[Functional expression of congenital long QT syndrome related HERG mutation A561V in vitro].
OBJECTIVE
To investigate the functional expression of HERG mutation A561V detected in a Chinese congenital long QT syndrome family.


METHODS
The mutation gene A561V was cloned into eukaryotic expressive vector pcDNA3 by quick site-directed mutagenesis PCR and restriction enzymes. The wild-type HERG, heterozygous type HERG and HERG mutation A561V were respectively cotransfected with pRK5-GFP into HEK293 cells by Suprefact transfection regent. The protein expression was measured by immunofluorescence method and Western blot. The electrophysiological characteristics of transfected cells were determined by whole cell patch-clamp technique.


RESULTS
Direct sequence analyses revealed a C to T transition at position 1682. A561V mutation was correctly combined to eukaryotic expressive vector pcDNA3 and expressed in HEK293 cells. The protein expression of mutation and heterozygosis were located in cytoplasm and cellular membrane. 155 kDa and 135 kDa protein bands were detected in wild type HERG channel while only 135 kDa protein band was shown in heterozygous and mutational channels. Significant HERG tail-current was recorded in wild type HERG channel but not in mutation and heterozygosis channels.


CONCLUSIONS
This study evidenced a functional dominant-negative current suppression in HEK293 cells transfected with HERG mutation A561V.
Li
Yu
Y
Department of Cardiology, First Hospital of Xi'an Jiaotong University, Xi'an 710061, China.
Cui
Chang-cong
CC
Zhao
Yong-hui
YH
Xue
Xiao-lin
XL
Zhang
Ai-feng
AF
Lian
Jiang-fang
JF
Huang
Chen
C
D002460:Cell Line; D004252:DNA Mutational Analysis; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D008133:Long QT Syndrome / Q000151:congenital / Q000235:genetics*; D009154:Mutation; D018408:Patch-Clamp Techniques; D014162:Transfection
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human