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Two novel Brugada syndrome-associated mutations increase KV4.3 membrane expression and function.

You T, Mao W, Cai B, Li F, Xu H - Int. J. Mol. Med. (2015)

Bottom Line: The two mutations slowed KV4.3/KChIP2‑encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2‑encoded channel inactivation.Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3‑WT.Reverse transcription‑polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China.

ABSTRACT
The human cardiac fast transient outward K+ channel is composed of the KV4.3 α subunit encoded by KCND3 and the K+ channel‑interacting protein 2 (KChIP2) β subunit, and determines the early repolarization of the action potential (AP). Two human mutations (G600R and L450F) in KV4.3 are associated with Brugada syndrome and they increase the KV4.3/KChIP2‑encoded fast transient outward K+ current (Ito,f) and cause the stable loss of the AP dome. However, the detailed mechanisms underlying the gain of Ito,f function by these two mutations are largely unknown. The experiments in the present study were undertaken to investigate the effect of these mutations and the underlying mechanism. Whole cell patch‑clamp recording was performed in HEK‑293 cells expressing KV4.3‑wild‑type (WT) and KV4.3 mutants with KChIP2. The two individual mutant‑encoded currents were significantly increased but the kinetics of the channels affected by the two mutations were different. The two mutations slowed KV4.3/KChIP2‑encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2‑encoded channel inactivation. Western blotting showed that total KV4.3 protein was significantly augmented in HEK‑293 cells expressing the two individual mutants with KChIP2. Furthermore, immunofluorescence confocal microscopy demonstrated that the KV4.3 channel protein was expressed more in the cell membrane compared to the cytoplasm in cells that expressed individual mutants with KChIP2. Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3‑WT. Reverse transcription‑polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2. Taken together, the present study revealed that the mutations cause a gain‑of‑function of KV4.3/KChIP2‑encoded channels by increasing membrane protein expression and slowing channel inactivation.

No MeSH data available.


Related in: MedlinePlus

Effects of G581R and L450F on membrane localization of KV4.3 channel protein. (A) Representative confocal images of HEK-293 cells transfected with KV4.3-WT, KV4.3-G581R and KV4.3-L450F in the presence of KChIP2. (B) An intensity profile from a scan along the indicated line is shown below each image. (C) In the presence of KChIP2, confocal image analysis showed that G581R (n=10) and L450F (n=15) significantly (*P<0.05) increased the cell membrane to cytoplasmic intensity ratios of KV4.3 channel protein, compared with WT (n=13) ratio. The error bars represent standard error of the mean for the indicated number of cells from each group. AU, artbitrary units.
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f5-ijmm-36-01-0309: Effects of G581R and L450F on membrane localization of KV4.3 channel protein. (A) Representative confocal images of HEK-293 cells transfected with KV4.3-WT, KV4.3-G581R and KV4.3-L450F in the presence of KChIP2. (B) An intensity profile from a scan along the indicated line is shown below each image. (C) In the presence of KChIP2, confocal image analysis showed that G581R (n=10) and L450F (n=15) significantly (*P<0.05) increased the cell membrane to cytoplasmic intensity ratios of KV4.3 channel protein, compared with WT (n=13) ratio. The error bars represent standard error of the mean for the indicated number of cells from each group. AU, artbitrary units.

Mentions: In order to determine if the two mutations affect the localization of the KV4.3 channel protein in HEK-293 cells expressing KV4.3-WT, KV4.3-G581R and KV4.3-L450F with KChIP2, confocal immunofluorescence microscopy was employed. The results revealed a significantly elevated (P<0.05) M/C ratio of KV4.3 channel in cells expressing the individual mutants and KChIP2, as compared with KV4.3-WT and KChIP2 (Fig. 5).


Two novel Brugada syndrome-associated mutations increase KV4.3 membrane expression and function.

You T, Mao W, Cai B, Li F, Xu H - Int. J. Mol. Med. (2015)

Effects of G581R and L450F on membrane localization of KV4.3 channel protein. (A) Representative confocal images of HEK-293 cells transfected with KV4.3-WT, KV4.3-G581R and KV4.3-L450F in the presence of KChIP2. (B) An intensity profile from a scan along the indicated line is shown below each image. (C) In the presence of KChIP2, confocal image analysis showed that G581R (n=10) and L450F (n=15) significantly (*P<0.05) increased the cell membrane to cytoplasmic intensity ratios of KV4.3 channel protein, compared with WT (n=13) ratio. The error bars represent standard error of the mean for the indicated number of cells from each group. AU, artbitrary units.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494594&req=5

f5-ijmm-36-01-0309: Effects of G581R and L450F on membrane localization of KV4.3 channel protein. (A) Representative confocal images of HEK-293 cells transfected with KV4.3-WT, KV4.3-G581R and KV4.3-L450F in the presence of KChIP2. (B) An intensity profile from a scan along the indicated line is shown below each image. (C) In the presence of KChIP2, confocal image analysis showed that G581R (n=10) and L450F (n=15) significantly (*P<0.05) increased the cell membrane to cytoplasmic intensity ratios of KV4.3 channel protein, compared with WT (n=13) ratio. The error bars represent standard error of the mean for the indicated number of cells from each group. AU, artbitrary units.
Mentions: In order to determine if the two mutations affect the localization of the KV4.3 channel protein in HEK-293 cells expressing KV4.3-WT, KV4.3-G581R and KV4.3-L450F with KChIP2, confocal immunofluorescence microscopy was employed. The results revealed a significantly elevated (P<0.05) M/C ratio of KV4.3 channel in cells expressing the individual mutants and KChIP2, as compared with KV4.3-WT and KChIP2 (Fig. 5).

Bottom Line: The two mutations slowed KV4.3/KChIP2‑encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2‑encoded channel inactivation.Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3‑WT.Reverse transcription‑polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology, The Second Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215004, P.R. China.

ABSTRACT
The human cardiac fast transient outward K+ channel is composed of the KV4.3 α subunit encoded by KCND3 and the K+ channel‑interacting protein 2 (KChIP2) β subunit, and determines the early repolarization of the action potential (AP). Two human mutations (G600R and L450F) in KV4.3 are associated with Brugada syndrome and they increase the KV4.3/KChIP2‑encoded fast transient outward K+ current (Ito,f) and cause the stable loss of the AP dome. However, the detailed mechanisms underlying the gain of Ito,f function by these two mutations are largely unknown. The experiments in the present study were undertaken to investigate the effect of these mutations and the underlying mechanism. Whole cell patch‑clamp recording was performed in HEK‑293 cells expressing KV4.3‑wild‑type (WT) and KV4.3 mutants with KChIP2. The two individual mutant‑encoded currents were significantly increased but the kinetics of the channels affected by the two mutations were different. The two mutations slowed KV4.3/KChIP2‑encoded channel inactivation; they did not increase the recovery from the KV4.3/KChIP2‑encoded channel inactivation. Western blotting showed that total KV4.3 protein was significantly augmented in HEK‑293 cells expressing the two individual mutants with KChIP2. Furthermore, immunofluorescence confocal microscopy demonstrated that the KV4.3 channel protein was expressed more in the cell membrane compared to the cytoplasm in cells that expressed individual mutants with KChIP2. Also, KChIP2 increased the amount of channel protein in the cell membrane of KV4.3 mutants significantly more than KV4.3‑WT. Reverse transcription‑polymerase chain reaction showed that KV4.3 mRNA was not significantly changed by individual mutations in the presence of KChIP2. Taken together, the present study revealed that the mutations cause a gain‑of‑function of KV4.3/KChIP2‑encoded channels by increasing membrane protein expression and slowing channel inactivation.

No MeSH data available.


Related in: MedlinePlus