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

Area of Science:

• Cardiovascular Physiology
• Molecular Biology
• Ion Channel Function

Background:

• The human cardiac fast transient outward K+ current (Ito,f) is crucial for cardiac action potential (AP) repolarization, primarily mediated by KV4.3 and KChIP2 subunits.
• Mutations G600R and L450F in KV4.3 are associated with Brugada syndrome, causing increased Ito,f and loss of the AP dome, but the underlying mechanisms remain unclear.

Purpose of the Study:

• To investigate the functional effects of G600R and L450F mutations in KV4.3 on Ito,f.
• To elucidate the molecular mechanisms responsible for the gain-of-function observed in these KV4.3 mutants.

Main Methods:

• Whole-cell patch-clamp recordings in HEK-293 cells co-expressing KV4.3 (wild-type or mutants) with KChIP2.
• Western blotting to assess total KV4.3 protein levels.
• Immunofluorescence confocal microscopy to determine subcellular localization of KV4.3.
• Reverse transcription-polymerase chain reaction (RT-PCR) to analyze KV4.3 mRNA expression.

Main Results:

• Both G600R and L450F mutations significantly increased Ito,f, with distinct kinetic alterations.
• Mutations slowed channel inactivation but did not affect recovery from inactivation.
• Western blotting revealed increased total KV4.3 protein levels for both mutants.
• Immunofluorescence showed enhanced membrane expression of KV4.3 mutants, with KChIP2 further increasing membrane localization compared to wild-type.

Conclusions:

• The G600R and L450F mutations confer a gain-of-function to KV4.3/KChIP2 channels.
• This gain-of-function is primarily mediated by increased KV4.3 protein expression at the cell membrane and slowed channel inactivation.
• These molecular changes provide a mechanistic basis for the association of these mutations with Brugada syndrome.