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OBJECTIVES@#To isolate potassium ion channel Kv4.1 inhibitor from centipede venom, and to determine its primary and spatial structure.@*METHODS@#Ion-exchange chromatography and reversed-phase high-performance liquid chromatography were performed to separate and purify peptide components of centipede venom, and their inhibiting effect on Kv4.1 channel was determined by whole-cell patch clamp recording. The molecular weight of isolated peptide Kv4.1 channel inhibitor was identified with MALDI-TOF, its primary sequence was determined by Edman degradation sequencing and two-dimensional mass spectrometry, its patial structure was established based on iterative thread assembly refinement online analysis.@*RESULTS@#A peptide SsTx-P2 was separated from centipede venom with the molecular weight of 6122.8, and its primary sequence consists of 53 amino acid residues, showed as NH2-ELTWDFVRTCCKLFPDKSECTKACATEFTGGDESRLKDVWPRKLRSGDSRLKD-OH. Peptide SsTx-P2 potently inhibited the current of Kv4.1 channel transiently transfected in HEK293 cell, with 1.0 μmol/L SsTx-P2 suppressing 95% current of Kv4.1 channel. Its spatial structure showed that SsTx-P2 shared a conserved helical structure.@*CONCLUSIONS@#The study has isolated a novel peptide SsTx-P2 from centipede venom, which can potently inhibit the potassium ion channel Kv4.1, and its spatial structure displays a certain degree of conservation.
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Background: Diabetic gastroparesis (DGP) is one of the most common complications of diabetes mellitus (DM), and its main symptoms include upper abdominal pain, nausea, vomiting, abdominal distension, etc. Gastric hypersensitivity is the main pathogenesis of DGP. Advanced glycation end products (AGEs) is the initiating factor of chronic complications of DM, and its relationship with gastric hypersensitivity has not yet been clear. Kv4.2 channel plays an important role in regulating visceral sensation. Subunit inactivation of Kv4.2 can reduce potassium current, enhance pain sensation, and increase gastric sensitivity. Aims: To investigate the mechanism of AGEs participating in gastric hypersensitivity by regulating the expression or activity of Kv4.2 channel in DM rat model. Methods: Fifty⁃four rats were randomly divided into control group, DM group and DM+AG group. Streptozocin (STZ) was intraperitoneally injected to induce DM rat model. Blood glucose, body weight, gastric sensitivity and gastric emptying rate were monitored. Western blotting and ELISA were used to detect CML content in stomach tissue and serum, respectively. The expression of RAGE and its co⁃expression with Kv4.2 in dorsal root ganglia (DRG) neurons were detected by immunofluorescence. Western blotting was used to detect RAGE expression and phosphorylation levels of ERK1/2 and Kv4.2 in DRG neurons. Results: Compared with control group, gastric sensitivity was significantly increased in DM group (P<0.01), gastric emptying rate was significantly decreased (P<0.05), AGEs marker CML content in serum and gastric tissue were significantly increased (P<0.05), co ⁃ expression rate of RAGE and Kv4.2 in DRG neurons was significantly increased (P<0.01), and phosphorylation levels of ERK1/2 and Kv4.2 were up⁃regulated (P<0.05). After intervention with AG, above⁃mentioned indices were significantly ameliorated (P<0.05). Conclusions: AGEs is an upstream factor leading to gastric hypersensitivity in DM rat model. AGEs increases the excitability of DRG neurons by inhibiting Kv4.2 channel, leading to gastric hypersensitivity. RAGE and ERK1/2 signal may be involved in the above process.
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The tarantula Chilobrachys jingzhao is one of the largest venomous spiders in China. In previous studies, we purified and characterized at least eight peptides from C. jingzhao venom. In this report, we describe the purification and characterization of Jingzhaotoxin-X (JZTX-X), which selectively blocks Kv4.2 and Kv4.3 potassium channels. Methods: JZTX-X was purified using a combination of cation-exchange HPLC and reverse-phase HPLC. The amino-acid sequence was determined by automated Edman degradation and confirmed by mass spectrometry (MS). Voltage-gated ion channel currents were recorded in HEK293t cells transiently transfected with a variety of ion channel constructs. In addition, the hyperalgesic activity of JZTX-X and the toxin´s effect on motor function were assessed in mice. Results: JZTX-X contained 31 amino acids, with six cysteine residues that formed three disulfide bonds within an inhibitory cysteine knot (ICK) topology. In whole-cell voltage-clamp experiments, JZTX-X inhibited Kv4.2 and Kv4.3 potassium channels in a concentration- and voltage-dependent manner, without affecting other ion channels (Kv1.1, 1.2, 1.3, 2.1, delayed rectifier potassium channels, high- and low-voltage-activated Ca2+ channels, and voltage-gated sodium channels Nav1.5 and 1.7). JZTX-X also shifted the voltage-dependent channel activation to more depolarized potentials, whereas extreme depolarization caused reversible toxin binding to Kv4.2 channels. JZTX-X shifted the Kv4.2 and Kv4.3 activities towards a resting state, since at the resting potential the toxin completely inhibited the channels, even in the absence of an applied physical stimulus. Intrathecal or intraplantar injection of JZTX-X caused a long-lasting decrease in the mechanical nociceptive threshold (hyperalgesia) but had no effect on motor function as assessed in the rotarod test. Conclusions: JZTX-X selectively suppresses Kv4.2 and Kv4.3 potassium channel activity in a concentration- and voltage-dependent manner and causes long-lasting mechanical hyperalgesia.(AU)
Subject(s)
Animals , Spider Venoms , Spiders , Shal Potassium ChannelsABSTRACT
[Objective]To forecast the sponge mechanism mediated by LOC389023 in patients with intractable temporal lobe epilepsy(TLE),through investigating the expression of microRNA interacted with dipeptidyl peptidase 10(DPP10)and LOC389023.[Methods]The expression of DPP10 and Kv4.3 were detected in 15 temporal neocortex from patients with brain trauma (control group)and in 26 temporal neocortex from patients with intractable TLE(epilepsy group)by western blot(WB)and immunohisto?chemical(IHC)staining. The location of DPP10 and voltage dependent potassium channel 4.3(Kv4.3)was detected by immunofluo?rescent(IF)staining. The interaction between DPP10 and Kv4.3 was testified by co-immunoprecipitation(Co-IP). The expression of microRNA obtained by softwares(miRanda,Pita,TargetScan and miRDB)was detected by qPCR.[Results]IHC and WB showed an increased expression of DPP10(P0.05). But decreased expression of LOC389023 and miR-140-5p and increased expression of miR-25-3p and miR-367-3p were found in epilepsygroup compared to control group (P < 0.05).[Conclusion]miR-25-3p and miR-367-3p may be regulated by LOC389023 through the sponge mechanism followed by altered expression of DPP10 in intractable temporal lobe epilepsy.
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Voltage-gated ion channels play an important role in cell's excitability.HCN is short for hyperpolarization-activated cyclic nucleotide-gated channels, and Kv4 family is the main ingredients of transient outward potassium current (IA) produced by hippocampal neuron, both of which belong to voltage-gated ion channels.HCN channel and its mediated Ih current could influence the resting potential of cell membrane, control the excitability of neurons, synaptic potential and synaptic transmission.They also participate in cardiac pacing and rhythmic activity of some neurons.IA is the main ingredients of outward potassium current in the early period of action potential repolarization, which mainly takes part in regulating the synaptic input and counterpropagation of action potential.Kv4 channel could reduce the excitability of neurons.Studies indicate that mutations of these two channels lead to epilepsy.This article focuses on the structure、 distribution and function of HCN and Kv4 in epilepsy.
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AIM To study the effects of propafenone on Kv 4 2 and Kv4 3 potassium current. METHODS Whole cell patch clamp technique was used to record changes of Kv4 2 and Kv4 3 potassium currents. RESULTS ① Propafenone redu ced the amplitude of Kv4 2 and Kv4 3 currents, in a concentration-dependent, IC 50 was 10 3 and 71 ?mol?L -1 respectively. ② Propafenone inhibi ted Kv4 2 and Kv4 3 current inactivation, 10 ?mol?L -1 propafenone acce lerates Kv4 2 current inactivation constant from (38 9?2 1) ms to (9 9?1 8) ms; and shifted the steady state inactivation curves of Kv4 2 current to negative potentials. 100 ?mol?L -1 Propaf enone accelerates Kv4 3 current inactivation constant from (144 8?20 8) ms t o (18 5?2 8) ms; and shifted the steady state inactivation curves of Kv4 2 c urrent to negative potentials. ③ propafenone inhibited Kv4 2 and Kv4 3 curren t activation, 10 ?mol?L -1 propafenone shifted the steady state activati on curves of Kv4 2 current to negative potentials. 100 ?mol?L -1 propafe none shifted the steady state activation curves of Kv4 2 current to negative po tentials. CONCLUSION Propafenone, maybe, also exerts its effects via blocking Kv4 2 and Kv4 3 current to antiarrhythmia.