Mitochondrial reactive oxygen species mediate hypoxic down-regulation
of hERG channel protein
Jayasri Nanduri
a,
*
, Ning Wang
a
, Pamela Bergson
a
, Guoxiang Yuan
a
, Eckhard Ficker
b
, Nanduri R. Prabhakar
a
a
Center for Systems Biology, Department of Medicine, University of Chicago, 5841S. Maryland Avenue, N603, Chicago, IL 60637, USA
b
MetroHealth Sciences, Case Western Reserve University, Cleveland, OH, USA
article info
Article history:
Received 6 June 2008
Available online 18 June 2008
Keywords:
hERG
Hypoxia
Mitochondria
ROS
NADPH oxidase
Mitochondrial reactive oxygen species
Proteasome
abstract
Previous studies suggest that reactive oxygen species (ROS) play an important role in physiological
responses to hypoxia. In the present study, we examined the effects of hypoxia on human ether-a-go-
go related gene (hERG) channel protein expression and assessed the role of ROS. Hypoxia, in a stimulus-
and time-dependent manner, decreased hERG protein with marked reduction in hERG K
+
conductance in
human embryonic kidney cells stably expressing the hERG
a
subunit. Down-regulation of hERG by
hypoxia was not due to increased proteasomal degradation or decreased transcription but due to
decreased synthesis of the protein. Hypoxia increased ROS in a time-dependent manner. Antioxidants
prevented hypoxia-evoked down-regulation of hERG protein and exogenous oxidants mimicked the
effects of hypoxia. Hypoxia-evoked down-regulation of hERG protein and elevation in ROS were absent
in p
O
cells, which are devoid of mitochondrial DNA. Inhibitors of NADPH oxidase failed to prevent the
effects of hypoxia. These results demonstrate that hypoxia enhances the production of ROS in the mito-
chondria, resulting in down-regulation of hERG translation and decreased hERG-mediated K
+
conductance.
Ó 2008 Elsevier Inc. All rights reserved.
Hypoxia, i.e. the decreased availability of oxygen, occurs under
a variety of physiological and pathophysiological conditions. K
+
channels account for a wide variety of cellular responses to hypox-
ia including cell depolarization, voltage-dependent calcium influx,
and secretion [1]. Acute hypoxia modulates K
+
channel activity in a
rapid, reversible manner and thereby alters cellular excitability.
Chronic hypoxia, on the other hand, affects functional expression
of K
+
channels by altering gene expression [2,3] and/or by post-
translational modification of one or more subunits [4].
Human ether-a-go-go (hERG) channels conduct a rapidly acti-
vating delayed rectifier K
+
current [5]. Unlike many K
+
channels,
hERG channels are open at relatively hyperpolarized membrane
potentials, providing an important control over resting membrane
potential and action potential repolarization. Impaired hERG chan-
nel function has been implicated in long QT syndrome type 2
(LQT2), a cardiac depolarization disorder that predisposes affected
individuals to ventricular arrhythmias and sudden cardiac arrest
[6]. We and others have shown that blockade of hERG-like currents
depolarizes carotid body glomus cells [7] and the depolarization is
enhanced by acute hypoxia in newborn rats [8]. Prolonged hypoxia,
lasting several days, alters gating properties of hERG currents in
neuroblastoma cells [9]. Although activity of hERG channel has
been studied extensively, very little is known as to how hypoxia af-
fects the functional expression of hERG channel protein or the
mechanisms involved in channel regulation by hypoxia.
Previous studies suggest that reactive oxygen species (ROS) play
a pivotal role in mediating physiological responses to prolonged
hypoxia. Studies by Schumacker and co-workers suggest that hy-
poxia increases ROS generation via inhibition of the mitochondrial
electron transport chain at complex III [10]. Acute application of
ROS mimetics alters the kinetics of hERG K
+
conductance [11–
14]. Whether ROS signaling also plays a role in hypoxia-evoked
alterations in hERG protein is not known. Consequently, in the
studies described below, we examined the effects of prolonged hy-
poxia on hERG protein expression in human embryonic kidney
(HEK293) cells stably expressing the hERG
a
subunit and assessed
the potential role of ROS. Our results demonstrate that prolonged
hypoxia down-regulates hERG protein via translational changes
in a time- and stimulus-dependent manner, and that ROS gener-
ated by mitochondria mediate the effects of low O
2
.
Materials and methods
Cell cultures and hypoxia exposure. HEK293 cells stably express-
ing the
a
subunit of the hERG channel (hERG/HEK) cells and human
Kv1.5 (hKv1.5) stably expressed in mouse L cells were cultured in
DMEM containing FBS, penicillin/streptomycin/geneticin at 37 °C
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved.
doi:10.1016/j.bbrc.2008.06.028
* Corresponding author. Fax: +1 773 834 5252.
E-mail address: jnanduri@medicine.bsd.uchicago.edu (J. Nanduri).
Biochemical and Biophysical Research Communications 373 (2008) 309–314
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Biochemical and Biophysical Research Communications
journal homepage: www.elsevier.com/locate/ybbrc