Single-channel analysis of functional epithelial sodium channel (ENaC)
stability at the apical membrane of A6 distal kidney cells
Ling Yu, My N. Helms, Qiang Yue, and Douglas C. Eaton
Center for Cell and Molecular Signaling, Department of Physiology, Emory University School of Medicine, Atlanta, Georgia
Submitted 20 December 2007; accepted in final form 5 September 2008
Yu L, Helms MN, Yue Q, Eaton DC. Single-channel analysis of
functional epithelial sodium channel (ENaC) stability at the apical
membrane of A6 distal kidney cells. Am J Physiol Renal Phys-
iol 295: F1519–F1527, 2008. First published September 10, 2008;
doi:10.1152/ajprenal.00605.2007.—Epithelial sodium channels (ENaC)
play an essential role in maintaining total body fluid and electrolyte
homeostasis. As such, abnormal expression of ENaC at the cell
surface is linked to several important human diseases. Although the
stability of ENaC subunits has been extensively studied by protein
biochemical analysis, the half-life of the functional channel in the
apical membrane remains controversial. Because the functional sta-
bility of the multisubunit channel may be more physiologically
relevant than the stability of individual subunit proteins, we performed
studies of functional ENaC channels using A6 epithelial cells, a
Xenopus laevis distal nephron cell line. We recorded single-channel
activity in over 400 cells with the translation blockers cycloheximide
(CHX) or puromycin, as well as the intracellular protein trafficking
inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-
channel recordings allow us to quantify the channel density in the
apical membrane, as well as to determine channel open probability
(Po) from control (untreated) cells and from cells at different times of
drug treatment. The data suggest that the half-life of ENaC channels
is ?3.5 h following puromycin, BFA, and nocodazole treatment.
Furthermore, these three drugs had no significant effect on the Poof
ENaC for at least 6 h after exposure. A decrease in apical channel
number and Po was observed following 2 h of CHX inhibition of
protein synthesis, and the apparent channel half-life was closer to
1.5 h following CHX treatment. Treatment of cells with the translation
inhibitors does not alter the expression of the protease furin, and
therefore changes in protease activity cannot explain changes in ENaC
Po. Confocal images show that BFA and nocodazole both disrupt most
of the Golgi apparatus after 1-h exposure. In cells with the Golgi
totally disrupted by overnight exposure to BFA, 20% of apical ENaC
channels remained functional. This result suggests that ENaC is
delivered to the apical membrane via a pathway that might bypass the
Golgi vesicular trafficking pathway, or that there might be two pools
of channels with markedly different half-lives in the apical membrane.
cell-attached patch clamp; brefeldin-A; ENaC half-life
IN MAMMALS AND VERTEBRATES, the tight control of extracellular
fluid homeostasis is mainly achieved by controlling salt and
water reabsorption in the kidneys. Only 1 or 2% of the filtered
load of sodium is reabsorbed in the distal nephron of the
kidney. Nonetheless, the distal nephron is the major point for
discretionary control of total body sodium balance. In the
collecting tubule, the predominant pathway mediating sodium
reabsorption is through highly selective epithelial sodium
channels (ENaC) (11). Unlike voltage- and ligand-gated chan-
nels, epithelial sodium channels (ENaC) are constitutively
active channels, and as such, one major mechanism for regu-
lating channel activity is by controlling expression of the
channels at the apical membrane.
The number of apical channels is determined by the balance
between insertion of new channels into the membrane and the
retrieval and degradation of channels from the membrane. The
lifetime of ENaC in the apical membrane ultimately determines
the rate of sodium reabsorption. Abnormal numbers of apical
ENaC (i.e., abnormal membrane stability or lifetime) are re-
lated to human inherited diseases, such as Liddle’s syndrome
(32, 33, 37). This disease is caused by mutations in the ?- or
?-ENaC subunit, which increase channel stability and thus
result in an overall increase in ENaC-mediated sodium reab-
Highly selective ENaC are composed of structurally con-
served ?-, ?-, and ?-subunits and belong to the ENaC/de-
generin family of proteins. It is clear that members of the
ENaC/degenerin family share several structural features, such
as two transmembrane-spanning domains (TM1 and TM2),
intracellular NH2 and COOH termini, as well as a large
extracellular domain (20). Recently, the crystal structure of
ASIC, an Acid-Sensing Ion Channel member of the ENaC/
degenerin family, has been described by Gouaux and col-
leagues (18) to be trimeric in the desensitized state. Since
ENaC and ASICs belong to the same family of channel
proteins, the crystal structure of ASIC suggests that ENaC may
similarly be composed of three subunits. However, despite the
structural composition of ENaC at the cell membrane, several
studies have suggested that a mature and functional ENaC
channel must undergo several posttranslational modifications,
which include formation of intrasubunit disulfide bonds (7, 35),
transformation of the N-glycan linkage on ENaC subunits from
endoglycosidase H (endo-H)-sensitive to endo-H-insensitive
forms (15, 17), as well as proteolytic cleavage of ?- and
?-subunits. However, not every ENaC subunit expressed in the
apical membrane is fully processed posttranslationally; both
mature and immature forms of the sodium channels have been
found to coexist in the plasma membrane (16). It has also been
reported that the immature channels expressed in the plasma
membrane have very low channel activity (5). Since both
mature and immature channels have been described in the
plasma membrane, perhaps the various different molecular
weights for ENaC subunits that have been reported (using
similar Western blotting techniques) are due to the differences
in subunit maturity and posttranslational processing. Because
there is uncertainty about the precise molecular weight of each
Address for reprint requests and other correspondence: L. Yu, Center for
Cell and Molecular Signaling, Dept. of Physiology, Emory Univ. School of
Medicine, Whitehead Biomedical Research Bldg. 615 Michael St., Atlanta,
GA 30322 (e-mail: Lyu@physio.emory.edu).
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 Renal Physiol 295: F1519–F1527, 2008.
First published September 10, 2008; doi:10.1152/ajprenal.00605.2007.
0363-6127/08 $8.00 Copyright © 2008 the American Physiological Societyhttp://www.ajprenal.orgF1519
subunit (based on standard Western blotting techniques), there
remain issues related to ENaC processing at the membrane:
1) how posttranslational modifications or proteolytic cleavage
can affect apparent subunit weight, 2) the precise stoichiometry
of the subunits necessary to form a functional channel, and
3) the stability of membrane surface ENaC. Another reason it
is difficult to correlate the biochemical data to the functional
stability of ENaC is that several studies suggest that changes in
?-, ?-, and ?-ENaC subunit levels at the cell surface are not
coordinately regulated (28, 40, 41), meaning that the half-life
of each subunit may differ from one another. Therefore, it is
difficult to accurately correlate the quantitative changes in the
expression levels of ENaC (determined using biochemical
approaches) to the actual functional stability of ENaC. How-
ever, determining the functional stability of ENaC is the most
relevant physiological parameter determining distal nephron
sodium reabsorption. In attempts to estimate the physiological
contribution of the channels and to overcome the technical
limitations of biochemical assays, a few groups have measured
the decrease in amiloride-sensitive whole-cell current in oo-
cytes or transepithelial current in cultured epithelial cells (9,
27, 36) under experimental conditions which prevent newly
synthesized channels from being delivered to the plasma mem-
brane. Posttranslational modifications and trafficking of ENaC
in heterologous expression systems sometimes differ from
channels in cells expressing endogenous ENaC (12, 41). Fur-
thermore, amiloride may inhibit transporters and channels
other than ENaC (10, 11), and therefore the half-life of ENaC
observed based solely on the decline of amiloride-sensitive
current may not actually reflect the half-life of individual
ENaC. Additionally, protein biochemical data suggest that the
half-life of apically expressed ?-ENaC is much shorter than
that of apically expressed ?- and ?-ENaC subunits (41). The
noncoordinated regulation of ENaC subunits may produce
functional channels composed of ? alone, or of ?- and ?-chan-
nels, rather than channels composed of all three ?-, ?-, and
?-subunits (4, 8). Although the sodium channels composed of
the ?-subunit only or ?- and ?-subunits are inhibited by
amiloride, their characteristics are different from the highly
Na?-selective ENaC that are believed to be composed of ?-,
?-, and ?-subunits (4). To overcome the technical limitations
described above and to gain a better understanding of ENaC
half-life at the apical membrane, we carefully measured single-
channel (ENaC) activity using the cell-attached patch-clamp
method and applied four different drugs to prevent either new
synthesis of channels or intracellular trafficking to the apical
membrane after synthesis. Single-channel measurements are
particularly useful for this investigation since there can be no
ambiguity about which channel we are measuring, and we can
separate the effect of the drugs on channel density from those
on open probability.
MATERIALS AND METHODS
A6 cell culture preparation. A6 cells are distal nephron cells that
are ideal for studying transepithelial Na transport because they ex-
press highly selective epithelial Na channels.
The 2F3 subclone of A6 cells was a gift from Dr. Dale Benos. They
were originally derived in the laboratory of Dr. Bernard C. Rossier,
University of Lausanne, Switzerland, by functionally selecting clonal
A6 cells for a high transepithelial resistance monolayer and respon-
siveness to aldosterone (39). All of the A6 cells used in this study
were this 2F3 subclone. A6 cells were maintained in plastic tissue
culture flasks as described previously (1). For single-channel patch-
clamp experiments, A6 cells were seeded onto permeable polyester
inserts until they reached confluency. A6 cell culture medium was
replaced 3 times/wk and consisted of 50/50 Dulbecco’s MEM/Ham’s
F-12 base media (GIBCO, Grand Island, NY), 5% fetal bovine
serum (GIBCO), 1.5 ?M aldosterone, 1.0% streptomycin, and
0.6% penicillin (Irvine Scientific, Santa Ana, CA) at pH of 7.4.
Patch-clamp experiments were carried out using A6 cells between
passages 98 and 106.
Single-channel recordings. The cell-attached configuration was
used in all patch-clamp studies. Micropipettes were pulled from
filamented borosilicate glass capillaries (TW-150F, World Precision
Instruments) with a two-stage vertical puller (Narishige, Tokyo,
Japan). The resistances of the pipettes were between 7 and 10 M?
when filled with and immersed in patch solution containing (in mM)
96 NaCl, 3.4 KCl, 0.8 MgCl2, 0.8 CaCl2, and 10 HEPES, with pH
adjusted to 7.4 by NaOH. Single-channel recordings were made
from individual cells for ?8–10 min at pipette holding potentials
of 0 or 20 mV.
Data analysis. Channel currents were recorded at 1 kHz with an
Axopatch 1-D amplifier (Molecular Devices) with a low-pass, 100-
Hz, eight-pole Bessel filter. Channel activity per patch was deter-
mined during an 8- to 10-min recording period. As a measure of
epithelial sodium channel activity (NPo), we used pCLAMP 9 soft-
ware (Molecular Devices). The channel NPo can be calculated from
the single-channel record without any assumptions about the total
number of channels in a patch or the Poof a single channel using the
where T is the total recording time, i is the number of channels open,
and ti is the time during the recording when there were i channels
open. The channel density per patch, N, presented in this paper, is
defined as the maximum number of unitary current transitions during
8–10 min of single-channel recording and was calculated for all
patches including those patches without any observable active chan-
nels. We have previously shown that if channels open independently
of one another, then the open probability of a single channel (Po) can
be calculated by dividing NPoby the number of channels in a patch
(26). The Powas calculated only for patches with active channels. For
patches with no activity, it is unclear whether there are no channels or
there are channels, but with zero open probability.
NPo. N or Povalues reported were averaged before and following
various drug-treatment time points. Data are reported as means ? SE.
Statistical analysis was performed with SigmaPlot and SigmaStat
software (Jandel Scientific). Differences between groups were evalu-
ated with one-way ANOVA with Holms-Sidak posttests, and results
were considered significant if P ? 0.05.
Drug treatments. Cycloheximide (CHX; 20 ?g/ml) and puromycin
(50 ?g/ml), drugs which interfere with normal protein synthesis, or
brefeldin A (BFA; 300 nM) and nocodazole (20 ?M), drugs which
inhibit protein trafficking, were added to both the apical and basolat-
eral sides of A6 cells during patch-clamp recordings. In general, cells
were not exposed to patch-clamp solution for more than a total of 2 h.
Drug treatments that were carried out for ?2 h were done so in the
presence of cell culture medium to maintain channel activity. All
chemicals (except where explicitly mentioned in the paper) were
purchased from Sigma.
Protein35S labeling and analysis. A6 cells were grown on 12-mm
permeable insert supports for 10 days, after which cells were incu-
bated with 0.5 ml cell culture medium containing 20 ?Ci/ml of
35S-labeled methionine and cysteine in the presence of 20 ?g/ml
CHX, or 50 and 75 ?g/ml puromycin for 30 min. After labeling, the
cells were washed three times with ice-cold phosphate-buffered saline
AJP-Renal Physiol • VOL 295 • NOVEMBER 2008 • www.ajprenal.org
7. Firsov D, Robert-Nicoud M, Gruender S, Schild L, Rossier BC.
Mutational analysis of cysteine-rich domains of the epithelium sodium
channel (ENaC)—identification of cysteines essential for channel expres-
sion at the cell surface. J Biol Chem 274: 2743–2749, 1999.
8. Firsov D, Schild L, Gautschi I, Merillat AM, Schneeberger E, Rossier
BC. Cell surface expression of the epithelial Na channel and a mutant
causing Liddle syndrome: a quantitative approach. Proc Natl Acad Sci
USA 93: 15370–15375, 1996.
9. Fisher RS, Grillo FG, SaribanSohraby S. Brefeldin A inhibition of
apical Na?channels in epithelia. Am J Physiol Cell Physiol 270: C138–
10. Frelin C, Barbry P, Vigne P, Chassande O, Cragoe EJ, Lazdunski M.
Amiloride and its analogs as tools to inhibit Na?transport via the Na?
channel, the Na?/H?antiport and the Na?/Ca2?exchanger. Biochimie 70:
11. Garty H, Palmer LG. Epithelial sodium channels: function, structure, and
regulation. Physiol Rev 77: 359–396, 1997.
12. Hanwell D, Ishikawa T, Saleki R, Rotin D. Trafficking and cell surface
stability of the epithelial Na?channel expressed in epithelial Madin-
Darby canine kidney cells. J Biol Chem 277: 9772–9779, 2002.
13. Harris M, Garcia-Caballero A, Stutts MJ, Firsov D, Rossier BC.
Preferential assembly of epithelial sodium channel (ENaC) subunits in
Xenopus oocytes—role of furin-mediated endogenous proteolysis. J Biol
Chem 283: 7455–7463, 2008.
14. Helms MN, Chen XJ, Ramosevac S, Eaton DC, Jain L. Dopamine
regulation of amiloride-sensitive sodium channels in lung cells. Am J
Physiol Lung Cell Mol Physiol 290: L710–L722, 2006.
15. Hughey RP, Bruns JB, Kinlough CL, Harkleroad KL, Tong QS,
Carattino MD, Johnson JP, Stockand JC, Kleyman TR. Epithelial
sodium channels are activated by furin-dependent proteolysis. J Biol Chem
279: 18111–18114, 2004.
16. Hughey RP, Bruns JB, Kinlough CL, Kleyman TR. Distinct pools of
epithelial sodium channels are expressed at the plasma membrane. J Biol
Chem 279: 48491–48494, 2004.
17. Hughey RP, Mueller GM, Bruns JB, Kinlough CL, Poland PA,
Harkleroad KL, Carattino MD, Kleyman TR. Maturation of the epi-
thelial Na? channel involves proteolytic processing of the alpha- and
gamma-subunits. J Biol Chem 278: 37073–37082, 2003.
18. Jasti J, Furukawa H, Gonzales EB, Gouaux E. Structure of acid-sensing
ion channel 1 at 19A resolution and low pH. Nature 449: 316-?, 2007.
19. Kabra R, Knight KK, Zhou R, Snyder PM. Nedd4-2 induces endocy-
tosis and degradation of proteolytically cleaved epithelial Na?channels.
J Biol Chem 283: 6033–6039, 2008.
20. Kellenberger S, Schild L. Epithelial sodium channel/degenerin family of
ion channels: a variety of functions for a shared structure. Physiol Rev 82:
21. Kemendy AE, Kleyman TR, Eaton DC. Aldosterone alters the open
probability of amiloride-blockable sodium-channels in A6 epithelia. Am J
Physiol Cell Physiol 263: C825–C837, 1992.
22. Lippincott-Schwartz J, Donaldson JG, Schweizer A, Berger EG,
Hauri HP, Yuan LC, Klausner RD. Microtubule-dependent retrograde
transport of proteins into the ER in the presence of brefeldin a suggests an
ER recycling pathway. Cell 60: 821, 1990.
23. Lippincottschwartz J, Yuan L, Tipper C, Amherdt M, Orci L, Klaus-
ner RD. Brefeldin-A’s effects on endosomes, lysosomes, and the Tgn
suggest a general mechanism for regulating organelle structure and mem-
brane traffic. Cell 67: 601–616, 1991.
24. Low SH, Wong SH, Tang BL, Tan P, Subramaniam VN, Hong WJ.
Inhibition by brefeldin-A of protein secretion from the apical cell surface
of Madin-Darby canine kidney cells. J Biol Chem 266: 17729–17732,
25. Lu C, Pribanic S, Debonneville A, Jiang C, Rotin D. The PY motif of
ENaC, mutated in Liddle syndrome, regulates channel internalization,
sorting and mobilization from subapical pool. Traffic 8: 1246–1264, 2007.
26. Marunaka Y, Eaton DC. Effects of vasopressin and camp on single
amiloride-blockable Na channels. Am J Physiol Cell Physiol 260: C1071–
27. Mohan S, Bruns JR, Weixel KM, Edinger RS, Bruns JB, Kleyman
TR, Johnson JP, Weisz OA. Differential current decay profiles of
epithelial sodium channel subunit combinations in polarized renal epithe-
lial cells. J Biol Chem 279: 32071–32078, 2004.
28. Planes C, Blot-Chabaud M, Matthay MA, Couette S, Uchida T,
Clerici C. Hypoxia and beta2-agonists regulate cell surface expression of
the epithelial sodium channel in native alveolar epithelial cells. J Biol
Chem 277: 47318–47324, 2002.
29. Planes C, Leyvraz L, Uchida T, Angelova MA, Vuagniaux G,
Hummler E, Matthay M, Clerici C, Rossier B. In vitro and in vivo
regulation of transepithelial lung alveolar sodium transport by serine
proteases. Am J Physiol Lung Cell Mol Physiol 288: L1099–L1109, 2005.
30. Pochynyuk O, Stockand JD, Staruschenko A. Ion channel regulation by
Ras, Rho, and Rab small GTPases. Exp Biol Med 232: 1258–1265, 2007.
31. Robert F, Gao HQ, Donia M, Merrick WC, Hamann MT, Pelletier J.
Chlorolissoclimides: new inhibitors of eukaryotic protein synthesis. RNA
12: 717–724, 2006.
32. Schild L. The ENaC channel as the primary determinant of two human
diseases: Liddle syndrome and pseudohypoaldosteronism. Nephrologie
17: 395–400, 1996.
33. Schild L, Canessa CM, Shimkets RA, Gautschi I, Lifton RP, Rossier
BC. A mutation in the epithelial sodium-channel causing Liddle disease
increases channel activity in the Xenopus laevis oocyte expression system.
Proc Natl Acad Sci USA 92: 5699–5703, 1995.
34. Sheng S, Carattino MD, Bruns JB, Hughey RP, Kleyman TR. Furin
cleavage activates the epithelial Na?channel by relieving Na?self-
inhibition. Am J Physiol Renal Physiol 290: F1488–F1496, 2006.
35. Sheng SH, Maarouf AB, Bruns JB, Hughey RP, Kleyman TR. Func-
tional role of extracellular loop cysteine residues of the epithelial Na?
channel in Na?self-inhibition. J Biol Chem 282: 20180–20190, 2007.
36. Shimkets RA, Lifton RP, Canessa CM. The activity of the epithelial
sodium channel is regulated by clathrin-mediated endocytosis. J Biol
Chem 272: 25537–25541, 1997.
37. Shimkets RA, Warnock DG, Bositis CM, Nelsonwilliams C, Hansson
JH, Schambelan M, Gill JR, Ulick S, Milora RV, Findling JW,
Canessa CM, Rossier BC, Lifton RP. Liddles syndrome—heritable
human hypertension caused by mutations in the beta-subunit of the
epithelial sodium channel. Cell 79: 407–414, 1994.
38. Staruschenko A, Patel P, Tong QS, Medina JL, Stockand JD. Ras
activates the epithelial Na?channel through phosphoinositide 3-OH
kinase signaling. J Biol Chem 279: 37771–37778, 2004.
39. Verrey F, Schaerer E, Zoerkler P, Paccolat MP, Geering K, Kraehen-
buhl JP, Rossier BC. Regulation by aldosterone of Na?, K?-ATPase
messenger RNAs, protein synthesis, and sodium transport in cultured
kidney cells. J Cell Biol 104: 1231–1237, 1987.
40. Weisz OA, Johnson JP. Noncoordinate regulation of ENaC: paradigm
lost? Am J Physiol Renal Physiol 285: F833–F842, 2003.
41. Weisz OA, Wang JM, Edinger RS, Johnson JP. Non-coordinate regu-
lation of endogenous epithelial sodium channel (ENaC) subunit expression
at the apical membrane of A6 cells in response to various transporting
conditions. J Biol Chem 275: 39886–39893, 2000.
42. Yu L, Eaton DC, Helms MN. Effect of divalent heavy metals on
epithelial Na?channels in A6 cells. Am J Physiol Renal Physiol 293:
AJP-Renal Physiol • VOL 295 • NOVEMBER 2008 • www.ajprenal.org