Hsp90 prevents interaction between CHIP and HERG proteins to facilitate maturation of wild-type and mutant HERG proteins.

AIMS: We examined the role of Hsp90 in expression and maturation of wild-type (WT) and mutant ether-a-go-go related gene (HERG) proteins by using Hsp90 inhibitors, geldanamycin (GA) and radicicol, and Hsp90 overexpression. METHODS AND RESULTS: The proteins were expressed in HEK293 cells or collected from HL-1 mouse cardiomyocytes, and analysed by western blotting, immunoprecipitation, immunofluorescence, and whole-cell patch-clamp techniques. GA and radicicol suppressed maturation of HERG-FLAG proteins and increased their immature forms. Co-expression of Hsp90 counteracted the effects of Hsp90 inhibitors and suppressed ubiquitination of HERG proteins. Overexpressed Hsp90 also inhibited the binding of endogenous C-terminus of Hsp70-interacting protein (CHIP) to HERG-FLAG proteins. Hsp90-induced increase of functional HERG proteins was verified by their increased expression on the cell surface and enhanced HERG channel currents. CHIP overexpression decreased both mature and immature forms of HERG-FLAG proteins in cells treated with GA. Hsp90 facilitated maturation of endogenous ERG proteins, whereas CHIP decreased both forms of ERG proteins in HL-1 cells. Mutant HERG proteins harbouring disease-causing missense mutations were mainly in the immature form and had a higher binding capacity to CHIP than the WT; Hsp90 overexpression suppressed this association. Overexpressed Hsp90 increased the mature form of HERG(1122fs/147) proteins, reduced its ubiquitinated form, increased its immunoreactivity in the endoplasmic reticulum and on the plasma membrane, and increased the mutant-mediated membrane current. CHIP overexpression decreased the immature form of HERG(1122fs/147) proteins. CONCLUSION: Enhancement of HERG protein expression through Hsp90 inhibition of CHIP binding might be a novel therapeutic strategy for long QT syndrome 2 caused by trafficking abnormalities of HERG proteins.

Effect of losartan and benzbromarone on the level of human urate transporter 1 mRNA.

Both an angiotensin II receptor blocker, losartan (CAS 124750-99-8) and a serum urate lowering agent, benzbromarone (CAS 3562-84-3) exert a uricosuric action by inhibiting urate transporter 1 (URAT1). A recent clinical trial indicated that losartan could reduce the level of serum urate in hypertensive patients treated with urate lowering agents, suggesting the different mode of action of losartan from benzbromarone. In the present study, the effect of losartan and benzbromarone on the level of URAT1 mRNA was determined in transfected HEK293 cells. Losartan caused a significant reduction of its mRNA level, whereas it was not affected by benzbromarone. These results indicate that losartan decreases the level of human URAT1 mRNA, which may underlie the uricosuric action of losartan in hypertensive patients treated with serum urate lowering agents.

Inhibition of beta-adrenergic signaling by intracellular AMP is independent of cell-surface adenosine receptors in rat cardiac cells.

We report a novel action of intracellular adenosine monophosphate (AMP) to inhibit beta-adrenergic signaling in isolated rat ventricular myocytes. Extracellular application of adenosine or AMP suppressed isoproterenol (Iso)-induced prolongation of action potential duration (APD). This effect was completely abolished by an A(1)-receptor antagonist, DPCPX. Intracellular application of AMP, but not adenosine, attenuated Iso-induced APD prolongation. Iso-induced increases in the L-type Ca(2+) current (I(Ca,L)) were also inhibited by intracellular AMP. These inhibitory effects were not affected by either DPCPX or glibenclamide. In vitro, AMP directly inhibited PKA activity via binding to its regulatory subunit. These results suggest that intracellular AMP attenuates beta-adrenergic signaling by directly inhibiting PKA activity, independently of A(1)-purinergic receptor.