[Effects of E23K polymorphism in KCNJ11 gene on membrane current].

Objective: E23K polymorphism in KCNJ11 gene is associated with cardiovascular disease and diabetes. In order to explore the mechanism of E23K correlation to related diseases, the effect of E23K polymorphism in KCNJ11 gene on membrane current was investigated.
Methods: The exon of KCNJ11 was obtained by PCR amplification and the G-->A mutation was completed by overlap extension PCR. The sequences of KCNJ11 exon contained 23E or 23K was inserted into pcDNA3.1/CT-GFP vector respectively. The recombinant plasmid, pcDNA3.1-KCNJ11(E) and pcDNA3.1-KCNJ11(K), were transfected into HEK293T cells by lipofectamine and the membrane current density was determined by whole-cell patch clamp technique.
Results: 1,173 bp sequences of KCNJ11 gene's exon were amplified by PCR and the recombinant expression plasmid, pcDNA3.1-KCNJ11(E) and pcDNA3.1-KCNJ11(K), were constructed successful. Positive and negative currents were detected in HEK293T cells transfected with difference plasmid by whole-cell patch clamp technique. Results showed that the reversed voltage was 50mV. The current in HEK293T cells with pcDNA3.1-KCNJ11(E) was significantly greater than that with pcDNA3.1-KCNJ11(K) (P < 0.05, n = 10).
Conclusion: The polymorphism of E23K in exon of KCNJ11 gene changed the membrane currents in HEK293T cells. It could be an experiment support for the possible mechanism between the locus and related diseases.