新生儿惊厥相关基因KCNQ2定点突变及其蛋白表达的研究

周熙惠; 惠智艳; 史瑞明; 宋红霞; 张葳; 刘俐

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中国当代儿科杂志 ›› 2011, Vol. 13 ›› Issue (8) : 611-616.

论著·临床研究

新生儿惊厥相关基因KCNQ2定点突变及其蛋白表达的研究

周熙惠，惠智艳，史瑞明，宋红霞，张葳，刘俐

作者信息 +

Site-directed mutagenesis and protein expression of KCNQ2 gene associated with neonatal convulsions

ZHOU Xi-Hui, HUI Zhi-Yan, SHI Rui-Ming, SONG Hong-Xia, ZHANG Wei, LIU Li

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摘要

目的：在收集的1个良性家族性婴儿惊厥家系中发现KCNQ2基因 c.812G>T（p.G271V）突变，探讨c.812G>T突变体及其真核表达载体的构建方法，并观察其在人胚肾(HEK)293细胞中的表达。方法：利用重叠延伸PCR和限制性内切酶法构建 KCNQ2 基因 c.812G>T突变体真核表达载体pcDNA3.0-c.812G>T-KCNQ2，用脂质体转染法将KCNQ2质粒（野生型pcDNA3.0-KCNQ2或突变型pcDNA3.0-c.812G>T-KCNQ2）与荧光真核表达载体pRK5-GFP共转染HEK293细胞，激光共聚焦显微镜下观察HEK293细胞，免疫荧光化学法检测蛋白质表达。结果：构建的突变体经DNA直接测序示 KCNQ2 基因cDNA第812位碱基G变为T,突变体在HEK293细胞中成功表达，野生型和突变型基因的蛋白质都在细胞膜上表达，提示KCNQ2钾通道孔区的突变c.812G>T并不影响蛋白的正常表达。结论：成功构建 KCNQ2 突变基因真核表达载体，并在HEK293细胞中表达KCNQ2蛋白，为突变型 KCNQ2 基因的功能研究及癫癎的分子发病机制研究奠定基础。

Abstract

OBJECTIVE: To study the protocol of construction of a KCNQ2-c.812G>T mutant and it′s eukaryotic expression vector, the c.812G>T (p.G271V) mutation which was detected in a Chinese pedigree of benign familial infantile convulsions, and to examine the expression of mutant protein in human embyonic kidney (HEK) 293 cells. METHODS: A KCNQ2 mutation c.812G>T was engineered on KCNQ2 cDNAs cloned into pcDNA3.0 by sequence overlap extension PCR and restriction enzymes. HEK293 cells were co-transfected with pRK5-GFP and KCNQ2 plasmid (the wild type or mutant) using lipofectamine and then subjected to confocal microscopy. The transfected cells were immunostained to visualize the intracellular expression of the mutant molecules. RESULTS: Direct sequence analysis revealed a G to T transition at position 812. The c.812G>T mutation was correctly combined to eukaryotic expressive vector pcDNA3.0 and expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both the wild type and mutant molecules on the plasma membrane, which suggested that the c.812G>T mutation at the pore forming region of KCNQ2 channel did not impair normal protein expression in HEK293 cells. CONCLUSIONS: Successful construction of mutant KCNQ2 eukaryotic expression vector and expression of KCNQ2 protein in HEK293 cells provide a basis for further study on the functional effects of convulsion-causing KCNQ2 mutations and for understanding the molecular pathogenesis of epilepsy.

导出引用

周熙惠，惠智艳，史瑞明，宋红霞，张葳，刘俐. 新生儿惊厥相关基因KCNQ2定点突变及其蛋白表达的研究[J]. 中国当代儿科杂志. 2011, 13(8): 611-616

ZHOU Xi-Hui, HUI Zhi-Yan, SHI Rui-Ming, SONG Hong-Xia, ZHANG Wei, LIU Li. Site-directed mutagenesis and protein expression of KCNQ2 gene associated with neonatal convulsions[J]. Chinese Journal of Contemporary Pediatrics. 2011, 13(8): 611-616

中图分类号： R722.1

参考文献

［1］Tang B, Li H, Xia K, Jiang H, Pan Q, Shen L, et al. A novel mutation in KCNQ2 gene causes benign familial neonatal convulsions in a Chinese family［J］. J Neuro Sci, 2004, 221(1-2): 31-34.

［2］周熙惠, 马爱群, 刘小红, 黄辰, 张艳敏, 史瑞明. 良性家族性婴儿惊厥家系KCNQ2基因新突变［J］. 中华儿科杂志, 2006, 44(7): 487-491.

［3］Miceli F, Soldovieri MV, Martire M, Taglialatela M. Molecular pharmacology and therapeutic potential of neuronal Kv7-modulating drugs［J］. Curr Opin Pharmacol, 2008, 8(1): 65-74.

［4］Volkers L, Rook MB, Das JH, Verbeek NE, Groenewegen WA, van Kempen MJ, et al. Functional analysis of novel KCNQ2 mutations found in patients with benign familial neonatal convulsions［J］. Neurosci Lett, 2009, 462(1): 24-29.

［5］Viadero MT, Rubín E, Amigo T, González-Lamu~no D. Three generations of hereditary long-QT syndrome with complete penetrance caused by the p.G316E KCNQ1 mutation［J］. Pediatr Cardiol, 2011, 32(1): 102-104.

［6］Otto JF, Yang Y, Frankel WN, White HS, Wilcox KS. A spontaneous mutation involving Kcnq2 (Kv7.2) reduces M-current density and spike frequency adaptation in mouse CA1 neurons［J］. J Neurosci, 2006, 26(7): 2053-2059.

［7］Heron SE, Cox K, Grinton BE, Zuberi SM, Kivity S, Afawi Z, et al. Deletions or duplications in KCNQ2 can cause benign familial neonatal seizures［J］. J Med Genet, 2007, 44(12): 791-796.

［8］Uehara A, Nakamura Y, Shioya T, Hirose S, Yasukochi M, Uehara K. Altered KCNQ3 potassium channel function caused by the W309R pore-helix mutation found in human epilepsy［J］. J Membr Biol, 2008, 222(2): 55-63.

［9］Charlier C, Singh NA, Ryan SG, Lewis TB, Reus BE, Leach RJ, et al. A pore mutation in a novel KQT-like potassium channel gene in an idiopathic epilepsy family［J］. Nat Genet, 1998, 18(1): 53-55.

［10］Sugiura Y, Nakatsu F, Hiroyasu K, Ishii A, Hirose S, Okada M, et al. Lack of potassium current in W309R mutant KCNQ3 channel causing benign familial neonatal convulsions (BFNC)［J］. Epilepsy Res, 2009, 84(1): 82-85.

［11］Seebohm G, Scherer CR, Busch AE, Lerche C. Identification of specific pore residues mediating KCNQ1 inactivation. A novel mechanism for long QT syndrome［J］. J Biol Chem, 2001, 276(17): 13600-13605.

［12］Seebohm G, Pusch M, Chen J, Sanguinetti MC. Pharmacological activation of normal and arrhythmia-associated mutant KCNQ1 potassium channels［J］. Circ Res, 2003, 93(10): 941-947.

［13］Dedek K, Fusco L, Teloy N, Steinlein OK. Neonatal convulsion and epileptic encephalopathy in an Italian family with a missense mutation in the fifth transmembrane region of KCNQ2［J］. Epilepsy Res, 2003, 54(1): 21-27.

［14］Borgatti R, Zucca C, Cavallini A, Ferrario M, Panzeri C, Castaldo P, et al. A novel mutation in KCNQ2 associated with BFNC, drug resistant epilepsy, and mental retardation［J］. Neurology, 2004, 63(1): 57-65.

［15］王家勤, 李红梅, 尹景岗, 张红亚, 李冲, 顾仁骏，等. 良性家族性婴儿惊厥一家系基因定位及钾离子通道基因测序研究［J］. 中华儿科杂志, 2002, 40(9): 513517.

［16］Ishii A, Fukuma G, Uehara A, Miyajima T, Makita Y, Hamachi A, et al. A de novo KCNQ2 mutation detected in nonfamilial benign neonatal convulsions［J］. Brain Dev, 2009, 31(1): 27-33.

［17］Neubauer BA, Waldegger S, Heinzinger J, Hahn A, Kurlemann G, Fiedler B, et al. KCNQ2 and KCNQ3 mutations contribute to different idiopathic epilepsy syndromes［J］. Neurology, 2008, 71(3): 177-183.

［18］Lange W, Geissend-rfer J, Schenzer A, Grtzinger J, Seebohm G, Friedrich T, et al. Refinement of the binding site and mode of action of the anticonvulsant Retigabine on KCNQ K+ channels［J］. Mol Pharmacol, 2009, 75(2): 272-280.