Identification of I_Kr and Its Trafficking Disruption Induced by Probucol in Cultured Neonatal Rat Cardiomyocytes

Abstract

The human ether-a-go-go-related gene (hERG) encodes a channel that conducts the rapidly activating delayed rectifier K ϩ current (I Kr ), which is important for cardiac repolarization. Mutations in hERG reduce I Kr and cause congenital long QT syndrome (LQTS). More frequently, common medications can reduce I Kr and cause LQTS as a side effect. Protein trafficking abnormalities are responsible for most hERG mutation-related LQTS and are recently recognized as a mechanism for druginduced LQTS. Whereas hERG trafficking has been studied in recombinant expression systems, there has been no reported study on cardiac I Kr trafficking at the protein level. In the present study, we identified that I Kr is present in cultured neonatal rat ventricular myocytes and can be robustly recorded using Cs ϩ as the charge carrier. We further discovered that 4,4Ј-(isopropylidenedithio)-bis-(2,6-di-t-butylphenol) (probucol), a cholesterol-lowering drug that induces LQTS, disrupted I Kr trafficking and prolonged the cardiac action potential duration. Probucol did not directly block I Kr . Probucol also disrupted hERG trafficking and did not block hERG channels expressed in human embryonic kidney 293 cells. We conclude that probucol induces LQTS by disrupting ether-a-go-go-related gene trafficking, and that primary culture of neonatal rat cardiomyocytes represents a useful system for studying native I Kr trafficking.

Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)

“…open versus closed) and the total number of functional channels at the plasma membrane. Studies have shown that mutations in hERG or drugs can decrease the hERG density at the plasma membrane, leading to a diminished I hERG , which causes long QT syndrome (7,41). However, it is unknown how the density of hERG channels on the plasma membrane is regulated.…”

“…Single cardiomyocytes were isolated from 1-day-old Sprague-Dawley rats of either sex using enzymatic dissociation methods as described previously (7). Cells were cultured in Dulbecco's modified Eagle's medium/Ham's F-12 medium (Invitrogen) with 10% FBS.…”

The Serum- and Glucocorticoid-inducible Kinases SGK1 and SGK3 Regulate hERG Channel Expression via Ubiquitin Ligase Nedd4-2 and GTPase Rab11

“…For electrophysiological experiments on transiently expressed channels, GFP cDNA (pIRES2-EGFP, Clontech) was co-transfected for selection of transfected cells. Cardiac myocytes were isolated from neonatal Sprague-Dawley rats using enzymatic dissociation as described previously (18).…”

“…hERG proteins display two distinct bands in the Western blot analysis: a mature fully glycosylated form in the plasma membrane with a molecular mass of 155 kDa and an immature coreglycosylated form with a molecular mass of 135 kDa (18,20). SGK1 or SGK3 overexpression significantly increased the expression of the mature hERG protein (155 kDa) but had no significant effect on the expression of the immature hERG protein (135 kDa) (Fig.…”

Effect of Terfenadine and Pentamidine on the hERG Channel and Its Intracellular Trafficking: Combined Analysis with Automated Voltage Clamp and Confocal Microscopy

“…7) Nevertheless, assessment of hERG-blocking activity has been proven to be valid for the risk evaluation of a majority of drugs. [8][9][10] However, there are some drugs which have no acute effect on the hERG channel current, but induce QT prolongation and ventricular arrhythmia such as pentamidine, [11][12][13] probucol 14,15) and cardiac glycosides. 16) These drugs have been reported to inhibit the intracellular trafficking of the hERG channel to the cell membrane.…”