From the Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan
Anion channels are activated by volume expansion in
most animal cell types and are known to be implicated
not only in regulatory volume decrease but also in many
other cell activities that are associated with changes in
cell volume and shape, including cell proliferation and
cell death (Okada, 1998 Table I). Among a variety of swelling-activated Cl
channels (ICl.swell), the most important and
frequently observed channel is a volume-sensitive, outwardly rectifying, ATP-dependent Cl
channel (Okada,
1997
; termed VSOR-ClC here). The functional properties of VSOR-ClC have so far been intensively and systematically studied in rat C6 glioma cells (Strange et al.,
1996
), human epithelial Intestine 407 cells (Okada,
1997
), and human and bovine endothelial cells (Nilius
et al., 1997
), and proved to share the same properties. The principal phenotypic properties are attributed to
the pore and regulatory characteristics, as summarized
in Table I. The regulatory properties of the channel
may be modified by association with putative regulatory
subunits. Thus, the "fingerprint" characteristics of the
channel, which distinguish it from other Cl
channels,
should be related, at least, to the pore properties, such
as its intermediate single-channel conductance, outward
rectification, low-field anion selectivity, a high open
probability (>0.95), voltage-dependent inactivation gating at large positive potentials, and open-channel block
by extracellular ATP.
Comparison of Properties of Human, Bovine, and Rat Volume-sensitive Outwardly Rectifying Cl Channels, Guinea Pig ClC-3-associated
Cl
Channels and Rat ClC-2-associated Cl
Channels
Although much work has been performed to identify
the VSOR-ClC protein, the molecular identity of the
channel has not as yet been determined. Purification of
the channel protein has been hindered by the lack of a
highly specific channel ligand (antagonist or agonist).
Expression cloning of the VSOR-ClC protein has been hampered by the endogenous expression and house-keeping activity of the channel in almost all cell types
(Nilius et al., 1994; Okada, 1997
), including Xenopus
oocytes (Ackerman et al., 1994
), and by possible
changes in volume sensitivity of the channel and/or the osmosensitivity of the cell produced by overexpression of exogenous proteins (Okada, 1997
).
From gene expression studies, three candidates for the
VSOR-ClC protein have been proposed: the MDR1 gene
product, P-glycoprotein (PGP; Valverde et al., 1992; Gill
et al., 1992
), a ubiquitous (principally cytoplasmic)
protein, pICln (Paulmichl et al., 1992
), and a member
of the cloned ClC family, ClC-3 (Duan et al., 1997
). However, the PGP hypothesis is no longer viable, as reviewed by Okada (1997)
. The most crucial evidence
against the PGP hypothesis was the observation that abolition of the endogenous PGP expression by antisense
oligonucleotides failed to affect the VSOR-ClC current in Intestine 407 cells (Tominaga et al., 1995
). The misleading conclusion in the original studies (Valverde et
al., 1992
; Gill et al., 1992
) may have been caused by exaggerated experimental conditions, because overexpression of PGP was found to augment volume sensitivity of
endogenous VSOR-ClC currents (Miwa et al., 1997
).
Now, the pICln hypothesis has also been discarded, as
summarized recently in two letters for Perspectives in
General Physiology (Strange, 1998
; Clapham, 1998
). Several lines of crucial evidence against the pICln hypothesis were provided by Voets et al. (1996
, 1998
), Buyse et
al. (1997)
, and Emma et al. (1998)
. It should be stressed
that overexpression of pICln in Xenopus oocytes resulted in activation of Cl
currents with pore properties (rectification, anion selectivity, and blocking) distinct from
the phenotypic properties of VSOR-ClC (Voets et al.,
1996
).
The ClC-3 hypothesis is at present viable, though not
completely tested. As summarized in Table I, the pore
properties, except the open probability, of guinea pig
cardiac ClC-3-associated Cl currents (Duan et al., 1997
)
are largely similar to those of VSOR-ClC. Interestingly,
the N579K mutant of guinea pig ClC-3 was found to
bring about changes in anion selectivity from I
> Cl
to Cl
> I
and in rectification from outward to inward
(Duan et al., 1997
). Enormous basal activity under isotonic conditions and sensitivity to an activator of protein kinase C of the ClC-3-associated Cl
currents are
distinct from the properties of human, bovine, and rat
VSOR-ClC currents. Therefore, there is a possibility
that VSOR-ClC has a pore formed by ClC-3 and regulatory subunit(s) that may contain the volume sensor and
PKC phosphorylation sites, and these regulatory subunits may differ between animal species. Alternatively,
it is also possible that VSOR-ClC is formed by the ClC-3
isoform alone, the cytosolic regulatory domains of
which may be different between animal species. For example, the NH2-terminal 58 amino acid residues of the
human and mouse ClC-3 isoforms are missing from the
rat and guinea-pig isoforms. However, a definite conclusion concerning the contribution of ClC-3 to VSOR-ClC awaits further studies by knocking out endogenous
ClC-3 expression and by heterologous expression of
the human, bovine, and rat isoforms of ClC-3. In CHO
cells stably expressing the rat kidney-derived ClC-3 isoform, quite distinct properties (such as maxi unitary conductance) of Cl
currents were observed (Kawasaki
et al., 1994
). However, it should be noted that the inside-out patch-clamp study was performed in the absence of intracellular ATP, which is indispensable for
normal activities of VSOR-ClC (Okada, 1997
). Also,
there remains the possibility that VSOR-ClC is formed
by a heteroligomer of ClC-3 and other ClC family members or solely by the latter protein(s).
Another cloned Cl channel, ClC-2 (Thiemann et al.,
1992
), has also been listed as a candidate of VSOR-ClC
because of its volume sensitivity and ubiquitous expression (Grunder et al., 1992
). However, as summarized in
Table I, the following pore properties of ClC-2 are
quite distinct from VSOR-ClC: the small unitary conductance (3-5 pS; Jentsch et al., 1995
), inward rectification, inactivation gating at negative potentials, and the
anion selectivity sequence of Cl
Br
> I
(Thiemann et al., 1992
). Thus, although the possibility remains that ClC-2 may contribute to VSOR-ClC function, it is unlikely that VSOR-ClC is formed solely by
ClC-2.
Taken together, it appears that we are still far from
the identification of VSOR-ClC protein. To avoid unnecessary repetition of premature conclusions, as was
the case with the PGP and pICln hypotheses, we propose the following criteria for the molecular identification of VSOR-ClC: (a) transfection with the gene for the candidate protein induces swelling-activated anionic currents with characteristics identical to those of
phenotypic properties of VSOR-ClC, (b) the cells functionally exhibiting the VSOR-ClC current express endogenously the candidate mRNA and protein, (c) abolition of expression of the candidate protein abolishes
the endogenous VSOR-ClC current, and (d) the mutation of the candidate gene gives rise to significant
changes in the pore properties of the Cl current. Before obtaining evidence fulfilling all the above criteria, the molecular identity of VSOR-ClC cannot be established. This is what we have learned from previous work
that evolved around the PGP and pICln hypotheses.
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FOOTNOTES |
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Address correspondence to Yasunobu Okada, Department of Cellular and Molecular Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan. Fax: 81-564-55-7735; E-mail: okada{at}nips.ac.jp
We thank A.F. James for reviewing the manuscript.
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