The G604S-hERG mutation alters the biophysical properties and exerts a dominant-negative effect on expression of hERG channels in HEK293 cells.
We have recently identified a missense mutation, G604S, in the human ether-a-go-go related gene (hERG) that results in a malignant phenotype in a full pedigree of a Chinese congenital long QT syndrome (LQTS) family. The present study characterized the pathophysiological consequences of the mutation at the cellular level. Mutant G604S-hERG channels were expressed in HEK293 cells using a lipofectamine method. hERG currents were recorded using the voltage clamp technique. The expression of hERG protein was detected by Western blotting, and the subcellular location of hERG channels in cell was analyzed by confocal microscopy. We found that the G604S mutation did not lead to any expression of detectable currents, which was consistent with Western blotting analysis that the G604S-hERG mutation only expressed a band at 135 kDa. When coexpressed with wild-type (WT)-hERG, G604S-hERG exhibited strong dominant-negative current suppression resulting in decreased current density and altered gating properties of the WT-hERG channel, as well as interference with the trafficking of WT-hERG channel protein. In addition, confocal microscopy demonstrated that G604S-hERG subunits could be inserted into the cell membrane when forming heteromultimeric channels with WT-hERG channel subunits. Our results suggest that G604S mutation causes a loss of function in hERG through a strong dominant-negative effect on WT-hERG channel function that caused by impaired trafficking of WT-hERG channels, and further accentuates this suppression by forming heteromultimeric functional channels with WT-hERG subunits.
Huo
Jianhua
J
Department of Cardiovascular Medicine, First Hospital of Xi'an Jiaotong University, Key Laboratory of Environment and Genes Related to Diseases, Xi'an Jiaotong University, Ministry of Education, No.277 Yanta West Road, Xi'an, Shaanxi, 710061, China.
Zhang
Yanmin
Y
Huang
Na
N
Liu
Ping
P
Huang
Chen
C
Guo
Xueyan
X
Jiang
Wenhui
W
Zhou
Nan
N
Grace
Andrew
A
Huang
Christopher L H
CL
Ma
Aiqun
A
D002460:Cell Line; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating; D008133:Long QT Syndrome / Q000235:genetics* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D018408:Patch-Clamp Techniques; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins