Electrophysiological Characteristics Of The LQT2 Syndrome Mutation KCNH2-G572S And Regulation By Accessory Protein KCNE2

ObjectiveMutations in hERG cause long QT syndrome type 2 which is characterized by a prolonged QT interval on electrocardiogram and predisposition to life-threatening ventricular tachyarrhythmia, syncope and sudden death. KCNH2 mutations lead to LQT2. G572S is one of pathogenic mutations for LQT2. KCNE2 regulates the hERG channel. This study sought to the mechanism of LQT2 induced by G572S and the possible regulatory role of KCNE2.MethodsHEK 293 cells were transiently transfected with KCNH2 construct, either WT or G572S,using Lipofectamin 2000. The CD8 gene was as a reporter gene. The expression level of Kv11.1 protein was monitored using Western blotting analyse. Subcellular localization of channel proteins in the cell surface were examined by Confocal microscopy examination, respectively. We built a cell system in which sodium (Nav1.5) channels,inward rectifier potassium channel (Kir2.1) and hERG potassium channels were co-expressed in HEK293 cells. Using this system, we explored the regulatory role of KCNE2. Currents and AP were recorded using the whole-cell patch-clamp configuration.APDso. APD90 were measured.Results1. Regulatory effect of KCNE2 on G572S mutant current.(1) The current amplitude and current density of G572S mutant channel decreased significantly. KCNE2 has a more significant effect on G572S mutation current. When G572S:KCNE2 was 1:3, the up-regulation effect of KCNE2 was most significant.(2) The ?-? curve of G572S currents was lower than that of WT mutation between-10mV mV and +20mV, and ?-? curve of G572S tail current was lower than that of WT above +10mV. KCNE2 had a more significan upregulation on G572S.(3) When coexpressing with KCNE2, V1/2 and k value of steady-state activation and inactivation curves and the rapid inactivation time constant of both G572S and WT channels didn't change significantly. Only slow inactivation time constant of G572S/WT was shortened by KCNE2.(4) KCHE2 could upregulated G572S mature membrane protein (155kDa) expression.This effect was most significant when G572S:KCNE2 was 1:3. At lower temperature,the regulatory role of KCNE2 on the G572S mutation might be more effective.2. The regulatory role of KCNE2 on recombinant AP.(1) By transfecting SCN5A, KCNJ2 and KCNH2-WT into HEK293 cells, AP-WT was recombined. The currents of SCN5A, KCNH2 and KCNJ2 channels were examined and AP-WT was recorded.(2) By transfecting SCN5A, KCNJ2 and KCNH2-G572S into HEK293 cells,AP-G572S was recombined.The action potential duration of AP-G572S was significantly prolonged.(3) KCNE2 could shorten action potential duration of G572S-AP. When G572S :KCNE2 was 1:1 or 1:3, APD50 was significantly shortened. Whatever G572S:KCNE2 was 3:1, 1:1 or 1:3, APD90 was significantly shortened. Regulatory role of KCNE2 was the most significant when G572S:KCNE2 was 3: 1?Conclusions1. KCNE2 had a more significant effect on G572S mutation current. Regulation of KCNE2 was most effective when G572S:KCNE2 was 1:3.2. Study on channel gating kinetics showed that KCNE2 played an important role in the inactivation process.3. The study on migration mechanism showed that KCHE2 could upregulated G572S mature membrane protein (155kDa) expression. When G572S:KCNE2 was 1:3, the up-regulation effect of KCNE2 was most significant. At lower temperature, the regulatory role of KCNE2 on the G572S mutation might be more effective.4. The study on recombination action potential showed that KCNE2 could shorten action potential duration of G572S-AP. Regulatory role of KCNE2 was the most significant when KCNH2-G572S:KCNE2 was 3: 1.