From the Institute for Molecular and Cellular
Regulation, Gunma University and the § Second Department of
Surgery and the ¶ Department of Emergency and Critical Care
Medicine, Gunma University School of Medicine, Maebashi 371-8512, Japan
Received for publication, January 16, 2001, and in revised form, March 1, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
MID-1 is a Saccharomyces
cerevisiae gene encoding a stretch-activated channel. Using
MID-1 as a molecular probe, we isolated rat cDNA
encoding a protein with four putative transmembrane domains. This gene
encoded a protein of 541 amino acids. We also cloned the human
homologue, which encoded 551 amino acids. Messenger RNA for this gene
was expressed abundantly in the testis and moderately in the spleen,
liver, kidney, heart, brain, and lung. In the testis, immunoreactivity
of the gene product was detected both in the cytoplasm and the nucleus.
When expressed in Chinese hamster ovary cells, the gene product
was located in intracellular compartments including endoplasmic
reticulum and the Golgi apparatus. When microsome fraction obtained
from the transfected cells, but not from mock-transfected cells, was
incorporated into the lipid bilayer, an anion channel activity was
detected. Unitary conductance was 70 picosiemens in symmetric
150 mM KCl solution. We designated this gene Mid-1-related
chloride channel (MCLC). MCLC encodes a new
class of chloride channel expressed in intracellular compartments.
Various types of chloride channels modulate diverse cellular
functions. Chloride channels expressed on the plasma membrane regulate
various cellular functions including control of cell volume,
transepithelial ion transport, modulation of membrane potential,
neurotransmission, and bone resorption (1). Chloride channels are also
expressed in intracellular membranes and may regulate acidification of
intracellular compartments and vesicle trafficking (2). Mutations in
the chloride channel genes cause various human diseases such as
cystic fibrosis (3), kidney stone disease (4), and congenital myotonias
(5).
Recently, the molecular nature of chloride channels was revealed by
gene cloning, and the functions of chloride channels have been
extensively studied at the molecular level. Four major types of
chloride channels have been identified to date: the cystic fibrosis
transmembrane conductance regulator (6), the ligand-gated receptor
channels (7), the ClC family (8), and the
CLIC1 family (9). Among them,
members of the CLIC family and some of the ClC family are expressed in
intracellular organelles, including endoplasmic reticulum, the nuclear
envelope, and endosomes, and regulate ion fluxes across the
intracellular membranes.
The MID-1 gene of yeast Saccharomyces cerevisiae
(10) was originally identified as a gene mutation that induces cell
death during the treatment of yeast with the mating pheromone.
Loss-of-function mutation in the MID-1 gene results in the
reduction of calcium entry into the yeast and thereby induces cell
death (10). Functional analysis of the gene product of MID-1
(Mid-1) revealed that the Mid-1 was a stretch-activated
calcium-permeable cation channel (11); Mid-1 functions as a
non-selective calcium-permeable channel, the opening of which is
regulated by stretch of the plasma membrane. Mid-1 is the first
eukaryotic stretch-activated channel whose primary structure was
identified by molecular cloning (11). Because stretch-activated
channels regulate diverse cellular functions in mammalian cells
(12), we attempted to identify mammalian stretch-activated channels
using Mid-1 as a molecular probe.
During the course of the present study, we identified a new class of
ion channel molecule expressed in intracellular compartments. This
protein functions as a chloride channel when incorporated in the planar
lipid bilayer and, therefore, we designated it the Mid-1-related
chloride channel (MCLC).
Cloning of Rat MCLC cDNA--
A BLAST search using the
partial sequence of Mid-1 led to the identification of a
Xenopus unknown transmembrane protein (GenBankTM
accession number X92871). To obtain mammalian homologues, the total
sequence of X92871 was used to search again, and this led to the
identification of an expressed sequence tag (EST accession number
H51262). A rat brain oligo(dT)-primed Cloning of Human MCLC cDNA--
A BLAST search using the
total sequence of rat MCLC led to the identification of
expressed sequence tags of the human homologue of MCLC (EST
accession numbers AA375206, AA906589, and AB018304). The cDNA
template was synthesized from MCF7 and HepG2 cells using a
gene-specific primer (5'-ATTCAACAACAGTTGCATGTCGCCTT), and a 5'
end-specific forward primer (5'-CCTTATACAGGATGCTGTGTTCTTTG) and a 3'
end-specific reverse primer (5'-GCTGGTGTTCCTCTAGCCACA) were utilized
for the amplification of cDNA. The PCR products were cloned and
sequenced as described above.
DNA Recombination Procedures and Transfection--
The
rat cDNA clone was subcloned into the BamHI and
XhoI sites of the vector pcDNA3 (Invitrogen). To
introduce the FLAG epitope tag into the carboxyl terminus of
MCLC, we amplified a fragment of MCLC
(nucleotides 1128-1623) by PCR using the sense primer (5'-AGATGACAGAAGACGACAGAAGGAACTTG) and the antisense primer
(5'-TTATTACTTGTCGTCATCGTCTTTGTAGTCGCCACACGGGCTGCTGACAAG); the PCR
products were then exchanged for native MCLC cDNA. The PCR was carried out using high fidelity enzyme Ultima (Roche Molecular Biochemicals), and the PCR-generated constructs were verified by
sequencing the amplified region. The MCLC expression vectors were purified using a concert high purity plasmid maxiprep system (Life
Technologies, Inc.) according to the manufacturer's instructions. Cells were transfected using LipofectAMINE Plus reagent (Life Technologies, Inc.). To establish stable cell lines, the cells were
replaced at a lower concentration in fresh medium containing 10% fetal
calf serum at 24 h after transfection. 48 h after
transfection, the transfected cells were selected using 1 mg/ml
neomycin analog G418. After 10-14 days, independent colonies were
picked up, grown in 35-mm dishes, and screened for a high level of
expression of MCLC and monoclonality by immunostaining.
Northern Blot Analysis--
RNA from various tissues was
isolated using a TRIZOL Reagent (Life Technologies, Inc.). 40 µg of
total RNA was electrophoresed on a 1.0% agarose gel containing 2.2 M formaldehyde, 40 mM MOPS (pH 7.0) and
transferred to a nylon membrane (Hybond-N+, Amersham
Pharmacia Biotech) using a capillary blotting technique with 20× SSC
(sodium citrate buffer). Northern blots were performed as described
elsewhere (15).
Cell Culture--
Chinese hamster ovary (CHO) cells provided by
the Riken cell bank (Tsukuba, Japan) were cultured in Ham's F12 medium
supplemented with 2 mM glutamine containing 10% fetal calf
serum (Life Technologies, Inc.) under humidified conditions of
95% air and 5% CO2 at 37 °C. A10, MCF-7, and HepG2
cells were cultured in Dulbecco's modified Eagle's medium containing
10% fetal calf serum.
Preparation of Microsomes and Immunoblotting--
For the
preparation of microsomes, transfected CHO cells and various rat
tissues were homogenized in lysis buffer (250 mM sucrose,
10 mM Tris/HCl (pH 7.4), 1 mM EDTA, 10 µg/ml
leupeptin, 40 komberg international units/ml aprotinin, 10 µg/ml pepstatin A, 0.2 mM phenylmethylsulfonyl fluoride),
and the lysate was cleared by centrifugation at 1000 × g for 2 min. The supernatant was taken and centrifuged for
20 min at 8000 × g. The supernatant was then centrifuged at 100,000 × g for 1 h to obtain microsomes.
A polyclonal antibody against rat MCLC was raised by immunizing rabbits
with peptide (C)RALEPDDRRRQKEL conjugated with keyhole limpect
hemocyanine. The MCLC peptide was synthesized and purified to 90-95%
homogeneity with high pressure liquid chromatography. The MCLC
antiserum was purified with a SulfoLink coupling gel column (Pierce)
coupled with the same peptide, according to the manufacturer's
instruction. Immunoblotting was performed as described elsewhere
(15).
Immunohistochemistry--
For immunostaining, cells were grown
on coverslips, fixed for 5 min in 3% paraform/phosphate-buffered
saline, washed two times with phosphate-buffered saline and two times
with 50 mM glycine/phosphate-buffered saline, then blocked
with Blocking Ace (Snow Brand, Tokyo, Japan), and incubated with
anti-MCLC anti-rabbit IgG antibody or anti-FLAG M2 antibody
at room temperature for 1 h. For colocalization experiments, MCLC-expressing cells were transfected with PEYFP-ER vector
(CLONTECH), and anti-green fluorescent protein
(GFP) antibody was used. Anti-calnexin (Santa Cruz Biotechnology) or
anti-Golgi 58K antibody (Sigma) was used in other experiments. After
washing with phosphate-buffered saline, the coverslips were incubated
with secondary antibodies obtained from Jackson ImmunoResearch (West
Grove, PA) conjugated with indocarbocyanine (Cy3), fluorescein
isothiocyanate, or tetramethyl rhodamine isothiocyanate (15).
Various rat tissues were excised. 4-µm formalin-fixed,
paraffin-embedded tissue sections were mounted on
poly-L-lysine-coated slides. The sections were
deparaffinized in xylene and rehydrated through a graded alcohol
series. Endogenous peroxidase activity was blocked by incubating the
sections in 0.3% hydrogen peroxidase in methanol for 30 min and
boiling in 10 mM citrate buffer for 5 min. Immunostaining
was performed using VECTASTAIN ABC kits (Vector Laboratories,
Burlingame, CA).
Reconstitution of Ion Channels in the Planar Lipid
Bilayer--
The planar lipid bilayer was formed by the method of
Montal and Mueller (13). A small droplet of the phospholipid solution was taken up into a 10-µl glass capillary pipette. After the inside of the capillary had been coated with the lipid, a bilayer was formed
by applying an air bubble from the capillary pipette to the aperture
(diameter, 150-240 µm) of a Teflon septum mounted vertically between
the two halves of a Teflon chamber. The aperture was pretreated with a
solution of 1% n-hexadecane in n-hexane before
forming the membrane. The solution in each compartment was connected to
an Ag-AgCl electrode via a saturated KCl-agar salt bridge for current
measurements under voltage clamp conditions. A 20-µl phospholipid
solution (a 7:3 (w:w) mixture of phosphatidylethanolamine and
phosphatidylcholine, 10 mg/ml in n-hexane) was placed on the surface of 0.5 ml of a 10 mM MOPS buffer (pH 7.0)
containing appropriate salts in each compartment. A few minutes later,
the solvent had evaporated, and a lipid monolayer was formed at the
air/water interface. Phospholipids were obtained from Avanti Polar
Lipids (Alabaster, AL).
The lipid bilayer separated the cis solution from the
trans solution (1.5 ml each). The cis and
trans solutions contained initially 150 and 5 mM
KCl, respectively, both in 10 mM MOPS-Tris (pH 7.0). Ion
channels were incorporated into the bilayer by flushing 9 µl of the
microsome suspension (containing 0.5 to ~1 µg of protein) directly
toward the bilayer from the cis side.
The solution on the cis and trans sides of the
bilayer were connected to a patch clamp amplifier (EPC-7, List,
Darmstadt, Germany) via 0.5 M KCl-agar bridges in series
with Ag-AgCl electrodes. To shield from electromagnetic and mechanical
interference, the bilayer chamber and the head stage of the patch clamp
amplifier were placed in a Faraday cage mounted on an antivibration
table. The electrical current across the bilayer was through an 8-pole low pass Bessel filter and was visualized on a storage oscilloscope.
The unfiltered single channel current was stored via a pulse code
modulator on a commercial videocassette recorder. Analysis of channel
activity was performed with program pCLAMP (Axon Instruments, Foster City, CA) on a personal computer. Measurements recorded on the
videocassette recorder were filtered at 500 Hz and digitized at a
frequency of 2,000 Hz, using an analog-to-digital converter. At each
holding voltage, channel activity for 30 s was analyzed.
Cloning of Rat MCLC--
A BLAST search using the partial sequence
of Mid-1 led to the identification of a Xenopus unknown
transmembrane protein (GenBankTM accession number X92871).
To obtain a mammalian homologue, the total sequence of X92871 was used
to search again, and this led to the identification of an expressed
sequence tag (EST accession number H51262). A rat brain cDNA
library was screened using the cDNA probe derived from H51262. The
full-length rat MCLC cDNA was obtained and sequenced
(Fig. 1A). The rat
MCLC cDNA contained an open reading frame of 1623 base
pairs and coded for a protein of 541 amino acids with a calculated
molecular mass of 61 kDa. There was no overall sequence similarity
between MID-1 and MCLC. MCLC was
identical to AK2-8, identified as a differentially expressed gene in the hypothalamus (14). A hydrophobicity profile of
the sequence of MCLC displayed four potential transmembrane segments (M1-M4) (Fig. 1B). Computer-based analysis
indicated that MCLC contained a consensus sequence for
nuclear localization signals (Fig. 1A). There was no
sequence similarity between MCLC and known channel families.
There was, however, similarity in the sequence of the third
transmembrane domain (Fig. 1C). The amino acid sequence of
this domain resembled those of members of the CLIC family.
Cloning of Human MCLC--
A BLAST search was performed on EST
data bases with the rat MCLC sequence. Three human sequence
tags were identified corresponding to the partial sequence of 5' and 3'
regions. A cDNA template was synthesized from MCF7 and HepG2 cells
using gene-specific primers, and PCR was performed to clone the
complete coding sequence of human MCLC (Fig. 1A).
Fig. 1D depicts the phylogenetic tree of rat, human, and
Xenopus MCLC.
Tissue Distribution of Rat MCLC mRNA--
Northern blot
analyses were carried out to examine the tissue distribution of
MCLC in the rat. Two hybridization band sizes were found in
the rat tissues, including the spleen, liver, testis, kidney, heart,
aorta, brain, and lung (Fig. 2). A strong
expression was found in the testis.
Expression of MCLC in CHO Cells--
To clarify subcellular
localization of MCLC protein, expression vector rat
MCLC-pcDNA3, a FLAG tagged-MCLC pcDNA3,
was constructed. Localization of MCLC protein was studied by indirect
immunofluorescence with anti-MCLC antibody or anti-FLAG antibody in CHO
cells stably expressing MCLC. A strong fluorescence staining was
detected at the perinuclear region as well as in a fine reticular
network extending through the cytoplasm (Fig.
3A). This pattern was also observed in all of the MCLC-expressing cells from multiple transient transfections (data not shown). This pattern was similar to that seen
with known endoplasmic reticulum (ER) markers such as calnexin (16)
(Fig. 3B). Localization of MCLC in ER was confirmed by colocalization of MCLC with ER-targeted enhanced yellow fluorescent protein (Fig. 3C). MCLC is also localized in the Golgi
apparatus. The colonization of a Golgi marker, anti-Golgi 58K (17) with MCLC was observed in the transfected CHO cells (Fig. 3D).
MCLC was not localized in mitochondria, as assessed by using
MitoTracker (data not shown). We also studied the localization of
endogenous MCLC. Endogenous MCLC was detected in PC12 cells and A10
smooth muscle cells. The pattern of immunoreactive MCLC was similar to that of transfected cells mentioned above (data not shown). Western blot analysis was carried out to verify the expression of MCLC protein
in transfected cells. Under nonreducing conditions, anti-FLAG antibodies detected a major band with a molecular mass of 120 kDa from
CHO cells transfected with a tagged MCLC (Fig.
4A). Under reducing
conditions, a molecular mass of 60 kDa was detected. A similar blot
profile was obtained by using affinity-purified anti-MCLC antibody
(Fig. 4B). No signal was obtained from control CHO cells.
The mass of 60 kDa was in good agreement with the predicted molecular
mass of MCLC.
Expression of Endogenous MCLC--
Western blot analyses were then
carried out to clarify endogenous MCLC protein expression in various
rat tissues. The strong expression of MCLC with a molecular mass of 60 kDa was detected in the testis, liver, and lung (Fig.
5). In the testis, a strong immunoreactivity was observed in primary spermatocytes (Fig.
6A). In these cells, staining
of the cytoplasm and the nucleus was observed, and distribution of MCLC
was different depending upon the stage of the spermatocyte. In some
cells, immunoreactivity was found predominantly in the cytoplasm (Fig.
6B), but in other cells MCLC was localized in the nucleus
(Fig. 6C). Note that immunoreactivity disappeared when the
antibody was preincubated with the antigen peptide. In hepatocytes,
cytoplasmic but not nuclear stainings were observed (data not
shown).
Channel Activity of MCLC--
The above findings indicated that
MCLC was predominantly expressed in the intracellular compartments such
as the endoplasmic reticulum and the Golgi apparatus. To clarify
whether or not MCLC had a channel activity, we incorporated microsomal
vesicles obtained from MCLC-transfected and mock-transfected CHO cells
into the planar lipid bilayer and measured the channel activities. We
confirmed the presence of MCLC protein in the microsomal fraction from
MCLC-transfected cells but not from mock-transfected cells
by Western blotting (data not shown). Channel activities were
consistently detected in microsomal vesicles obtained from
MCLC-overexpressing CHO cells but not in those from mock-transfected
cells. As shown in Fig. 7A, a
bursting current was observed. The burst current showed open and closed
states. Single channel conductance was determined by linear regression
analysis of the current-voltage relationships in symmetrical 100 mM KCl solutions (Fig. 7B). The slope
conductance of the unitary current was 70.2 ± 5.0 picosiemens
(mean ± S.D., n = 5). The channel was more
permeable to anions than to cations. Based on the current-voltage
relationship in asymmetrical KCl solutions
(cis/trans, 500/100 mM), the reversal
potential was
We then examined the effect of chloride channel blockers.
4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS), known to be a specific inhibitor of various anion channels (18), did not block
the channel activity (data not shown). ATP, which blocks the anion
channel in platelets (19), did not affect the channel activity. Note
that Gd3+, a blocker of stretch-activated channels, did not
affect the channel activity of MCLC.
In the present study, we identified a gene, MCLC, using
the MID-1 gene as a molecular probe. MCLC encoded
a transmembrane protein with four putative transmembrane domains, but
there was no overall similarity between MCLC and MID-1. MCLC
was identical to AK2-8, previously identified as a gene
expressed in the suprachiasmatic nucleus of the hypothalamus (14), the
expression of which changed according to diurnal rhythm. Fukuhara (14)
suggested that AK2-8 was involved in the regulation of circadian
rhythm. Xenopus unknown transmembrane protein, a
Xenopus homologue of MCLC, was expressed in the pituitary.
Yet the function of Xenopus unknown transmembrane protein or
AK2-8 has not been identified.
When MCLC was transfected in cultured cells, it localized in
intracellular compartments but not in the plasma membrane. In culture
cells, MCLC was expressed in the ER and the Golgi apparatus, as
assessed by colocalization by the ER and Golgi markers. Although MCLC
has a nuclear localization signal, MCLC is not targeted to the nucleus
when transfected in various types of culture cells. As shown in Fig.
6A, however, MCLC expressed in the testis was localized in
the nucleus as well as in the cytoplasm. In spermatocytes, strong
immunoreactivity was found in the nucleus, and immunoreactive MCLC was
condensed in the nucleus in some stages of the spermatocytes during
maturation. The nuclear localization of MCLC in the nucleus of
spermatocytes suggests that MCLC may play a critical role in spermatogenesis.
MCLC has four putative transmembrane domains and is expressed in
intracellular organelles. When microsomal vesicles obtained from
MCLC-expressing cells were incorporated into the planar lipid bilayer,
the chloride channel activity with a unitary conductance of 70 picosiemens was consistently observed. Such activity was not detected
in microsomal vesicles obtained from mock-transfected cells. Given the
primary structure of MCLC as a transmembrane protein, it is likely that
MCLC functions as a chloride channel. We cannot, however, rule out the
possibility that MCLC exerts channel activity by interacting with other
proteins in microsomal vesicles. Hence, we should await definitive
evidence clarifying the function of purified MCLC protein in the lipid
bilayer. Electrophysiologically, the MCLC channel permeates anions, and
the sequence of permeability ratios was
Br During the course of the study to identify a mammalian
stretch-activated channel, we obtained the cDNA of MCLC
using a sequence taken from a stretch-activated channel, Mid1-1.
Unexpectedly, MCLC functions as a chloride channel expressed in
intracellular membranes. A question then arises whether or not MCLC is
activated by stretch of the membrane. Because we analyzed the channel
activity of MCLC in the lipid bilayer, a direct answer to this question is not available. In this regard, Gd3+, which blocks
stretch-activated channels (20), did not affect the activity of MCLC.
Consequently, there is no information at present suggesting that MCLC
is regulated by stretch. Further studies are necessary to address this
issue. With regard to ion selectivity, unlike Mid-1, MCLC functions as
an anion channel. In many instances, ion selectivity is determined by
amino acid composition of the channel pore. Although we obtained
MCLC using MID-1 as a probe, there is no sequence
similarity between the transmembrane domains of the two gene products.
It is conceivable that these two channels permeate different ions.
In summary, we identified a new chloride channel family expressed in
intracellular compartments, including the endoplasmic reticulum, the
Golgi apparatus, and the nucleus. The physiological role of this
channel remains to be elucidated.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ZAP (Stratagene, La Jolla, CA)
cDNA library was screened using cDNA probes derived from
H51262. Replicate filters were prehybridized for 3 h at 37 °C
in the following solution: 5× SSPE (standard saline phosphate-EDTA
solution), 10× Denhardt's solution, 50% formamide, 0.1% SDS,
and 0.1 mg/ml denatured herring sperm DNA. The filters were then
hybridized in the same solution plus 5 × 105 cpm
32P-labeled randomly primed probe (Ready to Go DNA labeling
kit, Amersham Pharmacia Biotech). The hybridized filters were
washed three times at room temperature with 0.1× SSC plus 0.1% SDS
and then washed for 1 h at 42 °C with the same buffer. The
cDNA was excised from the
ZAP vector with the use of a helper
phage according to the manufacturer's instructions (Stratagene). The
entire sequence of both strands was determined using an ABI PRISM dye
terminator cycle sequencing FS ready reaction kit and an Applied
Biosystems DNA sequencer 373S (Applied Biosystems, Cambridge, MA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (78K):
[in a new window]
Fig. 1.
Primary structure of MCLC. A,
comparison of the amino acid sequence of rat, human, and
Xenopus MCLC. Characters in black boxes represent
common amino acids. , nuclear localization signal.
B, hydrophobicity of rat MCLC. C, comparison of
the amino acid sequence of rat MCLC with that of the members of the
CLIC family. D, phylogenetic tree of rat, human, and
Xenopus MCLC. The scale shows the evolutionary distances
calculated (21).
View larger version (96K):
[in a new window]
Fig. 2.
Expression of mRNA for MCLC
in various rat tissues. RNA was extracted from various rat
organs, and Northern blotting was performed. Ribosomal RNA (28 S) is
shown in the lower panel.
View larger version (42K):
[in a new window]
Fig. 3.
Localization of rat MCLC in CHO cells
transfected with rat MCLC. A, CHO cells were
transfected with FLAG-tagged rat MCLC. Localization of MCLC was
determined with anti-FLAG antibody. B, localization of MCLC
(green), calnexin (Caln, red), and
4',6-diamidino-2-phenylindole (DAPI, blue) in a
rat MCLC-transfected CHO cell. C, localization of MCLC
(red), ER-targeted enhanced yellow fluorescent
protein (green), and 4',6-diamidino-2-phenylindole
(blue) in a rat MCLC-transfected cell. D,
localization of MCLC (green), anti-Golgi 58K
(red), and 4',6-diamidino-2-phenylindole (blue)
in a rat MCLC-transfected CHO cell.
View larger version (22K):
[in a new window]
Fig. 4.
Western blotting of MCLC in CHO cells
transfected with MCLC. CHO cells were transfected with rat MCLC
(A) or FLAG-tagged rat MCLC (B), and Western
blotting was done in the presence or absence of -mercaptoethanol
(ME) with either anti-FLAG antibody or anti-MCLC
antibody.
View larger version (15K):
[in a new window]
Fig. 5.
Western blotting of endogenous MCLC protein
in various tissues. Expression of the MCLC protein in various
tissues was studied by Western blotting. An aliquot of protein (100 µg) was applied to each lane.
View larger version (95K):
[in a new window]
Fig. 6.
Localization of immunoreactive MCLC in
the testis. Immunohistochemistry was performed in tissue slices
obtained from rat testis using anti-MCLC antibody. Immunoreactivity of
MCLC is shown in brown. At lower magnification
(A), immunoreactivity of MCLC was found in the cytoplasm or
in the nucleus. At higher magnification, immunoreactivity was found in
the cytoplasm (B) or in the nucleus (C) depending
upon the stage of spermatocytes. Magnification was as follows:
A, × 400; B and C, × 1000.
22.4 ± 1.0 mV (mean ± S.D.,
n = 3). The permeability ratio
PCl/PK was calculated to
be 3.7 using the Goldman-Hodgkin-Katz current equation. The
permeabilities of various anions were also estimated from reversal
potentials in asymmetric solutions of 100 mM KCl in the
trans compartment and 100 mM KBr, 100 mM KF, or 50 mM K2SO4 in the cis compartment. The reversal potential for
Br
was
1.8 mV (n = 3) and the
PBr/PCl was 1.1. The
reversal potential for F
and the
PF/PCl value were +12.0
mV (n = 4) and 0.54, respectively. The reversal
potential for SO
View larger version (21K):
[in a new window]
Fig. 7.
Channel Activity of rat MCLC.
A, CHO cells were transfected with rat MCLC, and microsomes
were prepared and incorporated into the lipid bilayer. Channel activity
was measured as described under "Materials and Methods." The
membrane potential was 60 mV. B, a current-voltage
relationship was obtained.
, cis/trans (500/100
mM KCl);
, symmetric (100 mM KCl). Values
are the means ± S.D.; n = 3.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
Cl>F
>SO
![]() |
ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Masahiro Sokabe of Nagoya University and Dr. Hidetoshi Iida of Tokyo Gakugei University for various discussions and to Mayumi Odagiri for technical and secretarial assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by a grant-in-aid from the Ministry of Science, Education, Sports, and Culture of Japan and a grant from the Japan Space Forum.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB052922 and AB052915.
To whom correspondence should be addressed. Tel.:
81-27-220-8835; Fax: 81-27-220-8893; E-mail:
ikojima@showa.gunma-u.ac.jp.
Published, JBC Papers in Press, March 5, 2001, DOI 10.1074/jbc.M100366200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: CLIC, chloride intracellular channel; MCLC, Mid-1-related chloride channel; EST, expressed sequence tag; PCR, polymerase chain reaction; MOPS, 4-morpholinepropanesulfonic acid; CHO, Chinese hamster ovary; ER, endoplasmic reticulum.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Jentsch, T. J. (1994) Curr. Opin. Cell Biol. 6, 600-606[Medline] [Order article via Infotrieve] |
2. | Al-Awqati, Q. (1995) Curr. Biol. 7, 504-508 |
3. | Riordan, J., Rommens, J. M., Kerem, B. S., Alon, N., Rozmahel, R., Gtzelczack, Z., Zielenski, J., Lok, S., Plavsic, N., and Chou, J. I. (1989) Science 245, 1066-1073[Medline] [Order article via Infotrieve] |
4. | Lloyd, S. E., Pearce, S. H. S., Fisher, S. E., Steinmeyer, K., Schwappach, B., Scheinman, S. J., Harding, B., Bolino, A., Devoto, M., and Goodyer, P. (1996) Nature 379, 445-449[CrossRef][Medline] [Order article via Infotrieve] |
5. | Koch, M. C., Steinmeyer, K., Lorenz, C., Ricker, K., Wolf, F., Otto, M., Zoll, B., Lehmann-Horn, F., Grzschik, K. H., and Jentsch, T. J. (1992) Science 257, 797-800[Medline] [Order article via Infotrieve] |
6. | Welsch, M. J., and Smith, A. E. (1993) Cell 73, 1251-1254[Medline] [Order article via Infotrieve] |
7. | Hosie, A. M., Aronstein, K. S., Sattelle, D. B., and Constant, R. A. (1997) Trends Neurosci. 20, 578-583[CrossRef][Medline] [Order article via Infotrieve] |
8. | Jentsch, T. J., Gunther, W., Pusch, M., and Schwappach, B. (1995) J. Physiol. (Lond.) 482, 19-25 |
9. | Edwards, J. (1998) Am. J. Physiol. 276, F398-F408 |
10. | Iida, H., Nakamura, H., Ono, T., Okumura, M. S., and Anraku, Y. (1994) Mol. Cell. Biol. 14, 8259-8271[Abstract] |
11. |
Kanzaki, M.,
Nagasawa, M.,
Kojima, I.,
Sato, C.,
Naruse, K.,
Sokabe, M.,
and Iida, H.
(1996)
Science
285,
882-886 |
12. | French, A. S. (1992) Annu. Rev. Physiol. 54, 135-152[CrossRef][Medline] [Order article via Infotrieve] |
13. | Montal, M., and Mueller, P. (1972) Proc. Natl. Acad. Sci. U. S. A. 69, 3561-3566[Abstract] |
14. | Fukuhara, C. (1996) Sophia Life Sci. Bull. 15, 79-93 |
15. | Kanzaki, M., Mashima, H., Zhang, Y. Q., Lu, L., Shibata, H., and Kojima, I. (1999) Nat. Cell Biol. 1, 165-170[CrossRef][Medline] [Order article via Infotrieve] |
16. |
David, W.,
Hochstenbach, F.,
Rajagopalan, S.,
and Brenner, M. B.
(1993)
J. Biol. Chem.
268,
9585-9592 |
17. | Ktistakis, N. T. (1991) J. Cell Biol. 113, 1009-1014[Abstract] |
18. |
Bretag, A. H.
(1987)
Physiol. Rev.
67,
618-724 |
19. | Manning, S. D., and Williams, A. J. (1989) J. Membr. Biol. 109, 113-122[Medline] [Order article via Infotrieve] |
20. | Sukharev, S. I., Blount, P., Martinac, B., and Kung, C. (1997) Annu. Rev. Physiol. 59, 633-658[CrossRef][Medline] [Order article via Infotrieve] |
21. | Kyte, I., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132[Medline] [Order article via Infotrieve] |