(Received for publication, October 17, 1995; and in revised form, February 6, 1996)
From the
A new outwardly rectifying Cl channels (ORCC)
that belongs to ClC Cl
channel family has been
identified from rat kidney and designated as ClC-5. ClC-5 cDNA encodes
a polypeptide of 746 amino acids, which is indicated by hydrophobicity
analysis to have structural features that are common of the ClC family.
However, the amino acid sequence was weakly homologous to those of
other ClC Cl
channels except for ClC-3, which we
recently identified as a Ca
-sensitive ORCC. Northern
blot analysis of rat tissues showed that ClC-5 mRNA was predominantly
expressed in the kidney and colon. To characterize the functional
properties of ClC-5 by whole cell patch-clamp technique, we established
the stably transfected CHO-K1 cell line using intranuclear
microinjection technique. The transfected cells induced outwardly
rectifying and 4,4`-diisothiocyanostilbene-2,2`-disulfonic
acid-sensitive Cl
currents on whole cell
configuration. Following the identification of two highly homologous
ORCCs, ClC-3 and ClC-5, a new subfamily encoding ORCC has emerged in
the ClC family. Furthermore, ClC-5 was almost identical to a partial
sequence of human cDNA that is related to Dent's disease. The
molecular structure and functional properties of ClC-5 will provide an
important insight into ORCCs and the pathogenesis of Dent's
disease.
Many physiological studies have demonstrated the presence of
outwardly rectifying Cl channels (ORCCs) (
)in a variety of
cells(1, 2, 3, 4) . Single channel
recordings by patch-clamp technique have shown that they have strong
outward rectification in a positive membrane voltage and that their
conductance is about 40
picosiemens(1, 2, 3, 4) . Recently,
ORCC has attracted special interest in relation to cystic fibrosis
transmembrane conductance regulator
(CFTR)(5, 6, 7, 8, 9) .
Patch-clamp studies in the epithelial cells of patients with cystic
fibrosis have shown that cyclic AMP-dependent ORCC is not properly
regulated in these
patients(5, 6, 7, 8, 9) ,
indicating the possible importance of ORCC in the pathogenesis of
cystic fibrosis. When the CFTR gene was cloned in
1989(10, 11) , it soon became clear that CFTR encodes
a small linear Cl
channel of
10 picosiemens and
not ORCC(12) . This discrepancy has been puzzling
investigators, but recent new findings that CFTR acts as an
ATP-permeating channel may solve this problem. Schwiebert et al.(13) have shown that CFTR is able to permeate ATP in
addition to Cl
and that ATP transported from inside
to outside of the cell in turn activates the purinergic receptors on
the cell surface. Activation of the purinergic receptor then stimulates
ORCC. On the basis of these findings, subsequent attention has been
focused on the molecular structure of ORCC.
Recently, we have cloned
and characterized a Ca-sensitive ORCC, ClC-3, an
intriguing member of the ClC family(14, 15) . Only
20-24% of the amino acid sequence encoded by ClC-3 is identical
to those of other cloned ClC Cl
channels, i.e. ClC-0, -1, -2, -K1, and
-K2(16, 17, 18, 19, 20) .
These findings led us to the hypothesis that ClC-3 may belong to a new
subfamily encoding ORCCs in the ClC family. In the present study, we
examined whether a new member of ORCC exists in the ClC family using a
homology-based cloning strategy. Here, we report a new cDNA clone
encoding an ORCC, ClC-5, isolated from rat kidney. ClC-5 is highly
homologous to ClC-3 and ClC-4 and almost identical to a partial human
cDNA, which is recently reported as a strong candidate for Dent's
disease(21) . We further established the stably transfected
mammalian cells and characterized its channel properties by the
patch-clamp technique.
In
the study of anion selectivity, the relative anion permeabilities were
determined on the basis of the shift of reversal potential after the
half-replacement of Cl with other anions using
inside-out patch technique (n = 5). The pipette
solution contained 120 mM NaCl. The bath solution for fluorine
was 60 mM NaCl and 60 mM NaF; for iodine, 60 mM NaCl and 60 mM NaI. In the study where the effect of cAMP
on whole cell Cl
currents was examined, voltage was
applied by step pulse from -30 (100 ms) to 0 (2 s) to +30 mV
(100 ms) using a computer software (CLAMPEX, Axon Instruments).
Chemicals were obtained from Sigma unless otherwise noted. All experiments were performed at room temperature (20-25 °C). Data are expressed as means ± S.E. of n observations (where n is the number of individual cells used).
Figure 1: Amino acid sequence alignment of ClC-5 with ClC-3 and ClC-4. A, nucleotide and deduced amino acid sequence of ClC-5. B, conserved residues are filled in black blocks, putative transmembrane-spanning domains are underlined according to the original topology by Jentsch et al.(16) , potential N-linked glycosylation sites (28) are indicated by asterisks, and PCR primer sites are double-underlined. Sequence data have been deposited in GenBank(TM)/EMBL data library (accession number D50497). C, hydrophobicity profile of ClC-5. The mean hydrophobicity index was computed according to the algorithm of Kyte and Doolittle with a window of 15 residues(27) .
The protein sequence of ClC-5 is highly homologous to that
of rat ClC-3 (77%), to those of rat and human ClC-4 (78%) (32) (Fig. 1B), and to that of a partial human
cDNA (99%)(21) . In contrast, the overall amino acid identity
of ClC-5 with other members of the ClC family is very low (29% amino
acid sequence identity with Torpedo channel, ClC-0; 31% with ClC-1, 33%
with ClC-2, 27% with ClC-K1). To establish possible evolutionary
relationship among these ClC Cl channels, a
phylogenic tree was constructed using DNAsis computer software (Mac
version 3.2, Hitachi, Yokohama, Japan) (Fig. 2). According to
the phylogenic analysis, the eight members of the ClC Cl
channel family can be classified into two subfamilies: the
ClC-0/ClC-1/ClC-2/ClC-K1/ClC-K2 chloride channels and the
ClC-3/ClC-4/ClC-5 chloride channels.
Figure 2:
Evolutionary relationship between the ClC
Cl channels. The phylogenic tree shows the
relationship between different eight members of the ClC family derived
from rat except for ClC-0. The phylogenic tree was constructed using
the computer software DNAsis.
Figure 3:
Northern blot analysis of ClC-5 expression
in different rat tissues. Total RNA (20 µg/lane) (A) and
poly(A) RNA (approximately 2 µg/lane) (B)
from various rat tissues were loaded in each lane and subsequently
hybridized with the full-length ClC-5 cDNA probe. Equal loading of RNA
was confirmed by staining of 28 S ribosomal RNA by ethidium bromide (A) or hybridization with
-actin probe (B).
Markers of transcript size (in kilobases) are
indicated.
Figure 4:
Northern blot analysis of transfected
cells. Control indicates that total RNA was isolated from the
mock-transfected cells; DEX (+), pretreatment with 5 µM dexamethasone for 24 h; DEX(-), without the pretreatment.
Each lane contained 20 µg of total RNA, and equal loading was
confirmed by hybridization with -actin (bottom).
To
examine whether ClC-5 actually acts as a Cl channel,
the whole cell patch-clamp technique was applied to J2702 cells. Fig. 5shows a typical trace of the whole cell currents following
the changes in holding membrane potential between -75 and
+25 mV (A) and their I-V relationship (E). The transfected cells generated large, time-dependent and
outwardly rectifying currents (1, 562 ± 128 pA at + 25 mV
membrane potential, n = 12), in contrast to the
mock-transfected CHO-K1 cells (70 ± 8 pA at +25 mV, n = 10; Fig. 5, A and B). The I-V curve based on the currents at the end point of voltage
pulses revealed an outwardly rectifying current-voltage relationship (Fig. 5E). The extracellular partial Cl
replacement with gluconate reduced the overall current and
resulted in a shift of the reversal potential toward the positive
direction, thus indicating that the current was Cl
selective (Fig. 5E). Also the current was
inhibited by extracellular addition of Cl
channel
blockers; 1 mM 4,4`-diisothiocyanostilbene-2,2`-disulfonic
acid (DIDS) (65 ± 3%, at +25 mV, 5 min after the addition
of DIDS, n = 4) or 0.5 mM diphenylamine
carboxylic acid (DPC) (86 ± 4%, at +25 mV, 5 min after the
addition of DPC, n = 5) (Fig. 5, C and D). To determine anion selectivity of ClC-5, we preliminary
applied the inside-out patch technique to the wild-type and the ClC-5
transfected CHO-K1 cells. In the wild-type cells, patched membranes
occasionally contained Cl
channels (34%, 12 of 35),
and these channels did not show any rectification as shown
previously(15) . In the ClC-5 transfected cells the patches
always contained Cl
channels as a cluster (100%, 40
of 40), and they showed outward rectification. When the transfected
cells were treated with DEX, the number of Cl
channels contained in the patch increased (
10 per patch),
indicating that these Cl
channels were mediated by
ClC-5. Using these cluster Cl
channels, we determined
anion selectivity of ClC-5. When we replaced a half of Cl
with other anions in the bath solution, the reversal potential
for I
move to negative direction (-5 ± 2
mV, n = 5), and for F
it moved to
positive direction (10 ± 4 mV, n = 5). The
sequence of relative anion permeability was I
>
Cl
> F
.
Figure 5:
Electrophysiological characterization of
ClC-5 expressed in CHO-K1 cells. A and B, the
representative typical traces of whole cell currents from the cells
transfected with pMAM-ClC-5-neo (A) or pMAMneo alone (B). C, a representative typical trace of whole cell
currents 5 min after extracellular addition of 1 mM DIDS. The
holding potential was changed from -75 to +25 mV. Inset, voltage-clamp program. D, the percent
inhibition of whole cell currents with 1 mM DIDS and 0.5
mM DPC in the transfected cell. Currents were measured 5 min
after extracellular addition of the each blocker (n =
5, mean ± S.E., at +25 mV). E, current-voltage
relationships of conductance expressed from ClC-5 cDNA, in TEA-Cl
solution (130 mM Cl) (closed circle), in low
chloride (10 mM Cl
, 120 mM gluconate) (open triangle).
Effect of cAMP on
ClC-5 channel was examined in J2702 cells using the whole cell
patch-clamp technique. Fig. 6shows the effect of cAMP
(CTP-cAMP, 2 mM) on the whole cell Cl currents. The currents were measured in response to voltage pulse
of ±30 mV. When the patched cells were exposed to cAMP, the
magnitude of outward currents did not change as shown in Fig. 6(n = 4).
Figure 6: Representative trace of whole cell currents in ClC-5 expressing cells in response to cAMP treatment. Labeled bar indicates application of a membrane permeable cAMP (CTP-cAMP, 2 mM). The holding potential was clamped at ±30 mV.
The molecular structures of ORCCs have not yet been identified because of the lack of their molecular cloning. In this study, we successfully isolated ClC-5, a new ORCC which is highly homologous to ClC-3, by a sequence homology-based strategy. We further established the stably transfected mammalian cultured cell line expressing ClC-5 using the intranuclear microinjection technique.
The predicted amino acid sequence of ClC-5 is highly homologous with
not only ClC-3 (77%), but also with ClCN4 (78%), and its rat
counterpart ClC-4 (78%), which has recently been isolated from Xp22.3
region using positional cloning strategy (28, 32) (Fig. 1B). Phylogenic
analysis indicated the presence of two subfamily in the ClC family (Fig. 2). A partial deletion of ClCN4 is known to cause delays
in psychomotor functions and mental retardation. Furthermore, the amino
acid sequence of ClC-5 is almost identical (99%) to a human sequence
predicted from a partial cDNA (780 bp) recently isolated as a candidate
molecule of Dent's disease (an X-linked hereditary renal tubular
disorder) from Xp11.22 region using positional cloning strategy (21) . ()It is tempting to speculate that this
subfamily of ClC Cl
channel is related to human
disease. Further clarification of physiological function of these
channels is needed to elucidate the pathogenesis of these disease.
In the motif analysis, ClC-5 has two potential N-glycosylation sites. One of the N-glycosylation sites (Asn-408) is located in the segment between D8 and D9, and this glycosylation motif is well conserved among all ClC channels known so far. Kieferel et al.(34) have shown recently that ClC-0, ClC-1, ClC-2, and ClC-K1 channels are glycosylated at this segment in in vitro translation experiments. Therefore, this glycosylation site between D8 and D9 should be located outside of the cells. In addition, there are two consensus sequences for phosphorylation by cAMP-dependent protein kinase in ClC-5 cDNA (see ``Results''). This finding suggest that ClC-5 protein itself might be modulated by cAMP-dependent protein kinase-mediated phosphorylation. However, any change in the whole cell current was not induced in response to cAMP in the present study (Fig. 6). This result revealed that cAMP-mediated signaling does not directly modulate the channel properties. However, it cannot be neglected that cAMP-mediated signaling indirectly modulates the function of ClC-5. A lack of channel regulator(s) in the transfected cells may disrupt the pathway of cAMP-mediated signaling.
Now, eight members of the ClC
family have been cloned. Some of these Cl channels
have been functionally characterized by transient expression system
using Xenopus oocytes. However, the functional expression in Xenopus oocyte may not be ideal for characterization of ClC-3,
-4, and -5 channels. For unknown reasons, the expression of these
channels has been difficult in the oocytes except for ClC-3 (for
review, see (28) and (35) ). Also a
Ca
-dependent Cl
channel is present
in the oocytes (36, 37) and sometimes disturbs the
detection of the exogenously expressed Cl
currents.
We thought that mammalian cells were more suitable for proper
functional expression of ClC-5, which was obtained from the rat.
Accordingly, we stably transfected ClC-5 cDNA into a mammalian cultured
cell line, CHO cells, using the intranuclear microinjection technique.
This transfection system has the following advantages over others: 1)
the stable transformed cell line can be obtained more frequently and
more easily because the transfection efficiency of intranuclear
microinjection is higher than those of other indirect methods (
0.2 versus
0.001)(38, 39) , 2) the
dexamethasone-inducible expression vector (pMAM-neo) is useful to
induce the overexpression of the transformed full-length gene, 3) a
patch-clamp study has demonstrated that only a small linear
Cl
channel is endogenous in CHO cells(40) .
In the stably transfected mammalian cells with ClC-5, the whole cell
currents showed a typical outward rectification that was
time-dependently activated following depolarization of membrane voltage (Fig. 5E). The profile and amplitude of the currents
(1 nA at +25 mV, Fig. 5A) were different from
those of the endogenous small linear Cl
channel in
the wild-type CHO cells(40) . Several lines of evidence suggest
that these currents were induced by a Cl
-selective
channel. First, Cl
was the predominant
current-carrying ion under the conditions of patch-clamp experiments.
Second, the replacement of Cl
with gluconate in the
bath solution reduced the currents and caused a positive shift in the
reversal potential (Fig. 5E). Third, the currents were
inhibited by Cl
channel blockers, DPC and DIDS. They
act as open channel blockers probably by decreasing the open
probability of ORCC from outside of the cells(2) , so that
their inhibition of ClC-5 is also consistent with that of ORCC. In
addition, based on the shift of zero potential after the
half-replacement of Cl
with other anions, we
determined the anion permeability sequence as I
>
Cl
> F
(see
``Results''). This sequence is compatible with
Eisenman's series 1(41) , indicating that ClC-5 channel
may have the same anion selectivity as that of previously demonstrated
ORCCs(42) . These electrophysiological characteristics of ClC-5
are different from those of other ClC Cl
channels,
ClC-0, ClC-1, and ClC-2. They do not show outward rectification and
have relatively less I
permeability(28) . In
contrast, ClC-3 has outward rectification and a rather high
I
permeability, which are consistent with
ClC-5(15) , indicating that these two channels belong to same
category in terms of channel characteristics. However, Steinmeyer et al. most recently isolated the identical Cl
channel cDNA from rat brain and showed that it was not sensitive
to DIDS and DPC and also has a anion conductivity sequence of
Cl
> Br
> I
using Xenopus oocyte expression system. (
)Although reason for functional discrepancies between
theirs and the present study is not clear, the expression system (Xenopus oocyte versus mammalian cultured cells,
CHO-K1) and experimental conditions for anion selectivity study (whole
cell versus inside-out patch configuration) are different. It
is possible that the expressed Cl
channel is
modulated by endogenous factor(s) in whole cell system. Accordingly,
further studies are needed to characterize the electrophysiological
properties of ClC-5.
The tissue distribution of ClC-5 mRNA is different from that of this human partial clone. In Northern blot analysis of rat tissues, ClC-5 is abundantly expressed in the kidney and colon and in lesser amounts in the heart, brain, lung, and testis. Although the expression in the colon has not been examined, the human partial clone was expressed only in the kidney. This may represent some discrepancy in the tissue distribution among species, and a similar discrepancy was observed for ClC-4(28, 32) .
ClC-5
is almost identical to a human partial cDNA, which was recently
isolated as a strong candidate for Dent's disease(21) .
This disease is a renal tubular disorder which is associated with low
molecular weight proteinuria, hypercalciuria, nephrocalcinosis,
nephrolithiasis, and eventual renal
failure(43, 44, 45) . Although the primary
mechanism responsible for this disease has not been clarified, the
feature of low molecular weight proteinuria indicates the dysfunction
of proximal tubule. The previous physiological studies have shown that
the acidification of endosomal compartment is a limiting factor for the
uptake of filtered proteins via active endocytotic pathway in the
proximal tubule, in which the Cl conductance in
endosomal membrane is responsible for the acidification of endosomal
compartment(46, 47, 48, 49) . The
Cl
channel expressed in endosomes has been isolated
from rat kidney cortex and has been shown to be voltage-dependent and
DPC- and DIDS-sensitive(50) . The further studies on ClC-5 may
help to evaluate the hypothesis that the defect of the Cl
channel is responsible for Dent's disease.
In summary, a
new molecule of ORCC was isolated from rat kidney and functionally
characterized. Following the elucidation of two structurally and
functionally homologous ORCCs (ClC-3 and ClC-5), the existence of a
subfamily encoding ORCCs in ClC Cl channel family has
emerged. Although ClC-4 has not yet been functionally characterized as
a Cl
channel, it could belong to the same subfamily
as ClC-3 and ClC-5. The analysis of the structure and function of these
ORCCs proteins will provide us with the framework to evaluate their
detailed regulatory mechanisms at a molecular level.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D50497[GenBank].