SARS-CoV proteins decrease levels and activity of human ENaC via
activation of distinct PKC isoforms
Hong-Long Ji,1,2* Weifeng Song,1* Zhiqian Gao,1Xue-Feng Su,3Hong-Guang Nie,2Yi Jiang,4
Ji-Bin Peng,4Yu-Xian He,5Ying Liao,6Yong-Jian Zhou,7Albert Tousson,8and Sadis Matalon1,3
Departments of1Anesthesiology,3Physiology and Biophysics,4Medicine, and8Cell Biology, University of Alabama at
Birmingham School of Medicine, Birmingham, Alabama;2Department of Biochemistry, Texas Lung Injury Institute, University
of Texas Health Center at Tyler, Tyler, Texas;5Lindsley F. Kimball Research Institute, New York Blood Center, New York,
New York;6Nanyang Technological University, Singapore; and7Department of Gastroenterology and Hepatology, First
Municipal People’s Hospital of Guangzhou, Guangzhou, China
Submitted 8 August 2008; accepted in final form 21 December 2008
Ji HL, Song W, Gao Z, Su X, Nie H, Jiang Y, Peng J, He Y, Liao
and activity of human ENaC via activation of distinct PKC isoforms.
Am J Physiol Lung Cell Mol Physiol 296: L372–L383, 2009. First
published December 26, 2008; doi:10.1152/ajplung.90437.2008.—
Among the multiple organ disorders caused by the severe acute
respiratory syndrome coronavirus (SARS-CoV), acute lung failure
following atypical pneumonia is the most serious and often fatal
event. We hypothesized that two of the hydrophilic structural coro-
noviral proteins (S and E) would regulate alveolar fluid clearance by
decreasing the cell surface expression and activity of amiloride-
sensitive epithelial sodium (Na?) channels (ENaC), the rate-limiting
protein in transepithelial Na?vectorial transport across distal lung
epithelial cells. Coexpression of either S or E protein with human ?-,
?-, and ?-ENaC in Xenopus oocytes led to significant decreases of
both amiloride-sensitive Na?currents and ?-ENaC protein levels at
their plasma membranes. S and E proteins decreased the rate of ENaC
exocytosis and either had no effect (S) or decreased (E) rates
of endocytosis. No direct interactions among SARS-CoV E protein
with either ?- or ?-ENaC were indentified. Instead, the downregula-
tion of ENaC activity by SARS proteins was partially or completely
restored by administration of inhibitors of PKC?/?1 and PKC?.
Consistent with the whole cell data, expression of S and E proteins
decreased ENaC single-channel activity in oocytes, and these effects
were partially abrogated by PKC?/?1 inhibitors. Finally, transfection
of human airway epithelial (H441) cells with SARS E protein de-
creased whole cell amiloride-sensitive currents. These findings indi-
cate that lung edema in SARS infection may be due at least in part to
activation of PKC by SARS proteins, leading to decreasing levels and
activity of ENaC at the apical surfaces of lung epithelial cells.
Xenopus oocytes; voltage clamp; cell-attached patches; amiloride-
sensitive currents; severe acute respiratory syndrome coronavirus;
surface epithelial sodium channels; H441 cells
THE FLUID THAT FILLS the alveolar spaces in the fetal lung is
cleared shortly after birth, mainly as a consequence of active
transport of sodium (Na?) ions across the alveolar epithelium.
This transport establishes an osmotic gradient that favors
reabsorption of intra-alveolar fluid (18). Studies that demon-
strate the reabsorption of intratracheally instilled isotonic fluid
or plasma from the alveolar spaces of adult anesthetized
animals and resected human lungs, and the partial inhibition of
this process by amiloride and ouabain, indicate that adult
alveolar epithelial cells are also capable of actively transport-
ing Na?ions (reviewed in Refs. 34, 35).
A variety of studies have clearly established that active Na?
transport limits the degree of alveolar edema under patholog-
ical conditions in which the alveolar epithelium has been
damaged. For example, intratracheal instillation of a Na?
channel blocker in rats exposed to hyperoxia increased the
amount of extravascular lung water (51). Conversely, intratra-
cheal instillation of adenoviral vectors expressing Na?,K?-
ATPase genes increased survival of rats exposed to hyperoxia
(14). Moreover, patients with acute lung injury who are still
able to concentrate alveolar protein (as a result of active Na?
reabsorption) have a better prognosis than those who cannot
(47). Results from electrophysiological studies across both
confluent monolayers of alveolar type II (ATII) cells mounted
in Ussing chambers and alveolar epithelial cells patched in the
whole cell or cell-attached modes indicate that Na?ions
diffuse passively into ATII and ATI cells through apically
located amiloride-sensitive cation and sodium-selective chan-
nels (16, 19, 26, 52) and are extruded across the basolateral cell
membranes by the ouabain-sensitive Na?,K?-ATPase (36).
The cation channels on the apical surface usually constitute the
rate-limiting step in this process, offering more than 90% of the
resistance to transcellular Na?transport in either ATI or ATII
Acute respiratory viral infections cause significant morbidity
and mortality in both adults and children. For example, respi-
ratory syncytial virus (RSV), a member of the pneumovirus
genus of the Paramyxoviridae, is the most common cause of
lower respiratory tract infections in infants and children world-
wide and also causes community-acquired lower respiratory
tract infections among adults (39). Influenza viruses (types A
and B) account for more than 50% of all viral pneumonias in
adults. Influenza has a high morbidity, affecting 10–20% of the
U.S. population, accounting for up to 40,000 deaths annually.
There is also a continuing risk of more severe influenza
pandemics. Both of these viruses have been shown to impair
Na?transport, albeit by different mechanisms: RSV inhibits
Na?-dependent alveolar fluid clearance in Balb/c mice and
amiloride-sensitive currents across human airway (H441) cells
via increasing levels of UTP, which exit alveolar cells via
* H.-L. Ji and W. Song contributed equally to this work.
Address for reprint requests and other correspondence: S. Matalon, Dept. of
Anesthesiology, Univ. of Alabama at Birmingham, 901 19th St., Birmingham,
AL 35233-6810 (e-mail: email@example.com).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Lung Cell Mol Physiol 296: L372–L383, 2009.
First published December 26, 2008; doi:10.1152/ajplung.90437.2008.
1040-0605/09 $8.00 Copyright © 2009 the American Physiological Societyhttp://www.ajplung.orgL372
volume-activated anion channels and act on purinergic recep-
tors (5, 10–13). Viral replication is essential for the inhibition
of Na?transport. On the other hand, nonreplicating influenza
viruses inhibit epithelial Na?channels (ENaC) by activating
PKC (6, 29). In the case of RSV, amelioration of the
decrease of Na?-dependent alveolar fluid clearance in vivo
prevented both the RSV-induced hypoxemia and pulmonary
edema (10, 11).
Among the multiple organ disorders caused by the newly
emerged severe acute respiratory syndrome coronavirus
(SARS-CoV), acute lung failure following atypical pneumonia
is an often fatal event. The pathology of SARS virus-infected
lung tissues includes acute lung injury, characterized by hy-
poxemia and lung edema. SARS virus has been detected in
100% of the lung tissues from infected individuals. SARS-CoV
shares a high degree of sequence identity to group 1 corona-
viruses and encodes one polyprotein for virus replication, four
structural proteins (spike protein S, envelop protein E, mem-
brane protein M, and nucleocapsid protein N), and eight
additional polypeptides, such as the 3C-like protease (43). The
S protein, located at the viral surface, plays a key role in
cell-viral binding and membrane fusion. The E protein spans
the viral shell and is involved in viral envelope formation as
well as viral replication.
In the present study we have sought to determine whether
SARS-CoV proteins alter ENaC-mediated amiloride-sensitive
Na?transport and, if so, to identify putative mechanisms
responsible for this action. Because of well-known interactions
between the cystic fibrosis transmembrane regulator (CFTR)
with ENaC (3, 7, 21) and possible effects of SARS proteins on
either transporter, we opted to coexpress ENaC and SARS
proteins in Xenopus oocytes, which normally do not express
CFTR. Thus we coinjected human ?-, ?-, and ?-ENaC
(hENaC) cRNAs and cRNAs for either the SARS-CoV S or E
proteins into Xenopus oocytes and measured the following
variables: 1) whole cell and single-channel amiloride-sensitive
currents, 2) total and plasma membrane levels of ?- and
?-ENaC by Western blotting, and 3) rates of ENaC endocytosis
and exocytosis. We also tested whether expression of either the
S or E proteins increased the permeability of oocytes to a
variety of cations, as previously suggested (48). Our results
indicate that the coexpression of either S or E protein with
ENaC significantly decreases both the whole cell and single-
channel amiloride-sensitive currents and ENaC protein levels
at the plasma membrane and that these changes are partially
due to activation of PKC. Furthermore, our data reveal that, in
contrast to previous observations in Escherichia coli (48),
Fig. 1. Coexpression of severe acute respiratory syndrome coronavirus (SARS-CoV) spike (S) and envelop (E) proteins reduce ?,?,?-epithelial Na?channel
(ENaC) activity in Xenopus oocytes. A: representative current traces recorded across Xenopus oocytes injected with ?,?,?-human ENaC (hENaC) cRNAs alone
(top), ?,?,?-hENaC and SARS-CoV S protein (middle), and ?,?,?-hENaC and SARS-CoV E protein (bottom). Currents were measured following a change in
the membrane potential from the holding potential (?40 mV) to ?120 and ?100 mV for 500 mS. After the inward and outward currents became stable, oocytes
were perfused with 10 ?M amiloride, as indicated. Amil, amiloride. B: amiloride-sensitive Na?currents (IENaC) at ?120 mV in oocytes expressing ?,?,?-hENaC
alone (???) or ?,?,?-ENaC and either SARS-CoV S protein (?S) or SARS-CoV E protein (?E). IENaCwere calculated as described in MATERIALS AND METHODS.
Values are means ? SE; no. of oocytes is as follows: ???, n ? 21; ?S, n ? 29; ?E, n ? 7. **P ? 0.001 compared with the IENaC of ??? alone. C: resting
membrane potentials (in mV) were measured at the current-clamp mode for water (H2O)-injected oocytes and oocytes expressing ?,?,?-hENaC with or without
SARS-CoV S or E protein. Bars represent means ? SE; no. of oocytes is as follows: H2O, n ? 20; ???, n ? 21; ?S, n ? 29; ?E, n ? 7. Normal resting
membrane potential of uninjected oocytes is approximately ?25 mV in ND-96 medium. **P ? 0.001 for the indicated comparisons.
SARS PROTEINS REGULATE ENaC
AJP-Lung Cell Mol Physiol • VOL 296 • MARCH 2009 • www.ajplung.org
activity was the result of inhibition of ENaC synthesis due to
overexpression of viral proteins. To further exclude this pos-
sibility, we coexpressed SARS-CoV S or E proteins with
transient receptor potential vanilloid type 6 (TRPV6), a Ca2?
channel protein in Xenopus oocytes. TRPV6 activity was not
altered by the E protein and was significantly increased by the
S protein (data not shown). Together, our data indicate that the
SARS-CoV protein-mediated reduction in ENaC expression
and activity was not due to competition for protein synthesis
Previous studies have shown that both native and heterolo-
gously expressed ENaC activity is regulated by PKC phosphor-
ylation (44, 50). Our results confirmed that at least three
PKC-specific isozymes, namely, ?, ?1, and ?, are activated
when S and E proteins are expressed in Xenopus oocytes, albeit
to different extents: SARS-CoV S activated predominantly the
PKC?/?1 isoforms, whereas SARS-CoV E activated the PKC?
isoform. Davis et al. (13) recently reported that respiratory
syncytial virus activates PKC? and, in turn, inhibits amiloride-
sensitive Na?uptake across the distal lung epithelium in
RSV-infected Balb/c mice. The PKC pathway also plays a
critical role in initiating a series of events leading to down-
regulation of ENaC following the binding of hemagglutinin
residues of inactivated influenza virus to sialic acid residues of
ATII cells and airway cells (6, 28).
Protein trafficking as well as phosphorylation can be regu-
lated by PKC activity (33). Our results also demonstrate that
both the S and E proteins decrease ENaC protein trafficking to
the plasma membrane, resulting in reduced ?-hENaC expres-
sion at the plasma membrane. We were unable to detect ?- or
?-hENaC at the plasma membrane, most likely due to low
affinity of commercially available antibodies against these
subunits. However, Na?single channels recorded from cell-
attached patches of oocytes injected with either hENaC alone
or hENaC plus SARS-CoV proteins had similar conductances
(4 pS; Fig. 6). Since changes in ENaC stoichiometry are
accompanied by changes in single-channel conductance (4, 35,
37), our data suggest that expression of SARS-CoV protein in
oocytes did not alter ENaC stoichiometry at the cell surface.
Thus we expect that changes in ?-hENaC were accompanied
by similar changes in both ?-hENaC and ?-hENaC. It is
unlikely that the observed changes in surface ENaC can ac-
count for the almost complete loss of the whole cell amiloride-
sensitive currents noted in Fig. 1. Instead, we believe that
decreased ENaC activity was the result of activation of PKC
isoforms, as previously described (2, 6, 38).
Expression of the S and E proteins in Xenopus oocytes alone
did not alter the cation permeability of the oocyte membrane to
Na?, K?, H?, Ca2?, Mg2?or Zn2?. Although the E protein
can function as a cation channel in the planar lipid bilayer
system (48), our results indicate that E protein may not act as
a cation channel by itself in a model vertebrate cell, the
Xenopus oocyte. Our observations are not consistent with a
recent report in which E protein expression in E. coli cells
altered membrane permeability (32).
Protein-protein interactions have been suggested to be in-
volved in the regulation of ENaC in a variety of systems; for
example, a reciprocal interaction between homomeric CFTR
and ENaC has been reported, and the intracellular COOH- and
NH2-terminal tails of ENaC have been identified as interactive
domains regulated by CFTR (21). We also have reported that
?-ENaC subunit interacts with ?- and ?-subunits modifying
ENaC activity (24). Because of these findings, we sought to
identify possible intramolecular interactions between SARS-
CoV viral proteins and each ENaC subunit. Our results showed
that SARS-CoV E protein does not physically interact with ?-
and ?-ENaC subunits. On the other hand, SARS-CoV S and E
proteins activate PKC, which may contribute to their diverse
effects on exocytosis and endocytosis rates of ENaC proteins,
in turn, downregulating ENaC channel activity at the whole
cell and single-channel levels.
In summary, our studies suggest that SARS-CoV S and E
proteins downregulate ENaC expression and activity in both
Xenopus oocytes injected with hENaC and human airway cells
expressing native ENaC. Thus these studies provide new in-
sight into pathogenesis of pulmonary edema in SARS infec-
We thank Dr. Peter Smith (Dept. of Physiology, University of Alabama at
Birmingham) for providing ENaC cDNAs. We also acknowledge the editorial
assistance of Terese J. Potter (Dept. of Anesthesiology, University of Alabama
at Birmingham) for editorial assistance in preparing this article.
This work was supported by National Institutes of HealthGrants HL31197
and U54 ES017218 (to S. Matalon) and HL87017 (to H. L. Ji).
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