Abstract OBJECTIVE: To construct the sodium channel gene SCN5A-delQKP1507-1509 mutation associated with congenital long QT syndrome, and its eukaryotic expression vector, and to examine the expression of mutation protein in human embryonic kidney (HEK) 293 cells. METHODS: Eukaryotic expression vector PEGFP-delQKP-hH1 for SCN5A-delQKP1507-1509 mutation was constructed by rapid site-directed mutagenesis. HEK293 cells were transfected with the wild or mutant vector using lipofectamine, and then subjected to confocal microscopy. The transfected cells were immunostained to visualize intracellular expression of the mutant molecules. RESULTS: Direct sequence and electrophoresis analysis revealed 9 basic group absences at position 1507-1509. The delQKP1507-1509 mutation eukaryotic expression vector was expressed in HEK293 cells. Immunostaining of transfected cells showed the expression of both wild type and mutant molecules on the plasma membrane and there was no difference in the amount of protein, which suggested that the mutant delQKP1507-1509 did not impair normal protein expression in HEK293 cells. CONCLUSIONS: Successful construction of mutant SCN5AdelQKP1507-1509 eukaryotic expression vector and expression of SCN5A protein in HEK293 cells provides a basis for further study on the functional effects of congenital long QT syndrome as a cause of SCN5A mutation.
SHI Rui-Ming,QIANG Hua,ZHANG Yan-Min et al. Site-directed mutagenesis and protein expression of SCN5A gene associated with congenital Long QT syndrome[J]. CJCP, 2013, 15(3): 223-226.
SHI Rui-Ming,QIANG Hua,ZHANG Yan-Min et al. Site-directed mutagenesis and protein expression of SCN5A gene associated with congenital Long QT syndrome[J]. CJCP, 2013, 15(3): 223-226.
［1］Brenyo AJ, Huang DT, Aktas MK. Congenital long and short QT syndromes［J］. Cardiology, 2012, 122(4): 237-247.
［2］Shi R, Zhang Y, Yang C, Huang C, Zhou X, Qiang H, et al. The cardiac sodium channel mutation delQKP1507-1509 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.
［3］Hedley PL, Jorgensen P, Schlamowitz S, Wangari R, Moolman-Smook J, Brink PA, et al. The genetic basis of long QT and short QT syndromes: a mutation update［J］. Hum Mutat, 2009, 30 (11): 1486-1511.
［4］Horigome H, Nagashima M, Sumitomo N, Yoshinaga M, Ushinohama H, Iwamoto M, et al. Clinical characteristics and genetic background of congenital long-QT syndrome diagnosed in fetal, neonatal, and infantile life: a nationwide questionnaire survey in Japan［J］. Circ Arrhythm Electrophysiol, 2010, 3(1): 10-17.
［5］Roberts JD, Gollob MH. The genetic and clinical features of cardiac channelopathies［J］. Future Cardiol, 2010, 6(4): 491-506.
［6］Cerrone M, Napolitano C, Priori SG. Genetics of ion-channel disorders［J］. Curr Opin Cardiol, 2012, 27(3): 242-252.
［7］Makita N, Behr E, Shimizu W, Horie M, Sunami A, Crotti L, et al. The E1784K mutation in SCN5A is associated with mixed clinical phenotype of type 3 long QT syndrome［J］. J Clin Invest, 2008, 118(6): 2219-2229.
［8］Eckardt L. LQT3: who is at risk for sudden cardiac death?［J］. Heart Rhythm, 2009, 6(1): 121-122.
［9］Rivolta I, Abriel H, Tateyama M, Liu H, Memmi M, Vardas P, et al. Inherited Brugada and long QT-3 syndrome mutations of a single residue of the cardiac sodium channel confer distinct channel and clinical phenotypes［J］. J Biol Chem, 2001, 276(33): 30623-30630.
［10］Zimmer T, Surber R. SCN5A channelopathies-an update on mutations and mechanisms［J］. Prog Biophys Mol Biol, 2008, 98 (2-3): 120-136.
［11］Morita H, Wu J, Zipes DP. The QT syndromes: long and short［J］. Lancet, 2008, 372 (9640): 750-763.
