title: Berberine induces hERG channel deficiency through trafficking inhibition.
abstract: OBJECTIVE
  The human ether-a-go-go-related gene (hERG) encodes the α subunit of the IKr, which plays an essential role in repolarization of action potentials. hERG channels are targeted by various pro-arrhythmic drugs. Berberine (BBR) was previously found to acutely inhibit hERG currents and prolong action potential duration. The present study aimed to determine long-term effects of BBR on the expression of 135kDa/155kDa hERG and the mechanism.
  
  
  RESULTS
  hERG expression was assessed by western blot. Mature hERG (155 kDa) was reduced, whereas ER-located hERG (135 kDa) was increased by BBR. This indicated that hERG was restricted to the ER and that BBR disrupted channel trafficking. To determine the mechanism of trafficking inhibition, we performed western blot and immunoprecipitation to test folding of hERG by assessing interaction between hERG and Hsp90/Hsp70. Both the expression of Hsp90 and its interaction with hERG were strongly decreased by BBR. These data suggest that BBR reduces channel folding to induce trafficking inhibition. Western blot and confocal imaging were used to further detect whether the unfolded protein response (UPR) was activated. Active ATF6, a marker of the UPR, was activated by BBR. Calnexin and calreticulin, chaperones that are activated by ATF6 to assist channel folding, were also elevated and increasingly colocalized with hERG. These data also demonstrate that the UPR was activated. Immunoprecipitation and western blot assays were performed after BBR treatment to examine ubiquitination and degradation, common endpoints of the UPR. We found that the ER-restricted hERG was ubiquitinized and degraded in the lysosomes and proteasomes.
  
  
  CONCLUSIONS
  Our study demonstrates that BBR induces hERG channel deficiency by inhibiting channel trafficking after incubation for 24h. Trafficking inhibition activated the UPR, and the ER-restricted hERG was ubiquitinized and degraded in lysosomes and proteasomes.
authors.affiliation: Department of Pharmacology, Harbin Medical University, Ministry of Education, Harbin, China.
authors.forename: Kaiping
authors.initials: K
authors.lastname: Zhang
authors.forename: Duo
authors.initials: D
authors.lastname: Zhi
authors.forename: Ting
authors.initials: T
authors.lastname: Huang
authors.forename: Yan
authors.initials: Y
authors.lastname: Gong
authors.forename: Meng
authors.initials: M
authors.lastname: Yan
authors.forename: Chen
authors.initials: C
authors.lastname: Liu
authors.forename: Ting
authors.initials: T
authors.lastname: Wei
authors.forename: Zengxiang
authors.initials: Z
authors.lastname: Dong
authors.forename: Baoxin
authors.initials: B
authors.lastname: Li
authors.forename: Baofeng
authors.initials: B
authors.lastname: Yang
mesh_terms: D000818:Animals; D001599:Berberine / Q000494:pharmacology*; D015153:Blotting, Western; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels / Q000037:antagonists & inhibitors / Q000172:deficiency* / Q000378:metabolism; D005260:Female; D005455:Fluorescent Antibody Technique; D006168:Guinea Pigs; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D021381:Protein Transport
chemical_list: D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D001599:Berberine