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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 recovery from inactivation in KV4.3 with KChIP2. (A) Representative currents of recovery from inactivation recorded from G581R and L450F with KChIP2. (B) The stimulation protocol used for recovery from inactivation. (C) G581R and L450F do not promote the recovery of KV4.3/KChIP2 channel from inactivation compared with WT. The error bars represent standard error of the mean for ≥6 cells from each group.
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f3-ijmm-36-01-0309: Effects of G581R and L450F on recovery from inactivation in KV4.3 with KChIP2. (A) Representative currents of recovery from inactivation recorded from G581R and L450F with KChIP2. (B) The stimulation protocol used for recovery from inactivation. (C) G581R and L450F do not promote the recovery of KV4.3/KChIP2 channel from inactivation compared with WT. The error bars represent standard error of the mean for ≥6 cells from each group.

Mentions: Outward K+ currents in the HEK-293 cells were recorded in a voltage-clamp mode at room temperature (24°C). Experiments were conducted using a Axopatch 200B amplifier attached to a Dell desktop computer equipped with a DigiData 1322 series analog/digital interface and pClamp 10.0 software (all from Axon, Sunnyvale, CA, USA). Electrodes were pulled using a PC-10 vertical pipette puller (Narishige, East Meadow, NY, USA) and had a pipette resistance between 1.5 and 3.0 MΩ subsequent to filling with a recording pipette solution containing: 135 mM KCl, 1 mM MgCl2, 10 mM HEPES and 5 mM glucose (pH 7.2). The bath solution for the recording contained: 136 mM NaCl, 4 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES and 10 mM glucose (pH 7.4). Only the data acquired from cells with an input resistance >0.7 GΩ were analyzed. Current densities were obtained from peak amplitudes normalized to cell capacitances. The voltage-dependent inactivation and recovery from inactivation were measured using the protocols shown in the Figs. 2 and 3. The voltage dependence of steady-state inactivation of the KV4.3-WT, KV4.3-G581R and KV4.3-L450F-encoded K+ currents in the presence of KChIP2 evoked from each conditioning potential were measured and normalized to the current evoked from −70 mV (in the same cell). Each sweep was applied with 10 sec intervals. Data were obtained at different sampling frequencies and the current signals were filtered simultaneously at 5 kHz prior to digitization and storage.


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 recovery from inactivation in KV4.3 with KChIP2. (A) Representative currents of recovery from inactivation recorded from G581R and L450F with KChIP2. (B) The stimulation protocol used for recovery from inactivation. (C) G581R and L450F do not promote the recovery of KV4.3/KChIP2 channel from inactivation compared with WT. The error bars represent standard error of the mean for ≥6 cells from each group.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-ijmm-36-01-0309: Effects of G581R and L450F on recovery from inactivation in KV4.3 with KChIP2. (A) Representative currents of recovery from inactivation recorded from G581R and L450F with KChIP2. (B) The stimulation protocol used for recovery from inactivation. (C) G581R and L450F do not promote the recovery of KV4.3/KChIP2 channel from inactivation compared with WT. The error bars represent standard error of the mean for ≥6 cells from each group.
Mentions: Outward K+ currents in the HEK-293 cells were recorded in a voltage-clamp mode at room temperature (24°C). Experiments were conducted using a Axopatch 200B amplifier attached to a Dell desktop computer equipped with a DigiData 1322 series analog/digital interface and pClamp 10.0 software (all from Axon, Sunnyvale, CA, USA). Electrodes were pulled using a PC-10 vertical pipette puller (Narishige, East Meadow, NY, USA) and had a pipette resistance between 1.5 and 3.0 MΩ subsequent to filling with a recording pipette solution containing: 135 mM KCl, 1 mM MgCl2, 10 mM HEPES and 5 mM glucose (pH 7.2). The bath solution for the recording contained: 136 mM NaCl, 4 mM KCl, 1 mM CaCl2, 2 mM MgCl2, 10 mM HEPES and 10 mM glucose (pH 7.4). Only the data acquired from cells with an input resistance >0.7 GΩ were analyzed. Current densities were obtained from peak amplitudes normalized to cell capacitances. The voltage-dependent inactivation and recovery from inactivation were measured using the protocols shown in the Figs. 2 and 3. The voltage dependence of steady-state inactivation of the KV4.3-WT, KV4.3-G581R and KV4.3-L450F-encoded K+ currents in the presence of KChIP2 evoked from each conditioning potential were measured and normalized to the current evoked from −70 mV (in the same cell). Each sweep was applied with 10 sec intervals. Data were obtained at different sampling frequencies and the current signals were filtered simultaneously at 5 kHz prior to digitization and storage.

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