E3 ligase CHIP and Hsc70 regulate Kv1.5 protein expression and function in mammalian cells

Area of Science:

• Molecular Biology
• Cardiology
• Protein Degradation

Background:

• Kv1.5 channels are crucial for atrial action potential repolarization, and their reduced levels are observed in some atrial fibrillation patients.
• The ubiquitin-proteasome pathway regulates protein degradation, and carboxyl-terminus heat shock cognate 70-interacting protein (CHIP) acts as an E3 ubiquitin ligase for short-lived proteins.

Purpose of the Study:

• To investigate the role of CHIP in the degradation of Kv1.5 proteins.
• To elucidate the mechanisms underlying Kv1.5 protein decrease and explore potential therapeutic targets for atrial fibrillation.

Main Methods:

• Coexpression of CHIP and Kv1.5 in HEK293 cells to assess ubiquitination and protein levels.
• Immunofluorescence microscopy to determine Kv1.5 protein localization.
• siRNA-mediated knockdown of CHIP and use of CHIP mutants to evaluate functional domains.
• Immunoprecipitation to identify protein complexes involving CHIP, Kv1.5, and heat shock cognate protein 70 (Hsc70).
• Electrophysiological recordings (Kv1.5 currents) in HEK293 cells and HL-1 cardiomyocytes.

Main Results:

• CHIP overexpression increased Kv1.5 ubiquitination and decreased its protein level and functional currents, while CHIP knockdown had opposite effects.
• CHIP directly interacts with Kv1.5 and facilitates its degradation, a process enhanced by Hsc70.
• CHIP and Hsc70 reduced endogenous Kv1.5 levels and IKur in HL-1 cardiomyocytes, prolonging action potential duration.

Conclusions:

• CHIP, in conjunction with Hsc70, promotes the degradation of Kv1.5 proteins.
• This interaction leads to decreased functional Kv1.5 channels and altered cardiac action potentials.
• The CHIP-Hsc70-Kv1.5 pathway represents a potential therapeutic target for atrial fibrillation.