUSION： Morphine and OFQ can respectively reduce the amplitude of sodium channel current of somatosensory neurons in rat cerebral cortex, and OFQ can reverse the effect of morphine partly. It is indicated that OFQ can produce antiopioid activity in the central nervous system by influencing sodium channel current.

The anti-nociceptive effect of bufalin,an active ingredient from toad venom via modulating voltage-gated sodium channels

OBJECTIVE Toad venom(Venenum Bufonis)isalways used for analgesia in China from ancient to modern times,but the effective component of it remains unclear.METHODS In the present study,we investigated the anti-nociceptive effect and the underlying mechanism ofbufalin,an active ingredient fromtoad venom by animal behavior,patch clamp and calcium imaging.RESULTS Bufalin could significantly relieve formalin-induced spontaneous flinching and licking response as well as carrageenan-induced mechanical and thermal hyperalgesia.Using the whole-cel patch-clamp recording,bufalincaused remarkable suppressive effect on the peak currents of Na+channels in dorsal root ganglion neuroblastoma ND7-23 cel line in a U-shaped dependent manner.In addition,bufalinprompted the voltage-dependent activationand caused a negative shift of the fast-state inactivation of Na+channels.However,bufalin produced insignificant effect not onlyon voltage-dependent Kv4.2,Kv4.3 and BK channels,but also on the capsaicin induced Ca2+influx.CONCLUSION The present results indicate bufalin is capable of producing remarkable anti-nociceptive effects whichis probably ascribed to its specific modulation of voltage-gated Na+channels.

Cloning of Partial Sodium Channel Gene From Strains of Fenvalerate-Resistant and Susceptible Cotton Aphid(Aphis gossypii Glover)

The strain of fenvalerate-resistant cotton aphids was selected using fenvalerate insecticide in the laboratory, the resistance factor of the strain was 199.54. Three degenerate primers were designed and used to perform PCR amplification. A cDNA encoding partial sodium channel gene was cloned from the fenvalerate-resistant and -susceptible strains. There were two nucleotide acid differences between fenvalerate-resistant strain and -susceptible strain, resulting in an amino acid mutation, Met→Leu. It is predicted that the mutation is related to the cotton aphid resistance to fenvalerate.

Voltage-gated Sodium Channels and Blockers:An Overview and Where Will They Go?

Voltage-gated sodium(Nav)channels are critical players in the generation and propagation of action potentials by triggering membrane depolarization.Mutations in Nav channels are associated with a variety of channelopathies,which makes them relevant targets for pharmaceutical intervention.Sofar,the cryoelectron microscopic structure of the human Nav 1.2,Nav 1.4,and Nav 1.7 has been reported,which sheds light on the molecular basis of functional mechanism of Nav channels and provides a path toward structure-based drug discovery.In this review,we focus on the recent advances in the structure,molecular mechanism and modulation of Nav channels,and state updated sodium channel blockers for the treatment of pathophysiology disorders and briefly discuss where the blockers may be developed in the future.

Expression of hNav1.8 sodium channel protein in affected nerves of patients with trigeminal neuralgia

Objective: To explore the pathogenesis of trigeminal neuralgia (TN) and to provide a new target for the drug treatment of TN by studying the expression of tetrodotoxin-resistant hNav1. 8 sodium channel protein in affected nerves of patients with TN. Methods: Twelve affected inferior alveolar nerves were obtained from patients with idiopathic TN, to whom the drug therapy was not effective. As negative control, one nonnal inferior alveolar nerve was obtained from patients who accepted the combined radical neck dissection with glossectomy and mandibulectomy. One muscle sample was obtained as normal control. One dorsal root ganglion from rat was as positive control. These tissues and prepared hNav1. 8 antibody were conducted immunohistochemistry response. Results: hNav1.8 channel protein was expresses in all the 12 specimens of the affected nerves of patients with TN, but not in the muscle sample and the normal inferior alveolar nerve. Conclusion: The abnormal expression of hNav1. 8 channel protein in the affected nerves of patients with TN may play an important role in the pathogenesis of TN.

PROPERTIES OF VOLTAGE-GATED SODIUM CHANNELS IN DEVELOPING AUDITORY NEURONS OF THE MOUSE IN VITRO

Objective. To investigate the properties of voltage-gated sodium (Na+) channels in developing auditoryneurons during early postnatal stages in the mammalian central nervous system.Methods. Using the whole-cell voltage-clamp technique, we have studied changes in the electrophysi-ological properties of Na+ channels in the principal neurons of the medial nucleus of the trapezoid body (MNTB).Results. We found that MNTB neurons already express functional Na+ channels at postnatal day 1 (P1),and that channel density begins to increase at P5 when the neurons receive synaptic innervation andreach its maximum (～3 fold) at P11 when functional hearing onsets. These changes were paralleled byan age-dependent acceleration in both inactivation and recovery from inactivation. In contrast, there wasvery little alteration in the voltage-dependence of inactivation.Conclusion. These profound changes in the properties of voltage-gated Na+ channels may increase theexcitability of MNTB neurons and enhance their phase-locking fidelity and capacity during high-frequencysynaptic transmission.

Research Progress on the Influence of Structural Changes inμ-Conotoxins on Sodium Channel Receptors

The voltage-gated sodium channel(Na v)is widely present in mammals and can generate cell action potentials,which are related to many diseases.Theμ-Conotoxins(μ-CTx)isolated from the venom of cone snails can specifically block the voltage-gated sodium channel;it can be widely used as a necessary probe to distinguish the Na v channel subtypes.In this study,the effects of eightμ-CTx on different Na v channel isoforms were reviewed,and sequence alignment and protein homologous modeling were used to predict their biological activities,and the structure-activity relationship betweenμ-CTx and mutagenesis strategies.

The study of sodium channels involved in pain responses using specific modulators

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 α-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 β-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.

Molecular Docking Studies on Anticonvulsant Enaminones Inhibiting Voltage-Gated Sodium Channels

Epilepsy is described as the most common chronic brain disorder. A typical symptom of epilepsy results in uncontrolled convulsions caused by temporary excessive neuronal discharges. Although several new anticon-vulsants have been introduced, some types of seizures have still not been adequately controlled with these new and current therapies. There is an urgent need to develop new anticonvulsant drugs to control the many different types of seizures. Many studies have shown that the epilepsies involve more than one mechanism and therefore may be responsible for the various types of observed seizures. Recently reported studies have shown that a group of newly synthesized 6 Hz active anticonvulsant fluorinated N-benzamide enaminones exhibited selective inhibitions of voltage-gated sodium (Nav) channels. Nav channels are responsible for the initial inward currents during the depolarization phases of the action potential in excitable cells. The activation and opening of Nav channels result in the initial phases of action potentials. We hypothesize that there is an essential pharmacophore model for the interactions between these enaminones and the active sites of Nav channels. The research reported here is focused on molecular docking studies of the interactions that occur between the fluorinated N-benzamide enaminones and the Nav channels. These studies may open an avenue for designing anticonvulsant drugs by inhibiting Nav channels.

Design of artificial biomimetic channels with Na^(+)permeation rate and selectivity potentially outperforming the natural sodium channel

Artificial ion channels that enable high-efficiency ion transport have important implications in nanofluidics and biomedical applications such as drug delivery.Herein,we show a simulation-based chemical design of a biomimetic sodium channel that possesses permeation rate and selectivity potentially higher than those of the state-of-the-art natural vertebrate voltage-gated sodium channels.Importantly,our theoretical findings have undergone empirical testing,aligning well with the Arrhenius law as derived from a diverse range of experimental results.The high-efficiency ion transport is achieved by anchoring the carboxylate functional groups within the channel filter.A key chemical guiding principle underlying the ion channel design is that the free-energy barrier for the Na^(+)passage across the channel should be comparable to typical thermal energy at room temperature.With the implementation of the chemical design,we found that the relatively low free-energy barrier can be attributed to the compensation effect of the carboxylate groups to the partially lost oxygen shell of the ion within the ion channel,as well as to the consonant vibration of the ions inside and outside the channel.This mechanistic understanding brings new insight,at the molecular level,into the high-efficiency ion transport across the designed membrane channels.The proof of principle achieved from the simulations will stimulate future experimental confirmation and potential applications of the high-performance artificial channels in nanofluidics and in bioinspired iontronics.

Modulation of BmKAS-1 and BmK1-3-2 to sodium channel in rat dorsal root ganglion neurons

Objective To investigate what effects BmKAS 1 (a polypeptide purified from the Chinese scorpion Buthus martensi Karsch [BmK] and named as BmK activator of skeletal muscle ryanodine receptor) and its upstream mixture BmK1 3 2 have on Na + channels in dorsal root ganglion (DRG) small diameter neurons Methods The whole cell patch clamp technique was used to investigate the effects of BmKAS 1 and BmK1 3 2 on Na + current in rat small diameter DRG neurons Results About 50% peak Na + current was suppressed by 10?μg/ml of BmK1 3 2 1 62?μg/ml of BmKAS 1 also blocked 50% peak Na + current, and there was an obvious dose dependent relationship Conclusion Both BmK1 3 2 and BmKAS 1 have a blocking effect on Na + channels, and this may one of the mechanisms for the analgetic effect of BmK1 3 2 and BmKAS 1

Two novel sodium channel mutations associated with resistance to indoxacarb and metaflumizone in the diamondback moth, Plutella xylostella

Indoxacarb and metaflumizone belong to a relatively new class of sodium channel blocker insecticides （SCBIs）. Due to intensive use of indoxacarb, field-evolved indoxacarb resistance has been reported in several lepidopteran pests, including the dia- mondback moth Plutella xylostella, a serious pest of cruciferous crops. In particular, the BY 12 population of P. xylostella, collected from Baiyun, Guangdong Province of China in 2012, was 750-fold more resistant to indoxacarb and 70-fold more resistant to metaflu- mizone compared with the susceptible Roth strain. Comparison of complementary DNA sequences encoding the sodium channel genes of Roth and BY12 revealed two point mu- tations （F1845Y and V1848I） in the sixth segment of domain IV of the PxNav protein in the BY population. Both mutations are located within a highly conserved sequence region that is predicted to be involved in the binding sites of local anesthetics and SCBIs based on mammalian sodium channels. A significant correlation was observed among 10 field-collected populations between the mutant allele （Y 1845 or I 1848） frequencies （1.7% to 52.5%） and resistance levels to both indoxacarb （34- to 870-fold） and metaflumizone （1- to 70-fold）. The two mutations were never found to co-exist in the same allele of PxNav, suggesting that they arose independently. This is the first time that sodium channel muta- tions have been associated with high levels of resistance to SCBIs. F 1845Y and V 1848I are molecular markers for resistance monitoring in the diamondback moth and possibly other insect pest species.

Expression and Role of Voltage-Gated Sodium Channels in Human Dorsal Root Ganglion Neurons with Special Focus on Nav1.7,Species Differences, and Regulation by Paclitaxel

Voltage-gated sodium channels （Navs） play an important role in human pain sensation. However, the expression and role of Nav subtypes in native human sensory neurons are unclear. To address this issue, we obtained human dorsal root ganglion （hDRG） tissues from healthy donors. PCR analysis of seven DRG-expressed Nav subtypes revealed that the hDRG has higher expression of Navl.7 （,-~ 50% of total Nav expression） and lower expres- sion of Navl.8 （~ 12%）, whereas the mouse DRG has higher expression of Nav 1.8 （- 45%） and lower expression of Navl.7 （- 18%）. To mimic Nav regulation in chronic pain, we treated hDRG neurons in primary cultures with paclitaxel （0.1-1 μmol/L） for 24 h. Paclitaxel increased the Navl.7 but not Navl.8 expression and also increased the transient Na＋ currents and action potential firing frequency in small-diameter （〈50 ~tm） hDRG neurons. Thus, the hDRG provides a translational model in which to study ＂human pain in a dish＂ and test new pain therapeutics.

Role of Epithelium Sodium Channel in Bone Formation

Objective：To review the recent developments in the mechanisms of epithelium sodium channels （ENaCs） induced bone formation and regulation.Data Sources：Studies written in English or Chinese were searched using Medline,PubMed and the index of Chinese-language literature with time restriction from 2005 to 2014.Keywords included ENaC,bone,bone formation,osteonecrosis,estrogen,and osteoporosis.Data from published articles about the structure of ENaC,mechanism of ENaC in bone formation in recent domestic and foreign literature were selected.Study Selection：Abstract and full text of all studies were required to obtain.Studies those were not accessible and those did not focus on the keywords were excluded.Results：ENaCs are tripolymer ion channels which are assembled from homologous α,β,and γ subunits.Crystal structure of ENaCs suggests that ENaC has a central ion-channel located in the central symmetry axis of the three subunits.ENaCs are protease sensitive channels whose iron-channel activity is regulated by the proteolytic reaction.Channel opening probability of ENaCs is regulated by proteinases,mechanical force,and shear stress.Several molecules are involved in regulation of ENaCs in bone formation,including nitride oxide synthases,voltage-sensitive calcium channels,and cyclooxygenase-2.Conclusion：The pathway of ENaC involved in shear stress has an effect on stimulating osteoblasts even bone formation by estrogen interference.

Regulation of epithelial sodium channel ^-subunit expression by adenosine receptor A2a in alveolar epithelial ceils

Background The amiloride-sensitive epithelial sodium channel a-subunit （a-ENaC） is an important factor for alveolar fluid clearance during acute lung injury. The relationship between adenosine receptor A2a （A2aAR） expressed in alveolar epithelial cells and aα-ENaC is poorly understood. We targeted the A2aAR in this study to investigate its role in the expression of αa-ENaC and in acute lung injury.Methods A549 cells were incubated with different concentrations of A2aAR agonist CGS-21680 and with 100 μmol/L CGS-21680 for various times. Rats were treated with lipopolysaccharide （LPS） after CGS-21680 was injected. Animals were sacrificed and tissue was harvested for evaluation of lung injury by analysis of the lung wet-to-dry weight ratio, lung permeability and myeloperoxidase activity. RT-PCR and Western blotting were used to determine the mRNA and protein expression levels of α-ENaC in A549 cells and alveolar type II epithelial cells.Results Both mRNA and protein levels of α-ENaC were markedly higher from 4 hours to 24 hours after exposure to 100μmol/L CGS-21680. There were significant changes from 0.1 umol/L to 100 μmol/L CGS-21680, with a positive correlation between increased concentrations of CGS-21680 and expression of α-ENaC. Treatment with CGS-21680during LPS induced lung injury protected the lung and promoted α-ENaC expression in the alveolar epithelial cells.Conclusion Activation of A2aAR has a protective effect during the lung injury, which may be beneficial to the prognosis of acute lung injury

Effects of external stimuli on the pacemaker function of the sinoatrial node in sodium channel gene mutations models

Loss of function and gain of function mutations of the sodium channel were investigated using an intact two-dimensional rabbit sinoatrial node (SAN) and atrial cell model. The effects of three external stimuli (acetylcholine secretion by the vagal nerve, acid-base concentration, and tissue temperature) on cardiac pacemaker function and conduction were studied. Our results show that these two groups of mutations have different effects on pacemaker function and conduction. Furthermore, we found that the negative effects of these mutations could be altered by external stimuli. The bradycardic effects of mutations were magnified by an increase in acetylcholine level. Changes in acid-base concentration and tissue temperature increased the ability of the SAN to recover its pacemaker function. The results of this study increase our understanding of sodium channel disorders, and help to advance research on the treatment of these conditions.