Characterization of a Chinese KCNQ1 mutation (R259H) that shortens repolarization and causes short QT syndrome 2

Abstract

Objectives: To evaluate the association between a KCNQ1 mutation, R259H, and short QT syndrome (SQTS) and to explore the electrophysiological mechanisms underlying their association.

Methods: We performed genetic screening of SQTS genes in 25 probands and their family members (63 patients). We used direct sequencing to screen the exons and intron-exon boundaries of candidate genes that encode ion channels which contribute to the repolarization of the ventricular action potential, including KCNQ1, KCNH2, KCNE1, KCNE2, KCNJ2, CACNA1c, CACNB2b and CACNA2D1. In one of the 25 SQTS probands screened, we discovered a KCNQ1 mutation, R259H. We cloned R259H and transiently expressed it in HEK-293 cells; then, currents were recorded using whole cell patch clamp techniques.

Results: R259H-KCNQ1 showed significantly increased current density, which was approximately 3-fold larger than that of wild type (WT) after a depolarizing pulse at 1 s. The steady state voltage dependence of the activation and inactivation did not show significant differences between the WT and R259H mutation (P > 0.05), whereas the time constant of deactivation was markedly prolonged in the mutant compared with the WT in terms of the test potentials, which indicated that the deactivation of R259H was markedly slower than that of the WT. These results suggested that the R259H mutation can effectively increase the slowly activated delayed rectifier potassium current (I Ks) in phase 3 of the cardiac action potential, which may be an infrequent cause of QT interval shortening.

Conclusions: R259H is a gain-of-function mutation of the KCNQ1 channel that is responsible for SQTS2. This is the first time that the R259H mutation was detected in Chinese people.

Keywords: Ion channel; KCNQ1 gene; Mutation; Short QT syndrome; Slowly activated delayed rectifier potassium current.

Figure 1.. Pedigree of the short QT syndrome family.

(A): ECG of the proband; (B): family investigation. The proband (denoted by the arrow); circles indicate female family members; squares indicate male family members; solid symbols indicate a person with a short QT interval; symbols with a slash indicate deceased family members. ECG: electrocardiogram; QTc: corrected QT.

Figure 2.. The current densities of WT-KCNQ1 and R259H-KCNQ1.

(A): Schematic representation of the voltage-clamp protocol and the representative WT and R259H current traces; (B): The peak I-V relationships for I_Ks under WT and R259H conditions; (C): Tail current was recorded at −120 mV for 3000 ms; (D): The tail I-V relationships for I_Ks under WT and R259H conditions at a voltage of −40 mV; (E): The comparison of the current density of the peak and step between the WT and R259H condition under different conditions; (F): The tail I-V relations for I_Ks under WT and R259H conditions at a voltage of −120 mV. *P < 0.05. WT: wild type.

Figure 3.. The comparison of the current between the WT and R259H condition under the simulated time of 4 s and 1 s.

(A): I_Ks was elicited in simulated voltage-clamp experiments measured 1 s into the imposed voltage commands; (B): The peak I–V relationships for I_Ks under WT and R259H conditions at the simulated time of 1 s; (C): The density of the R259H current was approximately 3-fold larger than WT after a 1 s depolarizing pulse; (D): I_Ks was elicited in simulated voltage-clamp experiments measured 4 s into the imposed voltage commands; (E): The peak I–V relationships for I_Ks under WT and R259H conditions at the simulated time of 4 s; and (F): The density of the R259H current was approximately 2-fold larger than WT after a 4 s depolarizing pulse. *P < 0.05. WT: wild type.

Figure 4.. The steady-state activation, inactivation and deactivation of KCNQ1/KCNE1.

(A): I_Ks was activated under WT-KCNQ1 and R259H-KCNQ1 conditions; (B): SSA curves are shown under WT-KCNQ1 and R259H-KCNQ1 conditions; (C): time constants of SSA under WT-KCNQ1 and R259H-KCNQ1 conditions; (D): representative inactivation current traces recorded from WT and R259H; (E): SSI curves are shown under WT-KCNQ1 and R259H-KCNQ1 conditions; (F): time constants of SSI under WT-KCNQ1 and R259H-KCNQ1 conditions; (G): representative deactivation current traces recorded from WT and R259H; (H): time constants of deactivation under WT-KCNQ1 and R259H-KCNQ1 conditions; and (I): comparison of Tau value under WT-KCNQ1 and R259H-KCNQ1 conditions. *P < 0.05. SSA: steady-state activation; SSI: steady-state inactivation; WT: wild type.

Figure 5.. Analysis of the cellular distribution.

Western blot with anti-KCNQ1 (Kv7.1) after transient transfection in HEK-293 cells. The expression levels of the R295H mutant are similar as that of WT. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; WT: wild type.