[The mechanistic rote of KCNH2 gene L413P and L559H mutations in long QT syndrome].
OBJECTIVE
To investigate the molecular pathogenesis for two novel mutations L413P and L559H of KCNH2 found in Chinese patients with long QT syndrome.


METHODS
L413P and L559H mutant constructs were generated by site-directed mutagenesis using human wild-type (WT) pcDNA3-HERG cDNA as a template. WT and mutant constructs were transiently transfected into human embryonic kidney 293 cells using lipofectamine method. After transfection, the recording of HERG current was performed using patch clamp technique. The expression and cellular localization of HERG protein were studied with Western blot and immunofluorescence methods.


RESULTS
Electrophysiological recordings showed that L413P and L559H mutations did not express HERG current. Western blot analysis revealed that only 135 000 immature HERG protein was expressed in L413P and L559H-transfected cells, whereas both mature and immature forms of HERG protein were observed in WT-transfected cells. Immunofluorescence study showed that L413P and L559H mutant proteins were predominantly localized around the nucleus, suggesting that the mutant channels are retained in the endoplasmic reticulum. When L413P or L559H was co-transfected with equal amount of WT plasmids, both 135 000 and 155 000 forms of HERG protein were observed, and the HERG current was not significantly changed as compared with that of WT transfection alone. Low temperature and E-4031could not rescue these two mutant channels.


CONCLUSIONS
The L413P and L559H mutations resulted in protein trafficking defects with failure of mutant proteins to reach the plasma membrane. However, both biochemical and electrophysiological results showed that the mutations did not have a dominant-negative effect on WT, indicating that the mechanism of the L413P and L559H mutations might be haploinsufficiency.
Li
Cui-lan
CL
Department of Cardiology, Peking University People's Hospital, Beijing 100044, China.
Hu
Da-yi
DY
Liu
Wen-ling
WL
Qi
Shu-ying
SY
Wang
Hong-tao
HT
Li
Lei
L
Gong
Qiu-ming
QM
Zhou
Zheng-feng
ZF
D019943:Amino Acid Substitution; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008133:Long QT Syndrome / Q000235:genetics* / Q000503:physiopathology; D008564:Membrane Potentials / Q000502:physiology; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation; D018408:Patch-Clamp Techniques; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D021381:Protein Transport; D014162:Transfection
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D050505:Mutant Proteins; D024642:Potassium Channels, Voltage-Gated; D049452:Green Fluorescent Proteins