Molecular Determinants of Pentamidine-Induced hERG Trafficking Inhibition
- Rammelkamp Center for Education and Research, MetroHealth Campus (A.T.D., L.W., H.W., D.N., I.D., E.F.) and Department of Physiology and Biophysics (D.N., I.D.), Case Western Reserve University, Cleveland, Ohio
- Address correspondence to:
Eckhard Ficker, Rammelkamp Center, MetroHealth Medical Center, 2500 MetroHealth Dr., Cleveland, OH 44109. E-mail: eficker{at}metrohealth.org
Abstract
Pentamidine is an antiprotozoal compound that clinically causes acquired long QT syndrome (acLQTS), which is associated with prolonged QT intervals, tachycardias, and sudden cardiac arrest. Pentamidine delays terminal repolarization in human heart by acutely blocking cardiac inward rectifier currents. At the same time, pentamidine reduces surface expression of the cardiac potassium channel IKr/human ether à-go-go-related gene (hERG). This is unusual in that acLQTS is caused most often by direct block of the cardiac potassium current IKr/hERG. The present study was designed to provide a more complete picture of how hERG surface expression is disrupted by pentamidine at the cellular and molecular levels. Using biochemical and electrophysiological methods, we found that pentamidine exclusively inhibits hERG export from the endoplasmic reticulum to the cell surface in a heterologous expression system as well as in cardiomyocytes. hERG trafficking inhibition could be rescued in the presence of the pharmacological chaperone astemizole. We used rescue experiments in combination with an extensive mutational analysis to locate an interaction site for pentamidine at phenylalanine 656, a crucial residue in the canonical drug binding site of terminally folded hERG. Our data suggest that pentamidine binding to a folding intermediate of hERG arrests channel maturation in a conformational state that cannot be exported from the endoplasmic reticulum. We propose that pentamidine is the founding member of a novel pharmacological entity whose members act as small molecule antichaperones.
Footnotes
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The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
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This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grants T32-HL105338, 1R01-HL096962].
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
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ABBREVIATIONS:
- acLQTS
- acquired long QT syndrome
- hERG
- human ether à-go-go-related gene
- ER
- endoplasmic reticulum
- HEK
- human embryonic kidney
- WT
- wild type
- DMEM
- Dulbecco's modified Eagle's medium
- bEAG
- bovine ether-à-go-go
- NRVM
- Neonatal rat ventricular myocyte
- PAGE
- polyacrylamide gel electrophoresis
- fg
- fully glycosylated 150-kDa cell surface form of hERG
- cg
- core-glycosylated 135-kDa ER-resident form of hERG
- IKr
- rapidly activating delayed rectifier K current
- Hsp90
- 90-kDa heat shock protein
- PBS
- phosphate-buffered saline
- HA
- hemagglutinin
- MK-499
- (+)-N-[1′-(6-cyano-1,2,3,4-tetrahydro-2(R)-naphthalenyl)-3,4-dihydro-4(R)-hydroxyspiro(2H-1-benzopyran-2,4′-piperidin)-6-yl]methanesulfonamide] monohydrochloride.
- Received August 3, 2011.
- Accepted November 1, 2011.
- Copyright © 2012 The American Society for Pharmacology and Experimental Therapeutics
