|
|
- 2015
钠通道SCN5a突变体R104W的构建及验证
|
Abstract:
摘要：目的 构建并验证Brugada综合征相关钠通道SCN5a基因R104W突变体。方法 采用一步法PCR突变技术，以pEGFP-SCN5a为模板，体外定点诱变构建pEGFP-SCN5a-R104W突变体，进行基因测序，并用Lipofectamine TM3000脂质体转染法转入HEK293细胞，通过Western blotting和膜片钳记录检测蛋白质表达和电流加以验证。结果 测序结果显示SCN5a-R104W突变体基因序列上第310 C>T，其他序列碱基与野生型相比未发生改变；Western blotting结果显示SCN5a-R104W突变体蛋白表达量较野生型SCN5a降低；荧光显微镜下显示SCN5a-R104W突变体蛋白位于胞质内；SCN5a-R104W突变体转染后膜片钳记录未能检测到钠电流，且R104W突变体对野生型通道有负显性抑制作用。结论 成功构建并验证SCN5a基因R104W突变体。
ABSTRACT: Objective To construct and verify the R104W mutant of SCN5a channel. Methods The SCN5a-R104W mutant was constructed by rapid site-directed mutagenesis, and the expected mutation was confirmed by direct sequencing. The mutant DNA was transfected into HEK293 cells using Lipofectamine TM3000. Function of the SCN5a-R104W mutant was tested by Western blot analysis and whole-cell patch clamp recording. Results Sequencing results showed that the base on 310 was changed from C to T of SCN5a-R104W mutant DNA. Protein expression of SCN5a-R104W mutant was lower than that of wild-type SCN5a (SCN5a-WT) channel. SCN5a-WT channels were expressed on the cell surface and SCN5a-R104W channels were mainly expressed in the cytoplasm. Patch clamping result showed that no sodium current was recorded from the cells expressing SCN5a-R104W mutant channel, and SCN5a-R104W exerted dominant-negative effect on SCN5a-WT channel. Conclusion Trafficking deficient SCN5a-R104W mutant channel was successfully constructed and verified
| [1] | 薛小临，廉姜芳，崔长琮. 先天性长QT综合征家系的临床特点[J]. 西安交通大学学报：医学版, 2005, 25(6):610-613. |
| [2] | CLATOT J, ZIYADEH-ISLEEM A, MAUGENRE S, et al. Dominant-negative effect of SCN5A N-terminal mutations through the interaction of Na(v)1.5 alpha-subunits[J]. Cardiovasc Res, 2012, 96(1):53-63. |
| [3] | ATTWELL D, COHEN I, EISNER D, et al. The steady state TTX-sensitive (“window”) sodium current in cardiac Purkinje fibres[J]. Pflügers Archiv, 1979, 379(2):137-142. |
| [4] | PERRIN MJ, GOLLOB MH. Genetics of cardiac electrical disease[J]. Can J Cardiol, 2013, 29(1):89-99. |
| [5] | LI A, SABA MM, BEHR ER. Genetic biomarkers in Brugada syndrome[J]. Biomark Med, 2013, 7(4):535-546. |
| [6] | VALDIVIA CR, NAGATOMO T, MAKIELSKI JC. Late Na currents affected by alpha subunit isoform and beta1 subunit co-expression in HEK293 cells[J]. J Mol Cell Cardiol, 2002, 34(8):1029-1039. |
| [7] | GUILLEM MS, CLIMENT AM, MILLET J, et al. Conduction abnormalities in the right ventricular outflow tract in Brugada syndrome detected body surface potential mapping[J]. Conf Proc IEEE Eng Med Biol Soc, 2010, 2010:2537-2540. |
| [8] | YAN GX, ANTZELEVITCH C. Cellular basis for the Brugada syndrome and other mechanisms of arrhythmogenesis associated with ST-segment elevation[J]. Circulation, 1999, 100(15):1660-1666. |
| [9] | MASHAR M, KWOK AJ, PINDER R, et al. The Brugada syndrome revisited[J]. Trends Cardiovasc Med, 2014, 24(5):191-196. |
| [10] | TAN BH, VALDIVIA CR, SONG C, et al. Partial expression defect for the SCN5A missense mutation G1406R depends on splice variant background Q1077 and rescue by mexiletine[J]. Am J Physiol Heart Circ Physiol, 2006, 291(4):H1822-1828. |
| [11] | REMME CA, BEZZINA CR. Sodium channel (dys)function and cardiac arrhythmias[J]. Cardiovasc Ther, 2010, 28(5):287-294. |
| [12] | ZHANG Y, WANG J, CHANG S, et al. The SCN5A mutation A1180V is associated with electrocardiographic features of LQT3[J]. Pediatr Cardiol, 2014, 35(2):295-300. |
| [13] | SHI R, ZHANG Y, YANG C, et al. The cardiac sodium channel mutation delQKP 1507-1509 is associated with the expanding phenotypic spectrum of LQT3, conduction disorder, dilated cardiomyopathy, and high incidence of youth sudden death[J]. Europace, 2008, 10(11):1329-1335. |
| [14] | ELLINOR PT, NAM EG, SHEA MA, et al. Cardiac sodium channel mutation in atrial fibrillation[J]. Heart Rhythm, 2008, 5(1):99-105. |
| [15] | BRUGADA P, BRUGADA J. Right bundle branch block, persistent ST segment elevation and sudden cardiac death: a distinct clinical and electrocardiographic syndrome. A multicenter report[J]. J Am Coll Cardiol, 1992, 20(6):1391-1396. |
