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Halo-BRMS1 full length transiently expressed in HEK293 cells

ABSTRACT: Affinity purification of BRMS1 for proteomic analysis.

INSTRUMENT(S): LTQ

ORGANISM(S): Homo Sapiens (ncbitaxon:9606)

SUBMITTER: Mike Washburn

PROVIDER: MSV000086667 | MassIVE | Thu Jan 07 09:40:00 GMT 2021

SECONDARY ACCESSION(S): PXD023461

REPOSITORIES: MassIVE

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Project description:The human ether-a-go-go-related gene (hERG) potassium channel conducts rapid delayed rectifier potassium currents (IKr) and contributes to phase III cardiac action potential repolarization. Drugs inhibit hERG channels by binding to aromatic residues in hERG helixes. Berberine (BBR) has multiple actions, and its hydrogenated derivative dihydroberberine (DHB) is a potential candidate for developing new drugs. Previous studies have demonstrated that BBR blocks hERG channels and prolongs action potential duration (APD). Our present study aimed to investigate the effects and mechanism of DHB on hERG channels. Protein expression and the hERG current were analyzed using western blotting and patch-clamp, respectively. DHB inhibited the hERG current concentration-dependently after instantaneous perfusion, accelerated channel inactivation by directly binding tyrosine (Tyr652) and phenylalanine (Phe656), and decreased mature (155-kDa) and simultaneously increased immature (135-kDa) hERG expression, respectively. This suggests disruption of forward trafficking of hERG channels. Besides, DHB remarkably reduced heat shock protein 90 (Hsp90) expression and its interaction with hERG, indicating that DHB disrupted hERG trafficking by impairing channel folding. Meanwhie, DHB enhanced the expression of cleaved activating transcription factor-6 (ATF-6), a biomarker of unfolded protein response (UPR). Expression of calnexin and calreticulin, chaperones activated by ATF-6 to facilitate channel folding, were also increased, which indicating UPR activation. Additionally, the degradation rate of mature 155-kDa hERG increased following DHB exposure. In conclusion, we demonstrated that DHB acutely blocked hERG channels by binding the aromatic Tyr652 and Phe656. DHB may decrease hERG plasma membrane expression through two pathways involving disruption of forward trafficking of immature hERG channels and enhanced degradation of mature hERG channels. Furthermore, forward trafficking was disrupted by impaired channel folding associated with altered interactions between hERG proteins and chaperones. Finally, trafficking inhibition activated UPR, and mature hERG channel degradation was increased by DHB.

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Halo-BRMS1 full length transiently expressed in HEK293 cells

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