1 Renal Section, Boston University Medical Center, and Departments of 2 Medicine, 3 Physiology, and 4 Pathology, Boston University School of Medicine, Boston, Massachusetts 02118
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ABSTRACT |
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The trafficking of H+-ATPase vesicles to the apical membrane of inner medullary collecting duct (IMCD) cells utilizes a mechanism similar to that described in neurosecretory cells involving soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins. Regulated exocytosis of these vesicles is associated with the formation of SNARE complexes. Clostridial neurotoxins that specifically cleave the target (t-) SNARE, syntaxin-1, or the vesicle SNARE, vesicle-associated membrane protein-2, reduce SNARE complex formation, H+-ATPase translocation to the apical membrane, and inhibit H+ secretion. The purpose of these experiments was to characterize the physiological role of a second t-SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP)-23, a homologue of the neuronal SNAP-25, in regulated exocytosis of H+-ATPase vesicles. Our experiments document that 25-50 nM botulinum toxin (Bot) A or E cleaves rat SNAP-23 and thereby reduces immunodetectable and 35S-labeled SNAP-23 by >60% within 60 min. Addition of 25 nM BotE to IMCD homogenates reduces the amount of the 20 S-like SNARE complex that can be immunoprecipitated from the homogenate. Treatment of intact IMCD monolayers with BotE reduces the amount of H+-ATPase translocated to the apical membrane by 52 ± 2% of control and reduces the rate of H+ secretion by 77 ± 3% after acute cell acidification. We conclude that SNAP-23 is a substrate for botulinum toxin proteolysis and has a critical role in the regulation of H+-ATPase exocytosis and H+ secretion in these renal epithelial cells.
soluble N-ethylmaleimide-sensitive factor attachment protein target receptor; soluble N-ethylmaleimide-sensitive factor attachment protein; botulinum toxins; proton pumps; hydrogen ion secretion
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INTRODUCTION |
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INTERCALATED and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by an apical membrane H+-ATPase (2, 19). The rate of H+ transport in these cells is regulated in part by exocytic insertion or endocytic retrieval of H+-ATPase-laden vesicles (1, 2, 4). We have proposed that the trafficking of these vesicles to the apical membrane in IMCD cells utilizes a mechanism similar to that utilized by neurosecretory cells and involves soluble N-ethylmaleimide-sensitive factor attachment protein target receptor (SNARE) proteins (1, 2, 4).
Recent studies from this laboratory, in support of this proposal,
have demonstrated that IMCD cells express the vesicular targeting-fusion membrane proteins known as SNAREs and the soluble factors N-ethylmaleimide-sensitive factor (NSF) and - and
-soluble N-ethylmaleimide-sensitive factor attachment
protein (SNAP) (2). Clostridial neurotoxins that act as
specific SNARE proteases inhibit both H+ transport and the
exocytic amplification of apical membrane H+-ATPase
(2, 4). Consistent with these observations is the report
that in another tissue, the rat vas deferens, tetanus toxin also
inhibits H+ secretion and cleaves vesicle-associated
membrane protein (VAMP)-3 (5). We have also demonstrated
that we can immunoisolate, from IMCD cells, clostridial toxin-sensitive
complexes that consist of the vesicle (v-) SNARE, VAMP-2, the target
(t)- SNAREs, syntaxin-1, and SNAP-23 and the soluble factors NSF and
- and
-SNAP (4). These complexes resemble
the 20S complexes isolated from neurosecretory tissue in that they
consist of similar SNARE proteins, and for parsimony, we refer to these
complexes in the IMCD as 20S-like. Furthermore, it was found that the
31-kDa subunit of H+-ATPase had a close interaction with
the participating SNARE proteins and was an integral component of this
20S-like complex (4).
To document a functional role for the complex and for the SNAREs syntaxin-1 and VAMP-2, we demonstrated that toxins that specifically target one or the other of these proteins decrease the stability of the complex, inhibit insertion of H+-ATPase into the luminal membrane, and reduce the rate of H+ transport (1, 4). Another t-SNARE found in IMCD cells, SNAP-23, is a homologue of neuronal SNAP-25, is expressed in many tissues (11), and has been found to be colocalized in the kidney with vesicles that contain aquaporin-2 (20). Unlike SNAP-25, the role of SNAP-23 in exocytosis is controversial. SNAP-25, expressed by all species, has been found to be toxin sensitive (16, 17) and to enhance the affinity of syntaxin for VAMP (3, 11). In contrast to SNAP-25, human SNAP-23 is not sensitive to proteolysis by botulinum toxin (Bot) (7, 10, 15) and may not increase syntaxin affinity for VAMP (7). Thus any functional/regulatory role for SNAP-23 in exocytosis is not established.
The purpose of these experiments was to characterize the physiological role of SNAP-23 (presumed to be a participating t-SNARE) in the process of H+-ATPase translocation in acid secretion by the IMCD cell. Our experiments document that rat IMCD SNAP-23 is toxin sensitive, important in the formation and stability of complexes, and required for the translocation of H+-ATPase to the apical membrane. Thus these data document a critical role for SNAP-23 in the regulation of H+-ATPase exocytosis in rat IMCD cells.
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MATERIALS AND METHODS |
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Cell culture. IMCD cells were obtained from rat papillae as described previously (18). Cells from passages 6 to 12 were grown to confluence in 150-cm2 plastic culture dishes or on coverslips in multiwell dishes in DMEM with 10% fetal calf serum, penicillin, and streptomycin in an atmosphere of 95% air-5% CO2. Some IMCD monolayers were metabolically labeled with 35S by incubating them with [35S]methionine as previously described (12). In brief, the monolayers were incubated in methionine-free DMEM for 2 h, followed by an additional 2-h period in DMEM that contained 500 µCi/ml [35S]methionine. At the end of the loading period, the monolayers were washed and incubated for 45 min with DMEM that contained 10 mM cold methionine.
Acid loading of cells. IMCD monolayers were subjected to a reduction in cytosolic pH (pHi) from 7.2 to 6.5 by methods previously described (18). Briefly, IMCD monolayers were washed with PBS to remove any DMEM and then were washed once with choline chloride HEPES buffer [CHB; 110 mM choline chloride, 50 mM HEPES acid, 5 mM KCl, 1 mM MgCl2, 5 mM KH2PO4, 1 mM CaCl2, 5 mM glucose, and 10 mM potassium acetate (pH 7.2)]. The monolayers were then incubated with CHB for 20 min. During this time, the pHi rapidly declined to 6.5 due to reversal of the Na+/H+ exchanger and diffusion of acetic acid into the cell (2, 18).
Cell pH determination.
The method used for measuring cell pH was described earlier
(20). Quiescent cells grown on glass coverslips were
incubated for 1 h at 37°C in NaCl HEPES buffer (NHB; similar to
CHB, except having 110 mM NaCl instead of choline chloride and without
any potassium acetate) pH 7.2 that contained 10 µM of the
acetoxymethyl ester of
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF). The coverslip was then placed in a plastic cuvette that contained 1 ml
NHB and was secured by means of a device designed to hold the coverslip
at a 35° angle to the excitation beam. The monolayer was then washed
three times with NHB and suspended in 1 ml NHB. Fluorescence intensity
was measured in a Perkin Elmer LS 650-10 fluorospectrophotometer
equipped with a thermostatically controlled (37°C) cuvette holder, at
excitation wavelengths of 505 and 455 nm with a slit width of 2 nm, and
emission wavelengths of 560 nm with a slit width of 4 nm. At the end of
the experiment, the fluorescence intensity ratio (FIR) was calibrated
to cell pH (pHi) using potassium HEPES buffer that
contained 10 mg/ml nigericin. The FIR varied linearly with pH over the
range of 6.3-7.6. Autofluorescence of probe-free monolayers was
<10% of the fluorescent signal of BCECF-loaded monolayers at
excitation of both 505 and 455 nm, and correction for this was not
made. Na+-independent pHi recovery after a 20 mM NH4Cl-induced acid load when
incubated in CHB was determined as previously described (2,
18). After an initial control measurement of active
H+-ATPase proton transport (JH-act), the monolayers
were exposed to BotE or the vehicle, and JH-act was
redetermined. We have previously shown that successive measurements of
JH-act under control conditions are not significantly different
(2).
Effect of Bot on H+-ATPase translocation to the apical membrane. Just before study, confluent monolayers were incubated for 45 min in fresh DMEM that contained either 50 nM BotE or vehicle. Before BotE addition, the toxin was routinely incubated in 5 mM dithiothreitol (DTT) at room temperature to facilitate the separation of light and heavy chains. The control buffer was similarly incubated in 5 mM DTT. After incubating the cells with toxin or control buffer, the media was removed, and both control and toxin-treated monolayers were washed with fresh DMEM. The cells were then acid loaded (2).
Apical membranes were isolated from IMCD monolayers by a vesiculation method (18) modified in our laboratory for polarized epithelial cells (2). Control or acid-loaded cells, in which the pHi falls to 6.5, were incubated for 90 min at 37°C in a vesiculation medium (CHB) that contained 50 mM paraformaldehyde, 2 mM DTT, and protease inhibitors. Paraformaldehyde and DTT induced the formation of apical membrane vesicles that were released into the incubation medium. At the end of 90 min, the medium containing the released apical membrane vesicles was collected, filtered through 37 µM of nylon mesh to remove whole cells, and centrifuged at 25,000 rpm at 4°C in a Sorvall RC5B centrifuge for 1 h to pellet the vesicles. The pellet is aliquoted for protein determination and dissolved in SDS sample buffer for subjecting to SDS-PAGE and immunoblotting.Cell homogenization. Acid-loaded IMCD monolayers were scraped from the plastic plate with a spatula into a homogenizing buffer (CHB) that contained 1 mM phenylmethylsulfonyl fluoride, 1 mM aprotinin, and 1 mM leupeptin. The cells were pelleted at 800 g, and the pellet was resuspended in the homogenizing buffer, passed 20 times through a 23-gauge needle, and then further homogenized 10 times using Teflon-coated Dounce homogenizer. The homogenate was centrifuged at 1,000 g in a refrigerated Sorvall RC5B centrifuge for 20 min. The supernatant was further centrifuged at 13,000 g for 45 min. The pellet was resuspended in homogenizing buffer (membrane homogenate). In three studies, the pellet was detergent extracted. These pellets were suspended in 300 µl of detergent extraction medium (CHB with 1.5% Triton X-100 and 2% SDS). This suspension was sonicated with ten 0.5-s pulses in a Bronson sonicator, incubated at 20°C for 45 min, and then centrifuged for 20 min at 13,000 g. The supernatant was the detergent extract. Aliquots of the membrane homogenate and detergent extract were saved for protein determination, immunoprecipitation, or immunoblot analysis.
Effect of toxin on SNAP-23. Two approaches were used to assess the effects of BotA or BotE on SNAP-23. In the first, aliquots that contained 80 µg protein of membrane homogenate from IMCD cells were incubated with 50 nM of BotE or BotA (pretreated with 5 mM DTT), respectively, or with diluent as the control for 1 h at 37°C. At the end of the incubation, EDTA and EGTA (4 mM) were added to each incubating tube to inactivate the toxin, and they were immunoblotted with a rabbit polyclonal antibody against a COOH-terminal peptide of SNAP-23 (a gift from Dr. P. A. Roche).
In the second approach, [35S]SNAP-23 was immunoprecipitated with the same antibody as used above from a detergent extract of [35S]methionine-labeled IMCD cells. Rabbit antibody to SNAP-23 was cross-linked onto agarose beads with dimethyl pimelimidate (4). The antibody-coated beads (120 µl) were added to a 100-µg detergent extract of IMCD cells. The detergent extract was heated for 30 min to 56°C to induce dissociation of SNARE complexes before being subjected to immunoprecipitation. The beads were washed extensively with immunoprecipitation buffer (HB and 0.1% SDS) buffer, and the 35S-labeled SNAP-23 was eluted from the beads with glycine buffer (12). Two aliquots of the eluted [35S]SNAP adjusted to pH 7.4 with Tris base were incubated at 37°C for up to 1 h with either diluent or 50 nM BotE as described above. At timed intervals, samples were obtained for 15% gel electrophoresis, and [35S]SNAP was detected by autoradiography.Isolation of SNARE protein complex by immunoprecipitation and immunoblotting. Immunoprecipitation was performed on the cell membrane homogenate, and detergent extracts were prepared and treated with 50 nM BotE as described above. The homogenate was incubated with anti-rabbit or anti-mouse sera for 1 h, followed by incubation with either protein A- or G-agarose beads for another hour. The incubation mixture was then centrifuged, and the supernatant incubated overnight at 4°C with a rabbit antibody to the 31-kDa subunit of H+-ATPase (a gift from Dr. Dennis Brown) or a mouse monoclonal antibody to syntaxin/HPC-1 (Sigma), along with either protein A- or G-agarose beads. After overnight incubation, the immunoprecipitates were washed five times in CHB that contained 0.1% SDS and 0.1% Triton X-100 to eliminate any nonspecific protein binding. The washed immunoprecipitates were dissolved in SDS sample buffer, boiled, and resolved in 15% SDS-PAGE, followed by transfer onto nitrocellulose filters. After transfer, the nitrocellulose filters were incubated in 5% wt/vol nonfat powdered milk in Tris-buffered saline containing Tween 20 (TBST) for 1 h. The blots were washed in TBST and incubated with primary antibody (1:1,000) overnight at 4°C. After overnight incubation, the nitrocellulose filters were washed five times in TBST and incubated in secondary antibody (horseradish peroxidase-conjugated anti-rabbit or horseradish peroxidase-conjugated anti-mouse antibody) used in 1:3,000 dilution for 2 h at room temperature. After secondary antibody incubation, the blots were washed several times in TBST, and bound antibody was detected using an enhanced chemiluminescence system (Pierce). The images of immunoblot for all experiments presented are representative of at least four separate studies.
Antibodies. The following antibodies were used in these studies: mouse monoclonal anti-GP-135 (G. K. Ojakian); rabbit polyclonal COOH-terminal anti-SNAP-23 (P. Roche); rabbit polyclonal anti-31-kDa subunit of H+-ATPase (D. Brown); mouse monoclonal anti-syntaxin-1 (clone HPC-1; Sigma); and rabbit polyclonal NSF (Synaptic Systems).
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RESULTS |
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SNAP-23 cleavage by BotA and BotE.
Analyses of IMCD cell homogenates revealed a significant amount of
immunodetectable SNAP-23 without any detectable SNAP-25 (Fig.
1A). After incubation
in vitro with Bot (50 nM final concentration), the immunodetectable
SNAP-23 decreased by at least 70-75% from control when incubated
with BotE and by 60-65% when incubated with BotA (Fig. 1,
A and B). Even at a toxin concentration of 25 nM,
there was a marked decrease in the immunodetectable mass of SNAP-23,
but no change was noted when the toxin incubation was carried out at
4°C (data not shown).
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The effect of toxin on JH-act.
To determine whether SNAP-23 plays a physiological role in
H+ secretion by IMCD cells, the effect of SNAP-23 cleavage
with BotE on JH-act was measured. Each monolayer was initially acid loaded, allowed to recover, and the rate of alkalinization was determined. Only cells that had a recovery rate >0.025 U/min after acid load were utilized. The monolayers were allowed to recover and
were then acid loaded in the presence of toxin. The rate of alkalinization was again determined. Figure
3 is a representative tracing of the
effect of BotE (50 nM). During the control period after acute cellular
acidification, pHi increased at the rate of 0.056 ± 0.003 pH U/min. With the addition of BotE, pHi recovery was
significantly (P < 0.05, n = 5)
reduced to a rate of 0.013 ± 0.001 pH U/min.
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Effect of Bot on the translocation of the 31-kDa subunit of
H+-ATPase to the apical membrane.
The 31-kDa subunit of H+-ATPase translocates to the apical
membrane with the lowering of intracellular pH and elevation of Ca2+ (1). After IMCD cells are exposed to BotE
(50 nM) for 45 min at 37°C and are acid loaded, the immunodetectable
amount of 31-kDa in the apical membrane is markedly reduced, compared
with control acid-loaded monolayers. In four consecutive studies, the
amount of H+-ATPase in the apical membrane after cell
acidification was 52 ± 2% (P < 0.05) less in
BotE-treated IMCD cells compared with control. In contrast, the amount
of GP-135, a resident protein of the apical membrane, was unaffected by
toxin treatment (Fig. 4).
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Isolation of 20S-like protein complex and role of SNAP-23.
We have previously determined that IMCD cells (2, 4)
express the proteins that typically make up the 20S complex described in neuronal cells (8). By employing antibodies to the
31-kDa subunit of H+-ATPase and syntaxin to IMCD cell
homogenate for coimmunoprecipitation, v- and t-SNAREs, NSF, and -
and
-SNAP were detected. This indicates that, like the brain, IMCD
cells express similar SNAREs, with the exception of SNAP-25. Instead,
IMCD cells express SNAP-23 (Figs.
5A and
6A, lanes 1 and
2). In addition, they form an immunoprecipitable protein complex that is similar in composition to the neuronal 20S
complex. The stability and dependence of the isolated 20S-like complex
on SNAP-23 were determined by evaluating the effects of BotE. This
toxin cleaves a peptide from the COOH-terminal end of this t-SNARE
(Figs. 1 and 2). There was a >60% reduction in the amount of
coimmunoprecipitable protein with antibodies to either the 31-kDa
subunit of the H+-ATPase or syntaxin detected by
immunoblotting from the toxin-treated homogenate compared with the
control (Figs. 5 and 6). However, the amounts of 31-kDa and syntaxin
immunoprecipitated in the control and toxin-treated homogenates were
not different when immunoprecipitated with antibodies to the 31-kDa
subunit (Fig. 5A, lanes 2 and 3, and
Fig. 5B) or to syntaxin (Fig. 6A, lanes
2 and 3, and Fig. 6B). Only proteins that
are associated with the 31-kDa subunit or syntaxin and participate in
the complex formation are disturbed in some fashion, and, hence, result
in reduced recovery of the coimmunoprecipitated 20S-like complex.
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DISCUSSION |
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Cell pH-regulated trafficking of H+-ATPase vesicles in
IMCD cells share features with regulated trafficking of synaptic
vesicles in neurosecretory cells in that both rely on SNARE proteins.
Some of the SNARE proteins expressed by IMCD cells in culture include the v-SNARE, VAMP-2, the t-SNARE, syntaxin-1, SNAP-23, and the soluble
factors NSF and - and
-SNAP (2, 4). After induction of exocytosis of H+-ATPase vesicles by acutely lowering
pHi, an immunoisolated complex similar to the neuronal
fusion (20S) complex was identified that consists of these SNARE
proteins and also the H+-ATPase itself (4). To
evaluate the role of the specific SNAREs in the formation of this
complex and in the process of exocytosis, we determined the effect of
two different clostridial neurotoxins, highly specific Zn
metaloproteases that cleave specific SNAREs (17) on
complex formation, proton pump trafficking, and H+
transport (2, 4). Exposure of IMCD cells to BotC
resulted in the cleavage of its known target, syntaxin-1, and after
tetanus, toxin cleavage of its target, VAMP-2 (2, 4). In
addition to proteolysis of these specific SNAREs, BotC or tetanus toxin reduced complex stability; pHi induced translocation of
proton pumps to the apical membrane and H+ secretion
(4). Thus we have demonstrated that exocytosis of H+-ATPase by IMCD cells has a similar requirement for
syntaxin-1 and VAMP-2 as does exocytosis of synaptic vesicles. In
another tissue, the rat vas deferens, tetanus toxin has also been
documented to cleave its target protein, VAMP-3, and inhibit
H+ secretion (5). These studies utilizing
intact tissue are consistent with our observations in a tissue culture model.
Although it has been shown in vitro that the minimum requirement for docking is a t-v SNARE pair, i.e., syntaxin and VAMP, SNAP-25 is necessary for efficient fusion (3, 8, 22) of neurosecretory vesicles. SNAP-25 is part of the fusion complex in neuronal cells and has been shown to be essential, not only in the formation and function of the fusion complex (3, 11), but also for the delivery of syntaxin to the appropriate membrane domain (21). Furthermore, SNAP-25 is sensitive to cleavage by BotA and BotE, and after proteolysis by these toxins, synaptic vesicle exocytosis is inhibited (16, 17). However, this important t-SNARE is not expressed in nonneuronal cells (13). Instead of SNAP-25, a homologue of this t-SNARE, SNAP-23, is widely expressed in most somatic cells, including the kidney and in our cultured line of IMCD cells (4, 13, 20).
The role of SNAP-23 in exocytosis is controversial. Some species, such as human and mouse, express a SNAP-23 that has been reported to be resistant to proteolysis by both BotA and BotE (6, 10). It has also been reported that SNAP-23 does not affect the affinity of syntaxin for VAMP (7). Furthermore, from experiments testing the effects of toxin on exocytosis, SNAP-23 may not be involved in the docking fusion process in some systems (14).
In the present study, we provide evidence in this rat renal epithelial cell line that directly supports the regulatory role of the t-SNARE, SNAP-23, during targeting and fusion of H+-ATPase vesicles. Unlike human and mouse SNAP-23 (6, 10, 15), but similar to canine SNAP-23 (9), rat SNAP-23 is cleaved by both BotA and BotE (Figs. 1 and 2). The toxin action on SNAP-23 in rat IMCD cells has relevant physiological consequences. BotE prevents translocation of H+-ATPase to the apical membrane, suggesting disruption in the targeting and fusion of vesicles with the apical membrane (Fig. 4), and as a consequence, inhibits pHi recovery after an acute cell acid load (Fig. 3). The toxin action also interferes with the structural integrity of the 20S-like complexes that are present in cell homogenate (Figs. 5-7). This effect of toxin is consistent with a role for SNAP-23 in regulating the affinity of syntaxin-1 for its cognate pair, VAMP-2.
This study, therefore, documents that in this somatic, renal epithelial cell line, SNAP-23 is the t-SNARE that subserves the same functions as SNAP-25 in neural tissue for the regulation of exocytosis. It also provides additional evidence that the overall regulatory process of H+-ATPase exocytosis in this nonneuronal system utilizes a complex of SNAREs that includes syntaxin-1, VAMP-2, and SNAP-23.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-28164.
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FOOTNOTES |
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A. Banerjee was a recipient of the Joseph Shankman Award from the National Kidney Foundation of Massachusetts and Rhode Island.
Address for reprint requests and other correspondence: J. H. Schwartz, Evans Biomedical Research Center, 650 Albany St., Boston, MA 02118-2908 (E-mail: jhsch{at}bu.edu).
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.
Received 28 June 2000; accepted in final form 30 October 2000.
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