Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines.
We previously reported a transgenic rabbit model of long QT syndrome based on overexpression of pore mutants of repolarizing K(+) channels KvLQT1 (LQT1) and HERG (LQT2).The transgenes in these rabbits eliminated the slow and fast components of the delayed rectifier K(+) current (I(Ks) and I(Kr), respectively), as expected. Interestingly, the expressed pore mutants of HERG and KvLQT1 downregulated the remaining reciprocal repolarizing currents, I(Ks) and I(Kr), without affecting the steady-state levels of the native polypeptides. Here, we sought to further explore the functional interactions between HERG and KvLQT1 in heterologous expression systems. Stable Chinese hamster ovary (CHO) cell lines expressing KvLQT1-minK or HERG were transiently transfected with expression vectors coding for mutant or wild-type HERG or KvLQT1. Transiently expressed pore mutant or wild-type KvLQT1 downregulated I(Kr) in HERG stable CHO cell lines by 70% and 44%, respectively. Immunostaining revealed a severalfold lower surface expression of HERG, which could account for the reduction in I(Kr) upon KvLQT1 expression. Deletion of the KvLQT1 NH(2)-terminus did not abolish the downregulation, suggesting that the interactions between the two channels are mediated through their COOH-termini. Similarly, transiently expressed HERG reduced I(Ks) in KvLQT1-minK stable cells. Coimmunoprecipitations indicated a direct interaction between HERG and KvLQT1, and surface plasmon resonance analysis demonstrated a specific, physical association between the COOH-termini of KvLQT1 and HERG. Here, we present an in vitro model system consistent with the in vivo reciprocal downregulation of repolarizing currents seen in transgenic rabbit models, illustrating the importance of the transfection method when studying heterologous ion channel expression and trafficking. Moreover, our data suggest that interactions between KvLQT1 and HERG are mediated through COOH-termini.
Ren
Xiao-Qin
XQ
Cardiovascular Research Center, Division of Cardiology, Department of Medicine, Rhode Island Hospital, Alpert Medical School, Brown University, Providence, Rhode Island 02903, USA.
Liu
Gong Xin
GX
Organ-Darling
Louise E
LE
Zheng
Renjian
R
Roder
Karim
K
Jindal
Hitesh K
HK
Centracchio
Jason
J
McDonald
Thomas V
TV
Koren
Gideon
G
D000200:Action Potentials / Q000502:physiology*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000235:genetics / Q000502:physiology*; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels / Q000235:genetics* / Q000502:physiology*; D005260:Female; D017353:Gene Deletion; D006801:Humans; D051657:KCNQ1 Potassium Channel / Q000235:genetics* / Q000502:physiology*; D007668:Kidney / Q000166:cytology / Q000502:physiology; D009154:Mutation / Q000235:genetics*; D010053:Ovary / Q000166:cytology / Q000502:physiology; D018408:Patch-Clamp Techniques; D014162:Transfection
D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D051657:KCNQ1 Potassium Channel