The mechanistic rote of KCNH2 gene L413P and L559H mutations in long QT syndrome

Cui lan Li, Da yi Hu, Wen ling Liu, Shu ying Qi, Hong tao Wang, Lei Li, Qiuming Gong, Zhengfeng Zhou

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Pages (from-to)838-841
Number of pages4
JournalZhonghua nei ke za zhi [Chinese journal of internal medicine]
Volume46
Issue number10
StatePublished - Oct 2007
Externally publishedYes

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Long QT Syndrome
Mutation
Genes
Mutant Proteins
Fluorescent Antibody Technique
Transfection
Proteins
Western Blotting
Haploinsufficiency
Patch-Clamp Techniques
Protein Transport
Site-Directed Mutagenesis
Endoplasmic Reticulum
Plasmids
Complementary DNA
Cell Membrane
Kidney
Temperature

ASJC Scopus subject areas

  • Medicine(all)

Cite this

The mechanistic rote of KCNH2 gene L413P and L559H mutations in long QT syndrome. / Li, Cui lan; Hu, Da yi; Liu, Wen ling; Qi, Shu ying; Wang, Hong tao; Li, Lei; Gong, Qiuming; Zhou, Zhengfeng.

In: Zhonghua nei ke za zhi [Chinese journal of internal medicine], Vol. 46, No. 10, 10.2007, p. 838-841.

Research output: Contribution to journalArticle

Li, Cui lan ; Hu, Da yi ; Liu, Wen ling ; Qi, Shu ying ; Wang, Hong tao ; Li, Lei ; Gong, Qiuming ; Zhou, Zhengfeng. / The mechanistic rote of KCNH2 gene L413P and L559H mutations in long QT syndrome. In: Zhonghua nei ke za zhi [Chinese journal of internal medicine]. 2007 ; Vol. 46, No. 10. pp. 838-841.
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abstract = "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.",
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T1 - The mechanistic rote of KCNH2 gene L413P and L559H mutations in long QT syndrome

AU - Li, Cui lan

AU - Hu, Da yi

AU - Liu, Wen ling

AU - Qi, Shu ying

AU - Wang, Hong tao

AU - Li, Lei

AU - Gong, Qiuming

AU - Zhou, Zhengfeng

PY - 2007/10

Y1 - 2007/10

N2 - 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.

AB - 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.

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JF - Zhonghua nei ke za zhi [Chinese journal of internal medicine]

SN - 0578-1426

IS - 10

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