拉莫三嗪对酸敏感离子通道1a的调控作用
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摘要
目的探讨拉莫三嗪(lamotrigine, LTG)对酸敏感离子通道1a(acid-sensing ion channel 1a,ASIC1a)的调控作用。方法通过全细胞记录膜片钳技术研究LTG对CHO细胞转染酸敏感离子通道(acid-sensing ion channels,ASICs)不同亚型,尤其是酸敏感离子通道1a(acid-sensing ion channel 1a,ASIC1a)的调控作用及其调控方式;并通过免疫印迹定量检测LTG对ASIC1a蛋白表达的影响。结果在转染了不同的ASICs亚单位的CHO细胞系中,LTG对ASIC1a同聚体通道产生的内向电流与另外三种通道相比抑制作用显著增加(P<0.05)。这种抑制效应与胞外氢离子浓度相关呈剂量依赖性,pH为6.0时,抑制率为40%左右,pH为6.8时抑制率为20%左右;并且必须与酸共给的情况下才能对ASIC1a发挥抑制效应。四组不同浓度LTG直接作用原代培养的神经元24 h后ASIC1a的蛋白表达无统计学差异(P>0.05)。结论 LTG对ASIC1a同聚体通道产生的内向电流具有直接抑制作用,这可能是其治疗癫痫发作的作用机制之一。
Objective To explore the regulatory effect of lamotrigine(LTG)on acid-sensing ion channel 1 a(ASIC1 a). Method The whole cell patch-clamp recording technique was used to investigate the effect and mode of LTG's regulation on inward current of the different subtypes of acid-sensitive ion channels(ASICs), particularly acidsensing ion channel 1 a,(ASIC1 a) in CHO cells transfected with different ASICs subunits. Western blot was used to examine the protein expression of ASIC1 a. Results In the CHO cell lines transfected with different ASICs subunits, the inhibitory effect of LTG on the ASIC1 a homopolymer channels was significantly increased compared with the other three channels(P<0.05). The inhibitory effect was dose-dependent with the extracellular hydrogen ion concentration, the peak current amplitude of ASIC1 a was inhibited by LTG about 40% when pH was 6.0 or 20% when pH was 6.8. In addition,the inhibitory effect on ASIC1 a could only be exerted when it was co-fertilized with acid. There was no statistically significant difference in the protein expression of ASIC1 a in the four groups after 24 hours of primary culture neurons under direct action of LTG at different concentrations(P>0.05). Conclusion LTG has a direct inhibitory effect on the inward current generated by the homopolymer channel of ASIC1 a, suggesting that this may be one of the anti-epileptic mechanisms of LTG.
Objective To explore the regulatory effect of lamotrigine(LTG)on acid-sensing ion channel 1 a(ASIC1 a). Method The whole cell patch-clamp recording technique was used to investigate the effect and mode of LTG's regulation on inward current of the different subtypes of acid-sensitive ion channels(ASICs), particularly acidsensing ion channel 1 a,(ASIC1 a) in CHO cells transfected with different ASICs subunits. Western blot was used to examine the protein expression of ASIC1 a. Results In the CHO cell lines transfected with different ASICs subunits, the inhibitory effect of LTG on the ASIC1 a homopolymer channels was significantly increased compared with the other three channels(P<0.05). The inhibitory effect was dose-dependent with the extracellular hydrogen ion concentration, the peak current amplitude of ASIC1 a was inhibited by LTG about 40% when pH was 6.0 or 20% when pH was 6.8. In addition,the inhibitory effect on ASIC1 a could only be exerted when it was co-fertilized with acid. There was no statistically significant difference in the protein expression of ASIC1 a in the four groups after 24 hours of primary culture neurons under direct action of LTG at different concentrations(P>0.05). Conclusion LTG has a direct inhibitory effect on the inward current generated by the homopolymer channel of ASIC1 a, suggesting that this may be one of the anti-epileptic mechanisms of LTG.
引文
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[23]LENNOX W.The effect on epileptic seizures of varying the composition of the respired air[J].J Clin Invest,1929,4:23-24.
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[25]WENG JY,LIN YC,LIEN CC.Cell type-specific expression of acid-sensing ion channels in hippocampal interneurons[J].JNeurosci,2010,30(19):6548-6558.
[2]XIONG ZQ,SAGGAU P,STRINGER JL.Activity-dependent intracellular acidification correlates with the duration of seizure activity[J].J Neurosci,2000,20(4):1290-1296.
[3]BIAGINI G,BABINSKI K,AVOLI M,et al.Regional and subunit-specific downregulation of acid-sensing ion channels in the pilocarpine model of epilepsy[J].Neurobiol Dis,2001,8(1):45-58.
[4]ZIEMANN AE,SCHNIZLER MK,ALBERT GW,et al.Seizure termination by acidosis depends on ASIC1a[J].Nat Neurosci,2008,11(7):816-822.
[5]VOILLEY N,DE WEILLE J,MAMET J,et al.Nonsteroid antiinflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors[J].J Neurosci,2001,21(20):8026-8033.
[6]LIN J,CHU X,MAYSAMI S,et al.Inhibition of acid sensing ion channel currents by lidocaine in cultured mouse cortical neurons[J].Anesth Analg,2011,112(4):977-981.
[7]BOIKO N,KUCHER V,EATON BA,et al.Inhibition of neuronal degenerin/epithelial Na+channels by the multiple sclerosis drug 4-aminopyridine[J].J Biol Chem,2013,288(13):9418-9427.
[8]MULA M.New antiepileptic drugs:molecular targets[J].Cent Nerv Syst Agents Med Chem,2009,9(2):79-86.
[9]LI WG,LIU MG,DENG S,et al.ASIC1a regulates insular long-term depression and is required for the extinction of conditioned taste aversion[J].Nat Commun.2016,7:13770
[10]WALDMANN R,CHAMPIGNY G,BASSILANA F,et al.Aproton-gated cation channel involved in acid-sensing[J].Nature,1997,386(6621):173-177.
[11]AJMO CT JR,VERNON DO,COLLIER L,et al.Sigma receptor activation reduces infarct size at 24 hours after permanent middle cerebral artery occlusion in rats[J].Curr Neurovasc Res,2006,3(2):89-98.
[12]HERRERA Y,KATNIK C,RODRIGUEZ JD,et al.sigma-1 receptor modulation of acid-sensing ion channel a(ASIC1a)and ASIC1a-induced Ca2+influx in rat cortical neurons[J].J Pharmacol Exp Ther,2008,327(2):491-502.
[13]WU LJ,DUAN B,MEI YD,et al.Characterization of acidsensing ion channels in dorsal horn neurons of rat spinal cord[J].J Biol Chem,2004,279(42):43716-43724.
[14]DUAN B,WU LJ,YU YQ,et al.Upregulation of acid-sensing ion channel ASIC1a in spinal dorsal horn neurons contributes to inflammatory pain hypersensitivity[J].J Neurosci,2007,27(41):11139-11148.
[15]VERGO S,CRANER MJ,ETZENSPERGER R,et al.Acidsensing ion channel 1 is involved in both axonal injury and demyelination in multiple sclerosis and its animal mode[J].Brain,2011,134(Pt 2):571-584.
[16]ESCOUBAS P,DE WEILLE JR,LECOQ A,et al Isolation of a tarantula toxin specific for a class of proton-gated Na+channels[J].J Biol Chem.2000,275(33):25116-25121.
[17]ZHANG GC,MAO LM,WANG JQ,et al.Upregulation of acidsensing ion channel 1 protein expression by chronic administration of cocaine in the mouse striatum in vivo[J].Neurosci Lett,2009,459(3):119-122.
[18]SUMAN A,MEHTA B,GUO ML,et al.Alterations in subcellular expression of acid-sensing ion channels in the rat forebrain following chronic amphetamine administration[J].Neurosci Res,2010,68(1):1-8.
[19]WEMMIE JA,CHEN J,ASKWITH CC,et al.The acid-activated ion channel ASIC contributes to synaptic plasticity,learning,and memory[J].Neuron,2002,34(3):463-477.
[20]ASKWITH CC,WEMMIE JA,PRICE MP,et al.Acid-sensing ion channel 2(ASIC2)modulates ASIC1 H+-activated currents in hippocampal neurons[J].J Biol Chem,2004,279(18):18296-18305.
[21]XIONG ZG,ZHU XM,CHU XP,et al.Neuroprotection in ischemia:blocking calcium-permeable acid-sensing ion channels[J].Cell,2004,118(6):687-698.
[22]YERMOLAIEVA O,LEONARD AS,SCHNIZLER MK,et al.Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a[J].Proc Natl Acad Sci U SA,2004,101(17):6752-6757.
[23]LENNOX W.The effect on epileptic seizures of varying the composition of the respired air[J].J Clin Invest,1929,4:23-24.
[24]MITCHELL WG,GRUBBS RC.Inhibition of audiogenic seizures by carbon dioxide[J].Science,1956,123(3189):223-224.
[25]WENG JY,LIN YC,LIEN CC.Cell type-specific expression of acid-sensing ion channels in hippocampal interneurons[J].JNeurosci,2010,30(19):6548-6558.