Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines

Xiao Qin Ren, Gong Xin Liu, Louise E. Organ-Darling, Renjian Zheng, Karim Roder, Hitesh K. Jindal, Jason Centracchio, Thomas V. McDonald, Gideon Koren

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume299
Issue number5
DOIs
StatePublished - Nov 2010

Fingerprint

Down-Regulation
Mink
Cell Line
Rabbits
Cricetulus
Ovary
Long QT Syndrome
Surface Plasmon Resonance
Transgenes
Ion Channels
Transfection
Peptides

Keywords

  • Human ether-a-go-go-related gene
  • KCNH2
  • KCNQ1
  • Potassium channel

ASJC Scopus subject areas

  • Physiology
  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Cite this

Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines. / Ren, Xiao Qin; Liu, Gong Xin; Organ-Darling, Louise E.; Zheng, Renjian; Roder, Karim; Jindal, Hitesh K.; Centracchio, Jason; McDonald, Thomas V.; Koren, Gideon.

In: American Journal of Physiology - Heart and Circulatory Physiology, Vol. 299, No. 5, 11.2010.

Research output: Contribution to journalArticle

Ren, XQ, Liu, GX, Organ-Darling, LE, Zheng, R, Roder, K, Jindal, HK, Centracchio, J, McDonald, TV & Koren, G 2010, 'Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines', American Journal of Physiology - Heart and Circulatory Physiology, vol. 299, no. 5. https://doi.org/10.1152/ajpheart.00479.2009
Ren, Xiao Qin ; Liu, Gong Xin ; Organ-Darling, Louise E. ; Zheng, Renjian ; Roder, Karim ; Jindal, Hitesh K. ; Centracchio, Jason ; McDonald, Thomas V. ; Koren, Gideon. / Pore mutants of HERG and KvLQT1 downregulate the reciprocal currents in stable cell lines. In: American Journal of Physiology - Heart and Circulatory Physiology. 2010 ; Vol. 299, No. 5.
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abstract = "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.",
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AU - Liu, Gong Xin

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AU - Zheng, Renjian

AU - Roder, Karim

AU - Jindal, Hitesh K.

AU - Centracchio, Jason

AU - McDonald, Thomas V.

AU - Koren, Gideon

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AB - 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.

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