Effects of SCN5A mutation on intracellular calcium concentration
Objective: To construct the delQKP1507-1509 mutant of SCN5A channel, and to study effects of this mutation on intracellular calcium concentration. Methods: SCN5A delQKP1507-1509 mutation was engineered using site-directed mutagenesis. Wild-type (WT), mutant (MT) and mixed (the mixture of wild-type and mutant plasmids at a ratio of 1:1) Nav1.5 plasmids were transfected into human embryonic kidney 293 (HEK293) cells respectively. Intracellular free calcium concentration were determined by laser confocal scanning microscope (LCSM) after the cells loaded with the Ca2+ indicator dye Fluo-3/AM. Results: Gel electrophoresis and DNA direct sequencing proved a deletion of nine base pairs in the SCN5A gene at position 1507-1509. Protein expression and localization of wild-type and mutant SCN5A gene in HEK293 cells were detected under the LSCM. Fluorescence intensity in wild-type group, mutant group and co-expression group (1:1 expression of WT and MT sodium channel) mimicking the heterozygous state were 79.3683±3.051, 94.6165±8.383, 90.7463±6.421 respectively, and there was no significant difference among the three groups (P>0.05). Conclusions: SCN5A delQKP1507-1509 mutant is successfully constructed and transfected into HEK293 cells. The mutation does not affect calcium concentration and further study is required to describe the possible pathophysiological mechanisms of dilated cardiomyopathy phenotype in sodium channel overlap syndromes caused by this SCN5A delQKP1507-1509 mutation.
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