RNA Interference Targeting E637K Mutation Rescues The HERG Channel Currents And Restores Its Gating Properties

Bankground and Objective：Long QT syndrome (LQTS) is a monogenic proarrhythmicdisorder that prediposes affected individuals to sudden death from tachyarrhythmia. As an inheriteddisease, LQTS can not be completely cured by conventional treatment modalities. Individualizedgene therapy is a potentially promising therapeutic approach. Mutations in hERG can lead to Type2LQTS (LQT2), which is the second most common type in China. In this study，we investigatedthe role of siRNA on expression of E637K-hERG mutant and if it can be used to rescue themutant’s dominant-negative suppressive effects on hERG protein channel function. Furthermore，it’s useful for assessing the mechanism and value of clinic application of the RNAi in thedominant-negative effect of the LQTS.Methods： HEK293cells were transiently transfected with WT-hERG and/or E637K-hERGplasmids using Lipofectamine2000according to the manufacturer’s instruction. pRK5-GFPplasmid was co-transfected to monitor transfection efficacy. Patch-clamp technique was used to assessthe effect of siRNA on the electrophysiological characteristics of the rapidly activating delayed rectifier K+current (Ikr) of the hERG protein channel.Results： About40-60percent transfected HEK293cells were expressed greenfluorescence，in which Ikr could be recorded by whole-cell clamp technique.1. siRNA rescues the current amplitudes of the WT/E637K-hERG protein channel.In comparison with cells expressing WT/E637K-hERG protein channel without siRNA treatment, themaximum current and tail current amplitudes in cells treated with siRNA increased by68.71%and65.92%respectively. However, the addition of siRNA to WT-hERG only cells shows minimal differences inmaximal current amplitudes and tail currents without siRNA,. Furthermore，treatment with siRNA incells expressing only E637K-hERG has no effect on current generation. .2. siRNA restores gating properties of WT/E637K-hERG protein channelWith siRNA treatment, HEK293cells expressing WT/E637K-hERG protein channel shifted towardmore positive potentials for the half-maximal activation (V1/2) and the half-maximal steady-state inactivationvoltage (V1/2) compared with cells without siRNA treatment. Furthermore, inactivation in siRNA treatedWT/E637K-hERG protein channel occurs more slowly than those without siRNA treatment.However, siRNA treatment in cells expressing WT/E637K-hERG protein channel did not result insignificant changes in the recovery from inactivation or deactivation, relative to those cells without siRNAtreatment. Moreover, siRNA had no effect on the gating properties of WT-hERG protein channel.Conclusion：Our findings illustrated for the first time that siRNA can effectively inhibit hERGmutant restored the maximum current and tail current amplitudes. Furthermore, siRNA treatment rescued thegating properties of WT/E637K-hERG protein channel to a level comparable to that of WT-hERG proteinchannel.