| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 279, Issue 52, 54826-54832, December 24, 2004
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Department of Environmental Biochemistry and Toxicology, School of Pharmaceutical Sciences, **Department of Molecular Toxicology and the 21st Century COE program, School of Pharmaceutical Sciences, and ||Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan and the ¶Department of Pharmaceutical Physiology, Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2630 Sugitani, Toyama 930-0194, Japan
Received for publication, June 7, 2004 , and in revised form, October 15, 2004.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
TRV) and the alanine substitution (ARV and TRA) inhibited the association of PCLN-1 with ZO-1. Confocal immunofluorescence demonstrated that the wild-type PCLN-1 and the TAV mutant localized in the tight junction along with ZO-1, but the TRV, ARV, and TRA mutants were widely distributed in the lateral membrane including the tight junction area. Interestingly, monolayers of cells expressing the wild-type PCLN-1 and the TAV mutant showed higher activities of 45Ca2+ transport from apical to basal compartments, compared with those expressing the TRV, ARV, and TRA mutants and the mock cells. 45Ca2+ transport was inhibited by increased magnesium concentration suggesting that magnesium and calcium were competitively transported by PCLN-1. It was noted that a positive electrical potential gradient enhanced 45Ca2+ transport from apical to basal compartments without affecting the opposite direction of transport. Thus, PCLN-1 localizes to the tight junction followed by association with ZO-1, and the PCLN-1·ZO-1 complex may play an essential role in the reabsorption of divalent cations in renal epithelial cells. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Tight junction (TJ) forms apical junctional complexes in epithelial and endothelial cells and plays a central role in the maintenance of barrier and cell polarity (2, 3). These functions are facilitated by occludin and claudin family. So far, at least 22 mammalian isoforms of claudin have been identified, and their spatial distribution patterns in some epithelial tissues have been clarified. Paracellin-1 (PCLN-1), cloned by Simon et al. (4), is a most cogent protein that undertakes magnesium reabsorption, because (1) homozygous mutations in the PCLN-1 gene are associated with a rare familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC), and (2) PCLN-1 is exclusively expressed in the TJ of TAL. PCLN-1 belongs to the claudin family (named claudin-16), which composes a TJ strand and is the most distantly related member of this family. Amino acid homology between PCLN-1 and individual claudin is very low (10–18%), but these proteins have a consensus PSD95/DglA/ZO-1-like domain (PDZ)-binding motif at the C terminus (5).
Protein-protein interactions mediated by the PDZ-binding motif occur through the PDZ domains in multiple members of the membrane-associated guanylate kinases (6). Membrane-associated guanylate kinases contain several PDZ domains, an SH3 domain, and a C-terminal guanylate kinase homology region that may act as a protein-binding domain (7, 8). Among them, ZO-1 and ZO-2 are localized at the TJ area and they may anchor transmembrane proteins, such as occludin and claudin family. Thus, the interactions between transmembrane proteins and cytoplasmic molecules may modulate paracellular permeability.
The direct binding of ZO-1 with the C terminus (YV sequence) of claudin-1–8 has been revealed by an in vitro binding assay using glutathione S-transferase (GST) fusion protein and in vivo immunofluorescence analysis (9). However, the C terminus (TRV sequence) of PCLN-1 is different from that of other proteins in the claudin family, and the function of PCLN-1 has not been clarified yet. In the present study, we found the association between ZO-1 and the C-terminal TRV sequence of PCLN-1. Furthermore, the function of PCLN-1 was established by measurements of transepithelial electrical resistance (TER) and 45Ca2+ transport.
|
EXPERIMENTAL PROCEDURES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
Plasmid cDNA Constructs—Rat PCLN-1 cDNA containing the whole open reading frame was amplified by the reverse transcriptase-PCR from rat kidney mRNA using the set of primers, 5'-CGATATCCATGAAGGATCTTCTTC-3' (sense) and 5'-CCGGTCGACTTACACTCTCGTGTC-3' (antisense). The PCLN-1 cDNA was subcloned into the mammalian expression vector, pCMV-Tag2A (BD Biosciences Clontech), containing the FLAG epitope. Additional FLAG-PCLN-1 cDNA encoding PCLN-1 with point substitutions in the C-terminal PDZ-binding motif (substitutions are underlined, FLAG-PCLN-1-ARV, FLAG-PCLN-1-TAV, FLAG-PCLN-1-TRA) and with the deletion mutant (FLAG-PCL-N-1-TRV) were constructed by PCR using the above sense and the antisense primers (ARV, 5'-CCGGTCGACTTACACTCTCGCGTC-3'; TAV, 5'-CCGGTCGACTTACACTGCCGTGTC-3'; TRA, 5'-CCGGTCGACTTACGCTCTCGTGTC-3'; TRV, 5'-CGTCGACTTAGTCTACAGCGTAC-3'). Fusion proteins of the C terminus of PCLN-1 with GST were constructed by subcloning PCR amplified DNA fragments into pGEX-4T-1 (Amersham Biosciences) to yield GST-PCLN-1-TRV, GST-PCLN-1-TRA, GST-PCLN-1-TAV, and GST-PCLN-1-ARV, respectively. All wild-type and mutant PCLN-1 constructs were verified by DNA sequencing.
In Vitro Binding Assay—The plasmids of GST-PCLN-1-TRV, GST-PCLN-1-ARV, GST-PCLN-1-TAV, and GST-PCLN-1-TRA were transformed in Escherichia coli BL21. GST fusion proteins were purified with glutathione-Sepharose 4B beads (Amersham Biosciences). The beads were incubated with lysate of Madin-Darby canine kidney (MDCK) cells in a buffer composed of 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40, 2 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma) for 4 h at 4 °C. Then, bound proteins were eluted with a sample buffer and applied to the SDS-polyacrylamide gel. Proteins were blotted onto a polyvinylidene difluoride membrane and incubated for 1 h with each primary antibody followed by peroxidase-conjugated secondary antibody. The blots were visualized as described in the Western blotting section.
Cell Culture and Transfection—MDCK cells were obtained from the European Collection of Cell Cultures (#84121903, Wiltshire, UK). Cells were grown in Dulbecco's modified Eagle's medium (Sigma) supplemented with 5% Fetal Clone III (HyClone Laboratories, Logan, UT), 0.07 mg/ml penicillin-G potassium, and 0.14 mg/ml streptomycin sulfate in a 5% CO2 atmosphere at 37 °C. MDCK cells were transfected with expression plasmids using Lipofectamine 2000 as indicated in the manufacture's protocol. Stable transfectants were selected with 0.5 mg/ml G418 sulfate and maintained in the continuous presence of the selecting drug. Cell lines expressing FLAG-tagged protein were screened by Western blotting.
Preparation of Membrane Fraction and Immunoprecipitation— Whole membrane fraction was prepared from MDCK cells on day 7 in culture as described previously (10). The samples were solubilized in a lysis buffer containing 1% Triton X-100, 150 mM NaCl, 0.5 mM EDTA, and 50 mM Tris-HCl (pH 8.0) and incubated with protein G-Sepharose beads and an antibody specific for GST at 4 °C for 1 h with gentle rocking. After centrifugation at 6000 x g for 1 min, the pellet was washed four times with the lysis buffer. The pellet was solubilized in the sample buffer for SDS-polyacrylamide gel electrophoresis. Protein concentration was measured using the protein assay kit (Bio-Rad Laboratories) with bovine serum albumin as the standard.
SDS-Polyacrylamide Gel Electrophoresis and Western Blotting— SDS-polyacrylamide gel electrophoresis was carried out as described previously (11). In brief, whole membrane fractions (30 µg) or immunoprecipitants were applied to the SDS-polyacrylamide gel. Proteins were blotted onto a polyvinylidene difluoride membrane and incubated for 1 h with each primary antibody followed by a peroxidase-conjugated secondary antibody. Finally, the blots were stained with the ECL Western blotting kit from Amersham Biosciences.
Immunofluorescence Microscopy—The cells grown on cover glass for 7 days were washed twice with phosphate-buffered saline supplemented with 0.5 mM CaCl2 and 0.5 mM MgCl2 prior to fixation with 3% paraformaldehyde for 7 min at room temperature. After permeabilization with 0.3% Triton X-100 for 15 min and blocking with 5% skim milk in phosphate-buffered saline for 30 min, the cells were incubated with an anti-ZO-1 antibody and an anti-FLAG antibody (final dilution 1/100) for 60 min at room temperature. They were then washed three times with phosphate-buffered saline, followed by incubation with Texas Redlabeled anti-goat IgG and fluorescein isothiocyanate-labeled anti-rabbit IgG in a blocking solution (dilution 1/100) for 60 min. Immunolabeled cells were visualized on an LSM 510 confocal microscope (Carl Zeiss, Germany) set with the appropriate filter for Texas Red (543 nm excitation, 585–615 nm emission filter) and fluorescein isothiocyanate (488 nm excitation, 530 nm emission filter). Images were further processed using Adobe Photoshop (Adobe System, Inc).
Measurement of TER and Transepithelial Transport Assay—MDCK cells were plated at confluent densities on transwells with polyester membrane inserts (Corning Life Sciences, Acton, MA). TER was measured three times for each well at indicated times using a Millicell-ERS epithelial volt-ohmmeter (Millipore, Bedford, MA), and averaged values were collected. TER values (ohm x cm2) were normalized by the area of the monolayer and calculated by subtracting the blank values from the filter and the bathing medium. Transepithelial transport of 45Ca2+ across confluent MDCK cells was evaluated as described previously (12). Briefly, at time 0, a transport buffer containing 0.5 µCi/ml 45Ca2+ was poured into the filter well or the outside compartment. The transport buffer contained 140 mM NaCl, 5.8 mM KCl, 0.34 mM Na2HPO4, 0.44 mM KH2PO4, 1 mM CaCl2, 1 mM MgCl2, 25 mM glucose, and 20 mM Hepes, pH 7.4. As indicated, the concentration of MgCl2 was changed from 0 to 10 mM. The reverse compartment was filled with the transport buffer without 45Ca2+, CaCl2, and MgCl2. The filter plates were shaken horizontally at a frequency of 50 oscillations/min at room temperature. To evaluate the permeability of the monolayers, the opposite compartment media were collected after indicated periods. The amount of 45Ca2+ was determined with a liquid scintillation counter.
Electrophysiological Studies with the Ussing Chamber—Cells were plated at confluent densities on transwells with polyester membrane inserts. The filter rings were detached and mounted in Ussing chambers that were incubated in the transport buffer at 37 °C. The fluid volume on each side of the filter was 4 ml. 45Ca2+ transport was measured under short-circuit conditions with an amplifier (CEZ9100, Nihon Kohden, Tokyo, Japan). The transepithelial voltage was clamped at -10, 0, or +10 mV. A time 0, the transport buffer containing 0.5 µCi/ml 45Ca2+ was added to one side. After 30 min, 1 ml of the transport solution was collected from the trans compartment for liquid scintillation counting.
Statistics—Results are presented as means ± S.E. Differences between groups were analyzed by one-way analysis of variance, and correction for multiple comparison was made using Tukey's multiple comparison test. Significant differences were assumed at p < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
TRV) and the alanine substitution mutants for threonine (ARV) or valine (TRA) did not. Our point substitution studies indicated that C-terminal threonine and valine are critical residues in the association of PCLN-1 with ZO-1.
|
TRV, ARV, and TRA mutants. These results are similar to those of the GST pull-down assay (Fig. 1) indicating that the association of PCLN-1 with ZO-1 requires the C-terminal threonine and valine residues of PCLN-1.
|
TRV, ARV, and TRA. Furthermore, these mutants were widely distributed in the lateral membrane including the TJ area, as shown by xz-scan imaging, indicating that PCLN-1 is distributed in the plasma membrane without binding to ZO-1. These results suggest that the association of PCLN-1 with ZO-1 is necessary for the concentration of PCLN-1 at TJ.
|
TRV, ARV, and TRA mutants. These results indicated that the barrier function was enhanced by the expression of wild-type and TAV mutant at TJ.
|
TRV, ARV, and TRA mutants (Fig. 5C). These results indicate that the association of PCLN-1 with ZO-1 is necessary for the functional expression of PCLN-1 protein in MDCK cells. Next, we examined the effect of extracellular magnesium concentration on 45Ca2+ transport. The addition of MgCl2 in the apical compartment led to a decrease in 45Ca2+ transport in cells expressing the wild-type PCLN-1 (Fig. 6A). However, the change in 45Ca2+ transport by the addition of MgCl2 was not observed in the mock cells. It was noted that 45Ca2+ transport from basal to apical compartments was also unaffected by MgCl2. These results indicated that the PCLN-1·ZO-1 complex competitively transports calcium and magnesium from apical to basal compartments.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
The properties of claudin-1, -2, -4, -8, and -15 have been well characterized in the over-expression model of MDCK cells. Claudin-1 increases TER, and the flux of small solutes such as mannitol and fluorescein isothiocyanate-dextran (18). Claudin-4 decreases paracellular conductance resulting from a selective decrease in Na+ permeability (22). Claudin-8 increases TER via the reduction of paracellular permeability to monovalent inorganic and organic cations and divalent cations (16). In contrast, claudin-2 increases paracellular cation conductance without changing mannitol flux (23). Our present results showed for the first time that the PCLN-1·ZO-1 complex increases TER and the paracellular permeability of divalent cations. Thus, the effects of claudins on TER and paracellular ion selectivity are type-specific. Why did PCLN-1 expression cause increased divalent cation transport despite an increase in TER in MDCK cells? We hypothesize that PCLN-1 expression increases protein density in restricted TJ areas, because it did not change the expression level of endogenous occludin, and PCLN-1 constructs a pore that is selectively permeable to divalent cations. In the mock cells and cells expressing wild-type PCLN-1, the removal of Na+ or Cl- from the bathing media increased TER, but Mg2+ removal did not change TER (data not shown). Similarly, paradoxical increases in TER and the paracellular flux of mannitol and dextran were observed in MDCK cells expressing occludin (24) and claudin-1 (18).
PCLN-1 did not affect endogenous ZO-1 levels, consistent with previous reports on claudin-1 (18) and claudin-4 expression (22). The deletion of the PDZ-binding motif (YV) at the C terminus of claudin-1–8 was enough to inhibit the association of claudins with ZO-1, but the mutants could be still distributed at the TJ (9). On the contrary, PCLN-1 mutants with the substitution of Thr for Arg at the C terminus led to lysosomal mistargeting (21). We found that PCLN-1 mutants can be incorporated in TJ to some extent without binding to ZO-1 (Fig. 3) but could not record lysosomal mistargeting, because the mutants were widely distributed in the lateral membrane. The mutants unbound to ZO-1 did not increase both TER and divalent cation permeability. We suggest that ZO-1 is arranged by the dissociation of other complexes and concentrates PCLN-1 at the TJ. However, it is difficult to prove this hypothesis, because the stoichiometry of occludin and claudins in a single TJ fibril and their oligomerization into heteromeric and heterotypic assemblies are still unknown.
The reabsorption of magnesium and calcium in TAL occurs passively via the paracellular pathway under the apical-positive transepithelial potential difference (25, 26). Our present results indicate that calcium can be transported across monolayers of MDCK cells grown on permeable filters. PCLN-1 increased calcium transport from apical to basal compartments without affecting transport from basal to apical compartments. Furthermore, the apical-positive potential gradient enhanced calcium transport from apical to basal compartments. Interestingly, magnesium inhibited calcium transport in a dose-dependent manner, suggesting that PCLN-1 recognizes divalent positive charges. Negative residues on its extracellular loops are suggested to form paracellular channels selective for divalent cations (4). In the case of claudin-4, substitution of a negative charge for a positive one at position 65 in the first extracellular loop increases paracellular Na+ permeability (27). On the contrary, substitutions of positive for negative charges at three positions in the first extracellular loop of claudin-15 increase Cl- permeability. We hypothesize that the first extracellular loop of PCLN-1 looks toward the apical side and PCLN-1 can recognize divalent cations in the apical compartment. In addition, the elevation of divalent cations in the basal compartment may inhibit calcium transport via PCLN-1. The extracellular calcium/polyvalent cation-sensing protein is expressed in the basal membrane of epithelial cells in the proximal tubule, TAL, and distal tubule (28). It has been reported that the elevation of either basal calcium or magnesium decreases the reabsorption of calcium and magnesium in TAL (29). Further studies are required to clarify the residues involved in the recognition of divalent cations in PCLN-1.
In conclusion, the expression of the wild-type and TAV mutant of PCLN-1 increased TER and divalent cation permeability in accordance with the association between PCLN-1 and ZO-1, indicating that both Thr and Val residues at the C terminus of PCLN-1 are necessary for constructing the protein complex of functional TJ. Calcium transport from apical to basal compartments was regulated by the transepithelial potential gradient. In patients with FHHNC, mutations of PCLN-1 cause renal magnesium wasting, resulting in the development of nephrocalcinosis. The PCLN-1·ZO-1 complex at TJ may be essential for the reabsorption of divalent cations in renal epithelial cells.
|
FOOTNOTES |
|---|
To whom correspondence should be addressed: Dept. of Environmental Biochemistry and Toxicology, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Shizuoka 422-8526, Japan. Tel.: 81-54-264-5674; Fax: 81-54-264-5672; E-mail: ikari{at}u-shizuoka-ken.ac.jp.
1 The abbreviations used are: TAL, thick ascending limb; TJ, tight junction; PCLN-1, paracellin-1; SH, Src homology; GST, glutathione S-transferase; TER, transepithelial electrical resistance; MDCK, Madin-Darby canine kidney; FHHNC, familial hypomagnesemia with hypercalciuria and nephrocalcinosis.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Angelow, R. Ahlstrom, and A. S. L. Yu Biology of claudins Am J Physiol Renal Physiol, October 1, 2008; 295(4): F867 - F876. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Thumfart, S. Jung, S. Amasheh, S. Kramer, H. Peters, K. Sommer, J. Biber, H. Murer, I. Meij, U. Querfeld, et al. Magnesium stimulates renal phosphate reabsorption Am J Physiol Renal Physiol, October 1, 2008; 295(4): F1126 - F1133. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Alexander, J. G. Hoenderop, and R. J. Bindels Molecular Determinants of Magnesium Homeostasis: Insights from Human Disease J. Am. Soc. Nephrol., August 1, 2008; 19(8): 1451 - 1458. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Fujita, K. Sugimoto, S. Inatomi, T. Maeda, M. Osanai, Y. Uchiyama, Y. Yamamoto, T. Wada, T. Kojima, H. Yokozaki, et al. Tight Junction Proteins Claudin-2 and -12 Are Critical for Vitamin D-dependent Ca2+ Absorption between Enterocytes Mol. Biol. Cell, May 1, 2008; 19(5): 1912 - 1921. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Angelow, R. El-Husseini, S. A. Kanzawa, and A. S. L. Yu Renal localization and function of the tight junction protein, claudin-19 Am J Physiol Renal Physiol, July 1, 2007; 293(1): F166 - F177. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-T. Chang, C.-C. Hung, Y.-C. Tian, C.-W. Yang, and M.-S. Wu Ciclosporin reduces paracellin-1 expression and magnesium transport in thick ascending limb cells Nephrol. Dial. Transplant., April 1, 2007; 22(4): 1033 - 1040. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Charoenphandhu, K. Tudpor, N. Pulsook, and N. Krishnamra Chronic metabolic acidosis stimulated transcellular and solvent drag-induced calcium transport in the duodenum of female rats Am J Physiol Gastrointest Liver Physiol, September 1, 2006; 291(3): G446 - G455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. A. Appleton, Y. Zhang, P. Wu, J. P. Yin, W. Hunziker, N. J. Skelton, S. S. Sidhu, and C. Wiesmann Comparative Structural Analysis of the Erbin PDZ Domain and the First PDZ Domain of ZO-1: INSIGHTS INTO DETERMINANTS OF PDZ DOMAIN SPECIFICITY J. Biol. Chem., August 4, 2006; 281(31): 22312 - 22320. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Zhang, S. Yeh, B. A. Appleton, H. A. Held, P. J. Kausalya, D. C. Y. Phua, W. Lee Wong, L. A. Lasky, C. Wiesmann, W. Hunziker, et al. Convergent and Divergent Ligand Specificity among PDZ Domains of the LAP and Zonula Occludens (ZO) Families J. Biol. Chem., August 4, 2006; 281(31): 22299 - 22311. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Muller, P. J. Kausalya, D. Bockenhauer, J. Thumfart, I. C. Meij, M. J. Dillon, W. v. Hoff, and W. Hunziker Unusual Clinical Presentation and Possible Rescue of a Novel Claudin-16 Mutation J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3076 - 3079. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Ikari, S. Matsumoto, H. Harada, K. Takagi, H. Hayashi, Y. Suzuki, M. Degawa, and M. Miwa Phosphorylation of paracellin-1 at Ser217 by protein kinase A is essential for localization in tight junctions J. Cell Sci., May 1, 2006; 119(9): 1781 - 1789. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Muller, P. J. Kausalya, I. C. Meij, and W. Hunziker Familial hypomagnesemia with hypercalciuria and nephrocalcinosis: blocking endocytosis restores surface expression of a novel Claudin-16 mutant that lacks the entire C-terminal cytosolic tail Hum. Mol. Genet., April 1, 2006; 15(7): 1049 - 1058. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. M. T. Groenestege, J. G. Hoenderop, L. van den Heuvel, N. Knoers, and R. J. Bindels The Epithelial Mg2+ Channel Transient Receptor Potential Melastatin 6 Is Regulated by Dietary Mg2+ Content and Estrogens J. Am. Soc. Nephrol., April 1, 2006; 17(4): 1035 - 1043. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. F. Balkovetz Claudins at the gate: determinants of renal epithelial tight junction paracellular permeability Am J Physiol Renal Physiol, March 1, 2006; 290(3): F572 - F579. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. B. N. Lee, E. Huang, and H. J. Ward Tight junction biology and kidney dysfunction Am J Physiol Renal Physiol, January 1, 2006; 290(1): F20 - F34. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||
)-expressing cells were cultured on transwell inserts for 7 days. 45Ca2+ was added to apical (A) or basal (B) compartments, and then the contralateral compartment was collected at indicated periods. The calcium concentration of the transport buffer was 1 mM in the 45Ca2+-added side and nominally 0 mM in the opposite side, respectively. C, mock, wild-type, and mutants of PCLN-1 expressing cells were cultured for 7 days. 45Ca2+ transport was estimated 60 min after adding 45Ca2+. The value of 45Ca2+ transport from basal to apical compartments was subtracted from all values from apical to basal compartments. n = 4–5. **, significantly different from mock cells (p < 0.01).
