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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 KV4.3/KChIP2-encoded K+ currents. (A) In HEK-293 cells transfected with plasmids encoding KV4.3-WT, KV4.3-G581R or KV4.3-L450F together with KChIP2, KV4.3-G581R and KV4.3-L450F significantly increased the transient outward K+ current density compared with KV4.3-WT. (B) The current-voltage association for the transient outward K+ currents in cells expressing KV4.3-WT (n=12), KV4.3-G581R (n=11) or KV4.3-L450F (n=12) with KChIP2. Two mutations significantly increase transient outward K+ current densities in HEK-293 cells expressing KV4.3-G581 and KV4.3-L450F in the presence of KChIP2 at voltages from −40 to +60 mV. (C) Two mutations significantly slow the KV4.3-G581R/KChIP2-encoded (n=11) or KV4.3-L450F/KChIP2-encoded (n=7) channel inactivation at voltages from 0 to +60 mV compared to KV4.3-WT (n=12). •, KV4.3-WT/KChIP2; ■, KV4.3-G581R/KChIP2; ▲, KV4.3-L450F/KChIP2. (C–E) *P<0.05 vs. KV4.3-WT/KChIP2. The error bars represent standard error of the mean for the indicated number of cells from each group. WT, wild-type.
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f1-ijmm-36-01-0309: Effects of G581R and L450F on KV4.3/KChIP2-encoded K+ currents. (A) In HEK-293 cells transfected with plasmids encoding KV4.3-WT, KV4.3-G581R or KV4.3-L450F together with KChIP2, KV4.3-G581R and KV4.3-L450F significantly increased the transient outward K+ current density compared with KV4.3-WT. (B) The current-voltage association for the transient outward K+ currents in cells expressing KV4.3-WT (n=12), KV4.3-G581R (n=11) or KV4.3-L450F (n=12) with KChIP2. Two mutations significantly increase transient outward K+ current densities in HEK-293 cells expressing KV4.3-G581 and KV4.3-L450F in the presence of KChIP2 at voltages from −40 to +60 mV. (C) Two mutations significantly slow the KV4.3-G581R/KChIP2-encoded (n=11) or KV4.3-L450F/KChIP2-encoded (n=7) channel inactivation at voltages from 0 to +60 mV compared to KV4.3-WT (n=12). •, KV4.3-WT/KChIP2; ■, KV4.3-G581R/KChIP2; ▲, KV4.3-L450F/KChIP2. (C–E) *P<0.05 vs. KV4.3-WT/KChIP2. The error bars represent standard error of the mean for the indicated number of cells from each group. WT, wild-type.

Mentions: cDNA plasmids-encoding KV4.3 and KChIP2, respectively, were kindly provided by Dr Jeanne M. Nerbonne at Washington University School of Medicine in St. Louis (MO, USA). Alignment by NCBI Blast software (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that >90% of the amino acids of rat (NCBI protein, NP_001257891.1, 10-Aug-2014) and human (NCBI protein, NP_004971.2, 25-May-2014) KV4.3 are identical (Fig. 1B). Two targeted mutations in rat KCND3 were generated using the QuickChange II XL site-directed mutagenesis kit (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions. Rat KCND3, with two individual mutations corresponding to BrS in humans, was confirmed by sequencing of the constructs using the Big T3 terminator kit (Applied Biosystems, Foster City, CA, USA) (Fig. 1A) and they encode KV4.3-G581R and KV4.3-L450F, respectively. The following primers were used for polymerase chain reaction (PCR): rKV4.3-G581R, 5′AAAGCAGACGATCGACTGAGACCAA-3′ [nucleotides (nt) 1821–1855]; and rKV4.3-L450F, 5′GCGCAATGGACT CTTCAATGAAGCTCTGG-3′ (nt 1427–1455). The underlined letters (TC and T) refer to the base pair changes corresponding to the rat KV4.3 mutation G580R and L450F, respectively.


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 KV4.3/KChIP2-encoded K+ currents. (A) In HEK-293 cells transfected with plasmids encoding KV4.3-WT, KV4.3-G581R or KV4.3-L450F together with KChIP2, KV4.3-G581R and KV4.3-L450F significantly increased the transient outward K+ current density compared with KV4.3-WT. (B) The current-voltage association for the transient outward K+ currents in cells expressing KV4.3-WT (n=12), KV4.3-G581R (n=11) or KV4.3-L450F (n=12) with KChIP2. Two mutations significantly increase transient outward K+ current densities in HEK-293 cells expressing KV4.3-G581 and KV4.3-L450F in the presence of KChIP2 at voltages from −40 to +60 mV. (C) Two mutations significantly slow the KV4.3-G581R/KChIP2-encoded (n=11) or KV4.3-L450F/KChIP2-encoded (n=7) channel inactivation at voltages from 0 to +60 mV compared to KV4.3-WT (n=12). •, KV4.3-WT/KChIP2; ■, KV4.3-G581R/KChIP2; ▲, KV4.3-L450F/KChIP2. (C–E) *P<0.05 vs. KV4.3-WT/KChIP2. The error bars represent standard error of the mean for the indicated number of cells from each group. WT, wild-type.
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f1-ijmm-36-01-0309: Effects of G581R and L450F on KV4.3/KChIP2-encoded K+ currents. (A) In HEK-293 cells transfected with plasmids encoding KV4.3-WT, KV4.3-G581R or KV4.3-L450F together with KChIP2, KV4.3-G581R and KV4.3-L450F significantly increased the transient outward K+ current density compared with KV4.3-WT. (B) The current-voltage association for the transient outward K+ currents in cells expressing KV4.3-WT (n=12), KV4.3-G581R (n=11) or KV4.3-L450F (n=12) with KChIP2. Two mutations significantly increase transient outward K+ current densities in HEK-293 cells expressing KV4.3-G581 and KV4.3-L450F in the presence of KChIP2 at voltages from −40 to +60 mV. (C) Two mutations significantly slow the KV4.3-G581R/KChIP2-encoded (n=11) or KV4.3-L450F/KChIP2-encoded (n=7) channel inactivation at voltages from 0 to +60 mV compared to KV4.3-WT (n=12). •, KV4.3-WT/KChIP2; ■, KV4.3-G581R/KChIP2; ▲, KV4.3-L450F/KChIP2. (C–E) *P<0.05 vs. KV4.3-WT/KChIP2. The error bars represent standard error of the mean for the indicated number of cells from each group. WT, wild-type.
Mentions: cDNA plasmids-encoding KV4.3 and KChIP2, respectively, were kindly provided by Dr Jeanne M. Nerbonne at Washington University School of Medicine in St. Louis (MO, USA). Alignment by NCBI Blast software (http://blast.ncbi.nlm.nih.gov/Blast.cgi) showed that >90% of the amino acids of rat (NCBI protein, NP_001257891.1, 10-Aug-2014) and human (NCBI protein, NP_004971.2, 25-May-2014) KV4.3 are identical (Fig. 1B). Two targeted mutations in rat KCND3 were generated using the QuickChange II XL site-directed mutagenesis kit (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions. Rat KCND3, with two individual mutations corresponding to BrS in humans, was confirmed by sequencing of the constructs using the Big T3 terminator kit (Applied Biosystems, Foster City, CA, USA) (Fig. 1A) and they encode KV4.3-G581R and KV4.3-L450F, respectively. The following primers were used for polymerase chain reaction (PCR): rKV4.3-G581R, 5′AAAGCAGACGATCGACTGAGACCAA-3′ [nucleotides (nt) 1821–1855]; and rKV4.3-L450F, 5′GCGCAATGGACT CTTCAATGAAGCTCTGG-3′ (nt 1427–1455). The underlined letters (TC and T) refer to the base pair changes corresponding to the rat KV4.3 mutation G580R and L450F, respectively.

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