Roles Of KCNQ1-R259H Mutant In The Development Of SQT2 And Its Electrophysiological Mechanism

Background Short QT syndrome (SQTS) caused by mutation is an ion channelopathy that often leads to hereditary diseases and malignant arrhythmia. So far, it has been found seven kinds of SQTS subtypes (SQT1-SQT7). While the R259H mutant which leads to SQT2, has not yet identified the mechanism, even there is no witness among Chinese SQTS patients.Objective To evaluate the effect of a KCNQ1-R259H mutant on SQTS, we discovered the KCNQ1-R259H mutant from genetic screening of SQTS genes. And to explore the mechanisms of SQT2, we researched the characteristics of currents encoded by KCNQ1-R259H mutant and the effect on action potential by patch clamp.Method1. We collected SQTS patients and found a SQTS pedigree. After analyzing the clinical data, we conducted (KCNH2, KCNQ1, KCNJ2, CACNAlc, CACNB2b, CACNA2D1 and SCN5A) genetic screening, screening out the KCNQ1 gene mutation R259H.2. Wild type (WT) and the R259H mutation of KCNQ1 cDNAs were cloned in the eukaryotic expression vector pcDNA3.1. The analysis of KCNQ1 expression and localization was conducted after transient transfection by patch clamp technique, western blot and Confocal Image.Results1. A 20-year-old proband was found to be genopositive, who exhibited cardiac arrest (CA) at rest. The ECG showed that QTc was 310 ms with a high and symmetrical T wave and an ICD was implanted after CA.The main related parameters of the ECG were showed as follow, II-2:QTc= 300m;Ⅲ-1:QTc=310 ms;Ⅲ-2:QTc=310 ms. Further blood genetic screening identified KCNQ1 had a missense mutation at position 259 that leads to an amino acid change from arginine to histidine (R259H).2. The current of WT-KCNQ1 was similar to the triangle, while the current of R259H-KCNQ1 was typical rectangle. R259H-KCNQ1 showed significantly increase current density, which was approximately 3-fold larger than that of WT after a depolarizing pulse at 1 s. Therefore R259H is a gain-of-function mutation which can increase the current density of 7 IKs.3. The time constant of deactivation was markedly prolonged in the mutant compared with the WT (Tau R259H-KCNQ1:3113.0±175.0 ms, Tau WT-KCNQ1:1778.4± 193.4 ms) when the activation voltage exceeded-100 mV. It indicated that the deactivation of R259H was markedly slower than that of the WT, which may be the main reason for the increase of the current density.4. By study the dynamics of R259H channel and the expression of membrane protein, we could draw a conclusion that the increase of the current density and the mechanisms which induced SQT2, was caused by the change in voltage-gated property, not caused by changes in protein expression.5. We successfully built an action potential in HEK-293 cells. Furthermore, we found that R259H mutant reduced APD90 (R259H-AP:87.56±7.60 ms vs. WT-AP: 117.64±12.20 ms, n=9, P<0.01) significantly. While APD20 and APD50 did not show significant differences between the WT and R259H mutation.Conclusions1. The ECG of proband showed that QTc was 310 ms with a high and symmetrical T wave. Further blood genetic screening identified KCNQ1, encoding Kv7.1 channel protein, changed from arginine to histidine at position 259.2. R259H was a gain-of-function mutation of the KCNQ1 channel that increased IKs current density. And this phenomenon mainly relied on the extension of the deactivation. 4. We successfully built an action potential in HEK-293 cells. Furthermore, we found that R259H mutant reduced APD90 significantly.