From the Institut de Pharmacologie et de Toxicologie, Université de Lausanne, CH-1005 Lausanne, Switzerland
Amiloride-sensitive epithelial sodium channels constitute the rate-limiting step for sodium reabsorption in
the distal part of the renal tubule, in the distal colon, in
the ducts of several exocrine glands, and in the airways
(Rossier et al., 1994 ENaC is the major sodium-conducting pathway in the
distal nephron, participating in the fine control of
sodium balance, blood volume, and blood pressure.
ENaC also has a unique role in controlling lung fluid
clearance, especially at birth (Hummler et al., 1996 Awayda et al. (1995) Should we conclude from these studies that the epithelial sodium channel is mechanosensitive? Probably not.
In the present issue of The Journal of General Physiology, one
of the authors of the previous papers has now used the
Xenopus oocyte expression system to examine in a living
cell the effects of changes of membrane tension on In the oocyte model, the advantage is that one can
express well defined and purified molecules (i.e., mRNA
coding for the gene of interest) and measure their
function in the membrane of a living cell. Within one
experiment, hundreds of oocytes from the same female
can be injected, insuring a well-controlled statistical analysis of the data. The main disadvantages of the system are twofold. First, there is a large biological variability from one set of oocytes coming from an animal
to another, independent of the traditional factors invoked, such as season, water quality, or other ill-defined
and somewhat mythic components that make the life of
the oocyte fan sometimes so miserable. This intrinsic variability requires one to perform a large number of
independent experiments. The second pitfall is that
the oocyte can lack or express endogenous components that may be important physiologically to regulate
the activity of the channel under study.
The reconstitution of channel activity in planar lipid
bilayers also presents distinct experimental advantages.
It is a very well controlled experimental system, especially when the channel protein is biochemically pure
and its reconstitution into proteoliposomes at less than
one molecule per vesicle is achieved. This was, for instance, accomplished by Bear et al. (1992) Assuming that these parameters can be experimentally controlled, two possible observations can be made.
Either reconstituted channels in the planar bilayer recapitulate most, if not all, the biophysical properties of
the native channel measured in a cell expressing the
ionic transport of interest, or they do not. In the
present case, the properties of ENaC in apical membrane of native epithelial cells have been established by
Palmer and Frindt (1986) Some other basic properties, such as the consistent
appearance of multiple conductance states and sensitivity
to stretch activation are observed in planar lipid bilayers,
suggesting a triple barrel structure of the channel (Ismailov et al., 1996 At the present time, considering the respective limitations of each of the two experimental systems, I am inclined to believe that expression systems that reproduce
the properties of the native channels are more likely to
be more physiologically relevant. In my view, the more
"reductionist" approach of reconstituting ENaC in lipid
bilayers should be well suited to address questions about
detailed ion channel behavior. I am convinced that the
planar lipid bilayer system will be useful in the future
once the experimental conditions for reconstituting the
physiological activity of ENaC are fully defined. In this
respect, it will be of special interest to look whether the
properties of a biochemically purified ENaC channel protein can reproduce the biophysical properties of ENaC observed in the apical membrane of renal cells. The answer to this question should come sooner or later.
ARTICLE
Top
Article
References
). Until recently, the classification
of the epithelial sodium channels was exclusively based
on biophysical and pharmacological properties: sodium and potassium selectivity, single channel conductance, kinetics of gating and amiloride sensitivity. As
recently pointed out by Garty and Palmer (1997)
, the
molecular cloning of the three homologous channel subunits denoted
,
, and
epithelial sodium channels
(ENaC) has provided a molecular definition of at least one class of amiloride-blockable channels.
). The ENaC genes share striking homologies with a set of
genes that has been identified in Caenorhabditis elegans,
using a genetic screening for loss of mechanosensation
(Tavernarakis and Driscoll, 1997
). The high degree of
homology in the predicted transmembrane domains
and in the structure of the ectodomain of the ENaC
family suggested early on that ENaC may itself be involved in mechanosensitivity in mammalian cells.
have followed up on this idea.
They have reported that they were able to in vitro translate an
ENaC subunit from bovine kidney in a rabbit
reticulocyte lysate and reconstitute the polypeptide
product into a liposome (Awayda et al., 1995
). Incorporation into planar lipid bilayers leads to sodium channel activity with a single-channel conductance of 40 pS.
The channel was inhibited by a low concentration of
amiloride (Ki 150 nM) and was moderately sodium selective. Mechanosensitivity of the channel was demonstrated
by channel activation in response to small hydrostatic
pressure differences across the bilayer. Surprisingly, selectivity and amiloride affinity were drastically altered
by stretch activation. In a subsequent report (Ismailov et al., 1996
), the study was extended to the reconstitution of
,
,
ENaC subunits, either from in vitro translation in the presence of dog pancreas microsomes or
from a crude microsomal membrane fraction of Xenopus oocytes in which the subunits were heterologously
expressed. On incorporation into planar lipid bilayers, sodium channel activity was observed that was (a) sodium selective (PNa/PK = 10), (b) amiloride sensitive
(Ki 170 nM), (c) comprised of three different open-state conductance levels (13, 26, and 40 pS), and (d)
mechanosensitive. A hydrostatic pressure difference as
low as 0.26 mmHg induced a fourfold increase in open
probability (Po).
,
,
and
ENaC. In this setting, it is clear that ENaC is not
mechanosensitive at either the whole-cell or single-channel levels (Awayda and Subramanyam, 1998
). Are these
results really contradictory? or should they trigger one
of these long lasting controversies that we see (and
sometimes enjoy) in science? I do not believe so. However, they do serve to emphasize the advantages and
limitations of the experimental systems that were used.
for the cystic
fibrosis transmembrane conductance regulator (CFTR).
Upon incorporation, purified CFTR exhibited the basic biophysical and regulatory properties of the type of
chloride channel found in native cells and believed to
underlie cAMP-evoked secretion in epithelial cells (Bear
et al., 1992
). Reconstitution of channel activity in planar lipid bilayer suffers from different technical difficulties, however, when the channel protein studied is
not biochemically pure and fully characterized as it was
for CFTR. When using nonpurified membrane proteins,
as in the papers cited above (Awayda et al., 1995
; Ismailov et al., 1996
), one has to take great precautions to insure that the protein translated in vitro is normally
folded, properly oligomerized, and assembled in a
physiologically active form. The technical problem is
difficult enough for a homomeric channel (Awayda et
al., 1995
). The problem becomes even more severe when one wants to fold, oligomerize, and assemble an
heterotetrameric protein (Firsov et al., 1998
) such as
ENaC, which is made of three homologous subunits (
,
, and
). Experimental evidence was not provided
that this was achieved (Ismailov et al., 1996
). When crude microsomal membranes from oocytes expressing
ENaC subunits are used as the source of protein for reconstitution into planar bilayers, one also has to realize
that these membranes will contain monomeric, dimeric,
and heteromultimeric channel proteins in ill defined
proportions with different degrees of oligomerization and maturation. Nor should one overlook the problem
that microsomal membranes (dog pancreas microsomes
as well as oocyte microsomes) are likely to express various endogenous channel activities that will have to be
characterized separately in control experiments.
and Hamilton (1985). Thus
far, these basic biophysical properties have not been
fully reproduced in the planar lipid bilayer. Conversely,
no consistent and significant effects of pressure (10-60
mmHg applied to the patch pipette) could be documented in apical membranes of cortical collecting duct
cells (Palmer and Frindt, 1996
).
). These observations have never been
made in native cells or in oocytes expressing ENaC.
![]() |
REFERENCES |
---|
![]() ![]() ![]() |
---|
1. | Awayda, M.S., I.I. Ismailov, B.K. Berdiev, and D.J. Benos. 1995. A cloned renal epithelial Na+ channel protein displays stretch activation in planar lipid bilayers. Am. J. Physiol. 37: C1450-C1459 . |
2. |
Awayda, S.M., and
M. Subramanyam.
1998.
Regulation of the
epithelial Na channel by membrane tension.
J. Gen. Physiol.
112:
97-111
|
3. | Bear, C.E., C.H. Li, N. Kartner, R.J. Bridges, T.J. Jensen, M. Ramjeesingh, and J.R. Riordan. 1992. Purification and functional reconstitution of the cystic fibrosis transmembrane conductance regulator (CFTR). Cell. 68: 809-818 [Medline]. |
4. |
Firsov, D.,
I. Gautschi,
A.-M. Merillat,
B.C. Rossier, and
L. Schild.
1998.
The heterotetrameric architecture of the epithelial sodium
channel.
EMBO (Eur. Mol. Biol. Organ.) J.
17:
344-352
|
5. |
Garty, H., and
L. Palmer.
1997.
Epithelial sodium channels: function, structure, and regulation.
Physiol. Rev.
77:
359-396
|
6. | Hamilton, K.L., and D.C. Eaton. 1985. Single-channel recordings from amiloride-sensitive epithelial sodium channel. Am. J. Physiol. 249: C200-C207 [Abstract]. |
7. |
Hummler, E.,
P. Barker,
J. Gatzy,
F. Beermann,
C. Verdumo,
R. Boucher, and
B.C. Rossier.
1996.
Early death due to defective
neonatal lung liquid clearance in ![]() |
8. |
Ismailov, I.I.,
M.S. Awayda,
B.K. Berdiev,
J.K. Bubien,
J.E. Lucas,
C.M. Fuller, and
D.J. Benos.
1996.
Triple-barrel organization of
ENaC, a cloned epithelial Na+ channel.
J. Biol. Chem.
271:
807-816
|
9. | Palmer, L.G., and G. Frindt. 1986. Amiloride-sensitive Na channels from the apical membrane of the rat cortical collecting tubule. Proc. Natl. Acad. Sci. USA. 83: 2767-2770 [Abstract]. |
10. | Palmer, L.G., and G. Frindt. 1996. Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch. J. Gen. Physiol. 107: 35-45 [Abstract]. |
11. | Rossier, B.C., C.M. Canessa, L. Schild, and J.-D. Horisberger. 1994. Epithelial sodium channels. Curr. Opin. Nephrol. Hypertens. 3: 487-496 [Medline]. |
12. | Tavernarakis, N., and M. Driscoll. 1997. Molecular modeling of mechanotransduction in the nematode Caenorhabditis elegans. Annu. Rev. Physiol 59: 659-689 [Medline]. |