From the Laboratory of Cellular and Molecular Physiology, Departments of Anesthesiology and Pharmacology, Vanderbilt University Medical Center, Nashville, Tennessee 37232
Cells respond to swelling by activating anion and cation
channels that allow the passive loss of inorganic ions
and organic solutes. Net solute efflux accompanied by
osmotically obliged water functions to return cell volume towards its original value, a process termed regulatory volume decrease.
An apparently ubiquitous response to swelling in vertebrate cells is activation of an outwardly rectifying anion current termed ICl.swell. The general characteristics
of this current include an Eisenman type I anion permeability sequence (I The channel responsible for ICl.swell has been referred
to by a variety of names, including VRAC (volume regulated anion channel) and VSOR (volume expansion-sensing outwardly rectifying anion channel). Another
name for the channel is VSOAC (volume-sensitive organic osmolyte/anion channel), which was coined to
reflect the putative role it plays in the transport of organic
anions and electroneutral organic osmolytes (Strange
et al., 1996 The route to the molecular identification of the
ICl.swell channel has been an elusive and exceptionally
confusing one. The first purported molecular sighting
of the channel was made in 1992. Valverde et al. (1992) More recently, it has been suggested that P-glycoprotein functions to modulate or regulate ICl.swell (reviewed
by Wine and Luckie, 1996 A second purported sighting of the ICl.swell channel
was also made in 1992. Paulmichl et al. (1992) Paulmichl et al. (1992) Compelling evidence was presented to support the
hypothesis that pICln is a channel-forming protein. A
possible nucleotide binding site was identified in the
second Shortly after the publication of these studies, Krapivinsky et al. (1994) The controversy over whether pICln forms a transmembrane transport pathway, or whether it is a transport regulator, is similar to the controversies regarding
the functions of other transport proteins (see Tzounopoulos et al., 1995 Voets et al. (1996) Table I
INTRODUCTION
Top
Introduction
References
> Br
> Cl
> F
), modest outward rectification, voltage-dependent inactivation at
potentials above ECl, inhibition by a wide variety of compounds, including conventional anion transport inhibitors, and block by extracellular nucleotides such as
ATP (reviewed by Strange et al., 1996
; Okada, 1997
).
The degree of rectification, voltage sensitivity, and pharmacology can vary somewhat between different cell
types. It is not clear whether the differences observed reflect the existence of distinct channels, or whether they
are due to experimental and/or physiological variables.
For example, channel voltage sensitivity is altered by intracellular Mg2+ concentration (Okada, 1997
), the transmembrane Cl
gradient, and by the presence of foreign
anions in the extracellular solution (Meyer and Korbmacher, 1996
; Okada, 1997
; my unpublished observations). In this article, I will assume for the sake of simplicity that ICl.swell arises from the activity of a single channel
type or a family of closely related channels.
; Kirk and Strange, 1998
). To avoid confusion, I will refer to the channel in this paper simply as
the ICl.swell channel.
and Gill et al. (1992)
proposed that P-glycoprotein, the
product of the multidrug resistance-1 gene, functions
as both a drug transporter and the ICl.swell channel. However, numerous laboratories have been unable to
reproduce the findings of these investigators and additional experimental observations have failed to support
the original hypothesis (reviewed by Wine and Luckie,
1996
; Okada, 1997
). Because of this, the postulated role for P-glycoprotein as a volume-activated anion
channel is no longer considered to be a tenable idea by
most workers in the field.
; Okada, 1997
). As discussed
by Wine and Luckie (1996)
, the channel regulator concept may be stretched too far in the case of P-glycoprotein. It is not clear whether the apparent modulation of
ICl.swell by P-glycoprotein reflects a physiologically relevant function, or whether it is simply a consequence of
overexpression of the protein induced by transfection
or drug selection.
reported
the expression cloning of a cDNA encoding a protein
termed pICln. When expressed in Xenopus oocytes,
pICln induced an outwardly rectifying anion conductance that was blocked by extracellular nucleotides and inactivated by depolarizing voltages. Although the
pICln-associated current did not require swelling for
activation, its characteristics resembled, at least superficially, those of ICl.swell.
proposed initially that pICln
is an anion channel-forming protein with a novel structure. Hydrophobicity analysis indicated that pICln lacks
transmembrane helices believed to form the pore of
most vertebrate ion channels. It was instead proposed
that the pICln "channel" was a homodimer, and that
each monomer contained four
strands that contribute to the formation of an eight-stranded, antiparallel
barrel transmembrane pore. The putative
barrel
pore structure is reminiscent of that of bacterial and
mitochondrial porins (Jap and Walian, 1996
).
strand of each pICln monomer. This site has
poor homology with known nucleotide binding motifs
(see Saraste et al., 1990
), but its location within the putative channel pore was consistent with the inhibition
by extracellular nucleotides of the pICln-associated current (Paulmichl et al., 1992
). Mutation of the binding
site resulted in the apparent expression of an anion
current that was no longer inhibited by nucleotides. The "mutant" current also had altered voltage sensitivity and, unlike the current induced by wild-type pICln,
was inhibited by removal of extracellular Ca2+ (Paulmichl et al., 1992
).
proposed that pICln was not a channel, but was instead an anion channel regulator. Four
pieces of indirect evidence were presented to support
this idea. First, biochemical and immunofluorescence studies demonstrated that the protein was localized primarily in the cytoplasm rather than the plasma membrane as expected for an ion channel. Second, oocyte
swelling was shown to activate an endogenous ICl.swell
(see also Ackerman et al., 1994
) that superficially resembled the current induced by heterologous expression of pICln. This observation led the authors to conclude incorrectly (discussed below) that the current induced by expression of pICln was the same as that
induced by oocyte swelling. Third, oocytes express a
pICln homolog, and fourth, microinjection of an anti- pICln monoclonal antibody into the oocyte cytoplasm
inhibited swelling-induced activation of the endogenous ICl.swell slowly over a period of 8-20 h. These latter
two observations suggested that pICln was critical to the
function of the ICl.swell channel (Krapivinsky et al.,
1994
). Since pICln did not appear to be present in the
plasma membrane, the authors concluded that the protein was a regulator of the ICl.swell channel, and that heterologous overexpression of the protein activated the
channel in the absence of cell swelling. While intriguing, this hypothesis was troubling to many workers in
the field because it did not take into account the results
of mutagenesis studies described by the authors in their
first report on pICln (Paulmichl et al., 1992
). Thus, we proposed an alternate hypothesis in which pICln is a
channel activated by reversible insertion into the plasma
membrane (Strange et al., 1996
).
; Shimbo et al., 1995
). To investigators interested in cellular osmoregulation, the cloning of any protein, be it a volume-sensitive channel or
transporter, or accessory/regulatory molecule involved
in volume homeostasis, is interesting and important because it provides a new tool to explore the ever-vexing
and still unsolved puzzle of how cells sense volume
changes and transduce those signals into regulatory responses. However, recent work by Buyse et al. (1997)
,
as well as my laboratory, have suggested that, like P-glycoprotein, the postulated pICln-ICl.swell channel connection may be leading the field in the wrong direction.
recently compared the characteristics of the current induced by expression of human
pICln in Xenopus oocytes to the endogenous ICl.swell.
While the currents superficially resemble one another,
there are very clear differences when one looks closely
(Table I). The pICln-induced current is much more strongly rectified than ICl.swell, has a different anion permeability sequence, and is not activated by cell swelling.
Cyclamate permeates the ICl.swell channel, but blocks the
channel responsible for the pICln-induced current.
cAMP blocks both channels, but the block of the ICl.swell
channel is voltage independent, while the block of the
pICln-induced channel only occurs with depolarization above the reversal potential. Finally, both currents exhibit depolarization-induced inactivation, but inactivation of the ICl.swell channel is increased by elevation of
extracellular pH.
Comparison of ICl.swell and pICln-induced Current (ICln) in
Xenopus oocytes (from Voets et al., 1996)
Taken together, these observations cast some doubt
on the hypothesis that pICln is an anion channel regulator (Krapivinsky et al., 1994). If expression of pICln is
simply turning on an endogenous ICl.swell channel, why
should the characteristics of the current induced by
swelling and pICln cRNA injection differ? One might
argue that species differences, different activation modes
(i.e., swelling versus heterologous protein expression), or other factors account for this disparity. However,
such arguments seem moot in light of the demonstration by Buyse et al. (1997)
that (a) expression of an unrelated protein, ClC-6, induces the same current as that
induced by expression of pICln, and (b) the pICln-associated current is observed in ~5-6% of uninjected control oocytes (see also Paulmichl et al., 1992
). The simplest conclusion from these findings is that expression
of certain heterologous proteins "activates" an endogenous, outwardly rectifying anion current that is distinct
from the endogenous ICl.swell. This is not an unprecedented finding. Previous studies have shown that endogenous oocyte currents are activated by expression
of a variety of heterologous proteins (Tzounopoulos et
al., 1995
; Shimbo et al., 1995
).
If pICln and apparently unrelated proteins are simply activating an endogenous oocyte conductance, how
can the pICln mutagenesis experiments described by
Paulmichl et al. (1992) be explained? Recent evidence
indicates that the current ascribed to expression of the
pICln nucleotide binding site mutant is also due to the activity of an endogenous oocyte anion channel, possibly the well-described Ca2+-activated Cl
channel (e.g.,
Wu and Hamill, 1992
). Buyse et al. (1997)
have reported that uninjected oocytes express a current with
characteristics identical to those of the current associated with expression of the mutant pICln. In addition,
oocyte expression of the so-called "AAA" nucleotide
binding site mutant (Paulmichl et al., 1992
), which has
all three glycine residues comprising the putative nucleotide binding site mutated to alanine residues, induced a conductance with characteristics identical to
that induced by wild-type pICln (Voets et al., 1998
).
These characteristics include block by extracellular nucleotides, insensitivity to extracellular Ca2+, and inactivation at positive membrane potentials.
These observations indicate that the currents ascribed to the expression of pICln very likely arise from endogenous oocyte anion channels that are distinct from the ICl.swell channel. How can these results be reconciled with observations suggesting that there is a direct link between pICln and ICl.swell? In my opinion, there is no obvious way to do so. Instead, a critical re-evaluation of the data indicates that none of it provides a compelling basis for thinking that pICln plays a role in volume homeostasis.
Krapivinsky et al. (1994) proposed the regulator hypothesis in part because most of the pICln appeared to
be in the cytoplasm rather than localized to the plasma
membrane. Other investigators have confirmed this
finding. Buyse et al. (1997)
, using membrane fractionation techniques and confocal immunofluorescence
microscopy, demonstrated a largely cytoplasmic location
in control and swollen endothelial cells. We have made
similar observations in C6 glioma cells. In addition, we
were unable to detect membrane localization of pICln
in either control or swollen C6 cells that had been
transfected with carboxy- or amino-terminus green fluorescent protein (GFP)-pICln fusion constructs (Emma
et al., 1998
).
In contrast to these findings, studies carried out in
other laboratories have reported an apparent membrane localization of pICln. Schwartz et al. (1997) concluded that pICln was "concentrated in the membrane"
of red blood cells. However, the low resolution, confocal immunofluorescence images presented in their paper do not allow an accurate assessment of whether the
protein is truly localized to the membrane or whether it
is associated with submembranous cytoskeleton, as
their biochemical studies suggest (discussed below).
Fractionation studies carried out on MDCK cells (Laich et al., 1996
), embryonic skate heart cells (Musch et al.,
1997
), and neonatal rat myocytes (Goldstein et al.,
1997
) have revealed that swelling induces an increase
of pICln content in the particulate or membrane fraction. However, such findings must be interpreted cautiously. They certainly do not demonstrate that pICln
inserts into the plasma membrane in response to swelling. The apparent swelling-induced association with
the particulate fraction may represent an association of
pICln with membrane-attached cytoskeleton rather than
with the lipid bilayer. Immunoprecipitation (Krapivinsky et al., 1994
; Sanchez-Olea et al., 1997
), in vitro
protein binding assays (Krapivinsky et al., 1994
; Sanchez-Olea et al., 1997
), and yeast two-hybrid studies
(Schwartz et al., 1997
) have indicated that pICln interacts with cytoskeletal components including actin and
the nonmuscle isoform of the alkali myosin light chain.
pICln can be readily extracted from red cell ghost
membranes by treatment with low ionic strength buffers, which also extract the spectrin-actin cytoskeleton
(Schwartz et al., 1997
).
If the apparent swelling-induced association of pICln
with the membrane fraction represents an interaction
with the cytoskeleton, does this imply that the protein
is playing some regulatory role in volume homeostasis?
Not necessarily. Numerous studies have shown that cell
swelling alters the structure of the cytoskeleton, particularly F-actin (reviewed by Okada, 1997). Thus, changes in the interaction of pICln with the cytoskeleton may
reflect events that are not directly related to cell volume homeostasis.
The ability to reconstitute, from purified pICln, anion channel activity with the characteristics of the
ICl.swell channel would provide important support for
the pICln channel hypothesis. We have now carried out
extensive planar lipid bilayer reconstitution studies of
recombinant pICln and have been unable to detect anion channel activity with this protein (Li et al., 1998).
However, we have consistently observed a highly cation-selective channel in our bilayer experiments. Reconstitution of pICln in liposomes increases 86Rb flux but has
no effect on 36Cl transport, suggesting strongly that the
cation channel activity we see is due to pICln rather
than contaminant proteins. It must be stressed here
that the channel activity we observe in bilayers does not
imply that pICln functions in vivo as a cation channel. Many proteins that are clearly not ion channels give
rise to channel-like activity when reconstituted into artificial membranes.
As noted earlier, injection of monoclonal anti-pICln
antibodies into Xenopus oocytes inhibited activation of
the endogenous ICl.swell (Krapivinsky et al., 1994). This
inhibition occurred slowly over a period of 8-20 h.
While this effect is intriguing, it does not provide definitive or even compelling proof of a pICln-ICl.swell channel connection. Whatever pICln is, it is probably important to cell function. The protein is ubiquitous, abundant, and has a structure that is highly conserved
among evolutionarily divergent species (e.g., Krapivinsky et al., 1994
). The effects of long-term disruption of
pICln function on cell physiology are unknown. Activation of ICl.swell can be dramatically reduced by a variety
of seemingly unrelated parameters such as decreases in
cellular ATP levels (Strange et al., 1996
; Okada, 1997
),
increases in cytoplasmic ionic strength (Emma et al.,
1997
) and Mg2+ concentration (Okada, 1997
), dissociation of cells from their growth substrate (Han et al.,
1996
), disruption of the cytoskeleton (Levitan et al.,
1995
; Zhang et al., 1997
), and unidentified "environmental factors" (Jackson et al., 1996
). The ability of defolliculated oocytes to respond to swelling by activation
of ICl.swell declines slowly over a period of days when the
cells are maintained in relatively simple salt solutions
(Ackerman et al., 1994
; Hand et al., 1997
). Many of the
factors that inhibit ICl.swell do not prevent activation per
se, but instead simply shift the volume set-point of the
channel such that greater degrees of swelling are required to activate it (Emma et al., 1997
; Basavappa and Strange, 1998
). Taken together, these observations suggest that the inhibitory effect of anti-pICln on ICl.swell
could be a very indirect one. For example, one might
postulate that pICln is a protein involved in maintaining and regulating cytoskeletal function, and that experimental perturbation of pICln alters cytoskeletal architecture. Since disruption of the cytoskeleton alters
the volume sensitivity of ICl.swell (Levitan et al., 1995
;
Zhang et al., 1997
), concluding that pICln is a "regulator" of the channel is no more appropriate than concluding that drugs such as phalloidin and cytochalasin
are channel regulators. The studies of Gschwentner et
al. (1995)
, which demonstrated an ~40% inhibition of
ICl.swell in pICln antisense-transfected fibroblasts, are
subject to the same criticisms.
Hubert et al. (1998) have shown recently that transfection of pICln into tsA201a cells increases the rate of
ICl.swell activation 16-fold. Conversely, transfection with
pICln antisense decreases the rate of current activation.
These are interesting findings, but ones that must
again be viewed cautiously. Do these results indicate
that pICln is regulating ICl.swell, or do changes in its
expression affect swelling-induced current activation
through indirect mechanisms such as alterations in cytoskeletal structure?
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CONCLUSIONS AND PERSPECTIVE |
---|
There is some evidence to suggest a link between pICln
and the ICl.swell channel, but this evidence is neither definitive nor compelling. More importantly, it is difficult
to reconcile findings that argue for a pICln-ICl.swell
channel link with studies suggesting that the results of
initial investigations on pICln (Paulmichl et al., 1992)
were artifacts related to the use of the oocyte expression system (Buyse et al., 1997
; Voets et al., 1997). The
central question regarding pICln has shifted from a debate over whether the protein is a swelling-activated anion channel or channel regulator, to a debate about
whether it has anything at all to do with ICl.swell. pICln
may have functions related to cell volume homeostasis,
ion channels, and membrane transport processes, but
these may be quite indirect.
Identification of the protein(s) responsible for ICl.swell
will continue to be a very difficult and challenging
problem. There are two reasons for this. First, expression cloning and characterization of heterologously expressed ion channels requires a cell system with minimal or no background expression of the channel of interest. ICl.swell is expressed ubiquitously in mammalian
cells and is also present in Xenopus oocytes (Ackerman
et al., 1994; Hand et al., 1997
). Cells types such as insect Sf9 cells may lack ICl.swell, but they possess other
swelling-activated anion currents (C.E. Bear, personal
communication) that will confound expression studies. Second, the native ICl.swell channel has only recently
come under detailed study. Our understanding of its
biophysical characteristics is therefore far from complete. Importantly, despite intense efforts, we know almost nothing about how ICl.swell is activated by swelling. Without such detailed knowledge, the chances for false
starts and confusion are increased when molecular candidates for the channel itself or channel regulators are
put forth. Clearly, much additional rigorous cellular
and biophysical characterization of ICl.swell is needed
and warranted.
Currently, there are, in my opinion, only two molecular candidates for volume regulated anion channels
that are backed up by persuasive experimental evidence, ClC-2 and ClC-3. ClC-2 was shown to be swelling-activated by expression in Xenopus oocytes (Grunder et
al., 1992; Jordt and Jentsch, 1997
). cRNA injections were performed on defolliculated oocytes that were
maintained in simple salt solutions, conditions that dramatically suppress the endogenous ICl.swell (see Ackerman et al., 1994
; Hand et al., 1997
). The characteristics
of ClC-2 current are substantially different from ICl.swell,
indicating that it is unlikely to be the ICl.swell channel.
Furthermore, the role of ClC-2 in volume homeostasis
is uncertain since similar swelling-activated currents are
not readily observable in mammalian cells (reviewed by
Strange et al., 1996
; see Carew and Thorn, 1996
, for an
exception to this generalization). Nevertheless, studies
of volume-dependent gating of ClC-2 may provide important clues about ICl.swell regulation and the more
general problem of how cells sense volume changes.
ClC-3 cloned from guinea pig heart gives rise to an
outwardly rectifying anion current that is activated by
cell swelling when it is expressed in NIH/3T3 cells
(Duan et al., 1997). Experiments on ClC-3 were performed using a slow rate of cell swelling and a patch pipette solution with high ionic strength. Both of these maneuvers will reduce the rate of ICl.swell activation
(Emma et al., 1997
; Basavappa and Strange, 1998
). The
ClC-3 current has characteristics remarkably similar to
those of ICl.swell with one major exception
ClC-3 is inhibited by activation of protein kinase C (Duan et al.,
1997
; Kawasaki et al., 1994
). In rabbit atrial myocytes,
ICl.swell is also inhibited by PKC activation (Duan et al.,
1995
). This is very different from ICl.swell in other cells
where PKC has no inhibitory effect (e.g., Szücs et al.,
1996
; reviewed by Okada, 1997
), and may actually stimulate channel activity (Jackson and Strange, 1993
). The
findings of Duan et al. (1997)
need to be reproduced
and confirmed with additional studies. If they can be, it
will be interesting and important to determine if a
non-PKC-regulated ClC-3 isoform, a ClC-3 heteromultimer (Lorenz et al., 1996
), or another member of the
ClC family accounts for ICl.swell in other cells types.
Given the confusion that exists over the molecular nature of ICl.swell, it will be important to subject this hypothesis to cautious and rigorous scrutiny.
![]() |
FOOTNOTES |
---|
Received for publication 1 December 1997 and accepted in revised form 16 March 1998.
I thank Drs. Christine Bear, Jan Eggermont, Al George, Kevin Foskett, and Bernd Nilius for critically reviewing this manuscript.This work was supported by National Institutes of Health grants NS-30591 and DK-51610.
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