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Originally published In Press as doi:10.1074/jbc.M402829200 on April 1, 2004

J. Biol. Chem., Vol. 279, Issue 24, 25582-25589, June 11, 2004
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The N-terminal Extracellular Domain Is Required for Polycystin-1-dependent Channel Activity*

Victor Babich{ddagger}§, Wei-Zhong Zeng{ddagger}§, Byung-Il Yeh{ddagger}, Oxana Ibraghimov-Beskrovnaya¶, Yiqiang Cai||, Stefan Somlo||, and Chou-Long Huang{ddagger}**

From the {ddagger}Division of Nephrology, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, Texas 75390, Genomics and Genetics, Genzyme Corp. Framingham, Massachusetts 01701, and the ||Section of Nephrology, Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut 06519

Received for publication, March 12, 2004 , and in revised form, April 1, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autosomal dominant polycystic kidney disease (PKD) is caused by mutation of polycystin-1 or polycystin-2. Polycystin-2 is a Ca2+-permeable cation channel. Polycystin-1 is an integral membrane protein of less defined function. The N-terminal extracellular region of polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now report that expression of polycystin-1 alone in Chinese hamster ovary (CHO) cells and in PKD2-null cells can confer Ca2+-permeable non-selective cation currents. Co-expression of a loss-of-function mutant of polycystin-2 in CHO cells does not reduce polycystin-1-dependent channel activity. A polycystin-1 mutant lacking ~2900 amino acids of the extracellular region is targeted to the cell surface but does not produce current. Extracellular application of antibodies against the immunoglobulin-like PKD domains reduces polycystin-1-dependent current. These results support the hypothesis that polycystin-1 is a surface membrane receptor that transduces the signal via changes in ionic currents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Autosomal dominant (AD) polycystic kidney disease (PKD)1 is characterized by progressive enlargement of fluid-filled cysts in kidney and other tissues such as liver and pancreas, leading to loss-of-function in the kidneys and occasional mass effects in the liver (1). ADPKD is caused by mutations in one of the two genes, PKD1 and PKD2, which are responsible for ~85 and ~15% of cases, respectively (2, 3). Elucidation of function of polycystin-1 and polycystin-2, encoded by PKD1 and PKD2, respectively, is critical for understanding how mutations in these genes cause cyst formation.

Polycystin-1 is a large protein consisting of 4,302 amino acids (4). The predicted structure of polycystin-1 includes a large N-terminal extracellular region (~3,109 amino acids), eleven predicted transmembrane (TM) domains (~993 amino acids), and a small C-terminal cytoplasmic tail (~200 amino acids) (4, 5). The N-terminal extracellular portion contains two leucinerich repeats, a C-type lectin domain, 16 copies of unique immunoglobulin (Ig)-like PKD domains, an LDL-A-related motif, and a region of homology with a sea urchin receptor for egg jelly (suREJ) (5). The extracellular leucine-rich repeat, C-type lectin, Ig-like PKD domains, and LDL-A-related motif are potential sites for protein-protein and protein-carbohydrate interactions (5). The area of homology of polycystin-1 with the suREJ protein extends over ~1,000 amino acids from the last Ig-like PKD domain to the first TM domain (5). The suREJ protein is located on the sperm head and is involved in the influx of Ca2+ ions from the extracellular space and triggering of the acrosome reaction (6).

Polycystin-2 is a 968 amino acid protein with six predicted membrane-spanning domains. The region of six TM domains of polycystin-2 has significant sequence homology with the voltage-gated Ca2+ and Na+ channels, and transient receptor potential (TRP) channels (3, 5, 7). The TM region of polycystin-2 also share ~50% sequence homology with the last 6 TM domains of polycystin-1 (3, 5). Several studies have reported that polycystin-2 may function as a Ca2+-permeable non-selective channel in the surface or intracellular membrane depending on experimental systems (8–11). Recent data show that polycystin-2 is expressed on the cilia of renal tubular cells, and it has been suggested that this may be a site of its surface channel activity (12–14).

The function of polycystin-1 is less defined. Polycystin-1 is believed to participate in cell-cell/matrix interaction, regulation of cell proliferation, apoptosis, and cation transport, and G protein-coupled signaling (6, 7, 15–19). However, the precise function of polycystin-1 in normal and cystic state remains elusive. Mutations in PKD1 and PKD2 cause virtually indistinguishable clinical presentations, suggesting that polycystin-1 and -2 function in the same pathway. The near identity of cystic phenotypes in mouse knockouts of Pkd1 (20) and Pkd2 (21) as well as studies of the Caenorhabditis elegans orthologs of both proteins (22) further support this hypothesis. The existence of recognized motifs for protein-protein and protein-carbohydrate interaction suggests that the extracellular domain of polycystin-1 may be involved in sensing the environment, a hypothesis that is again supported by studies of the C. elegans orthologs (22).

The homology between polycystin-2 and the last 6 TM domains of polycystin-1 raises the possibility that function of polycystin-1 may also involve ion channel activity. Indeed, it has been suggested that polycystin-1 and -2 interact to form channels in the cell surface (11). In the present study, we investigate whether polycystin-1 can produce channel activity independent of polycystin-2 and the role of the extracellular domain of polycystin-1 in the channel activity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Molecular Biology—CHO-K1 clone (from ATCC) were cultured in F12-K medium (Invitrogen) containing 10% fetal calf serum. PKD2-/- cells, isolated as previously described (23), were grown in a culture medium containing Dulbecco's modified Eagle's medium/Ham's F-12 supplemented with 2% fetal bovine serum, insulin (8.3 x 10-7 M), prostaglandin E1 (7.1 x 10-8 M), selenium (6.8 x 10-9 M), transferrin (6.2 x 10-8 M), triiodothyronine (2 x 10-9 M), dexamethasone (5.09 x 10-8 M), and recombinant {gamma}-interferon (10 units/ml, Sigma) at the permissive temperature (33 °C).

Cells (at ~50% confluence) were transfected with combinations (as indicated individually) of cDNA for pEGFP (0.5 µg), CD4 (0.1 µg), PKD1 (2 µg), and PKD2 (1 µg). Expression constructs for full-length PKD1 (24), Nhe-{delta} mutant of polycystin-1 (deletion of amino acids 290–2960) (25), full-length wild-type PKD2 (26) and D511V-PKD2 mutant (10) have been described. The expression construct for N-terminal GFP-tagged polycystin-2 was generated by PCR-based molecular cloning and confirmed by direct sequencing.

Electrophysiological Recording—24 h after transfection, CHO or PKD2-/- cells were dissociated by limited trypsin treatment and kept in complete serum-containing medium at room temperature until recording. For each recording, an aliquot of cells were transferred to a new culture dish containing the initial bath solution (see below) and allowed to settle for 5–10 min. We found that only healthy cells reattached to the culture dish. Unattached cells were removed by solution changes. Whole cell currents were recorded in the ruptured whole cell configuration as previously described (27). Identical currents were recorded from undissociated cells grown on culture dish (not shown), indicating that dissociation of cells by trypsin treatment did not affect polycystin-1-dependent currents. The pipette solution contained (in mM) 135 sodium aspartate (NaAsp), 15 NaCl, 0.2 EGTA, 0.12 CaCl2, 5 glucose, and 5 HEPES (pH 7.4 adjusted by NaOH). The initial bath solution contained (in mM) 150 Na aspartate, 1 MgCl2, 1 CaCl2, 5 glucose, 5 HEPES at pH 7.4. For ion substitution studies, after establishing ruptured whole cell configuration in the initial bath solution, bath solution was changed to either (in mM) 150 NaAsp, 150 NaCl, 15 NaAsp, 15 NaCl, 50 CaCl2, or 5 CaCl2 containing 5 glucose and 5 HEPES at pH 7.4. Osmolarity was maintained by addition of mannitol. The permeability of monovalent cations relative to that of Na+, and Ca2+ relative to that of Na+ were estimated from Equation 1,

(Eq. 1)
and Equation 2,

(Eq. 2)
respectively (28). Data are shown as mean ± S.E. Statistical analysis was performed using an unpaired Student's t test.

Immunofluorescent Staining and Confocal Immunofluorescent Imaging—Transiently transfected cells (24–30 h later) were fixed (4% formalin in PBS for 10 min), permeabilized (0.1% Triton X-100 in PBS for 10 min), blocked by 5% bovine serum albumin in PBS at 37 °C for 30 min. Specimens were incubated with rabbit anti-polycystin-1 polyclonal antibodies (anti-BD3 antibody, see Ref. 23; 1:200 dilution), goat anti-polycystin-2 polyclonal antibodies (YC513, see below; 1:100 dilution), and mouse anti-CD4 monoclonal antibodies (1:400 dilution; Calbiochem) at 37 °C for 1 h, followed by Rhodamine Red-X-conjugated antirabbit, Cy2-conjugated anti-goat, and Cy5-conjugated anti-mouse (secondary polyclonal antibodies all from donkey), respectively. The goat anti-polycystin-2 antibody (YC513) was raised against a glutathione S-transferase fusion protein containing the C terminus of human polycystin-2.2 In double labeling of polycystin-2 and CD4 in Fig. 3b, rabbit polyclonal anti-polycystin-2 antibodies were used (29). Confocal fluorescent images were visualized through a Zeiss x100 objective lens using Zeiss LSM-410 laser-scanning confocal microscope. Fluorescence of Rhodamine Red, Cy2, and Cy5 were detected using excitation laser (wavelength in nm) of 568, 488, and 633 and an emission filter of 590 long-pass filter, 510–560 band-pass filter, and 670–810 band-pass filter, respectively. In Figs. 3, 4, and 6, the image of CD4 was assigned a pseudocolor of either blue, red, or green.



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  		FIG. 3.
Subcellular distribution of polycystin-1 and/or -2. Cells were transfected with cDNAs for PKD1 and CD4 (a), for PKD2 and CD4 (b), for PKD1, PKD2, and CD4 (c). Immunostaining was performed using respective antibodies (see "Experimental Procedures"). Images of CD4 were assigned pseudocolors of either blue, red, or green as indicated by the text color in each panel.

 



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  		FIG. 4.
Interaction of D511V mutant with polycystin-1 and -2, and its effect on polycystin-1-dependent current. A, cells were transfected with cDNAs for PKD1, D511V, and CD4. B, co-immunoprecipitation of D511V with GFP-tagged polycystin-2. See text for details. The identity of GFP-polycystin-2 in the Western blot was also confirmed using anti-GFP antibodies. C, effect of D511V on polycystin-1-dependent current. Cells were transfected with either PKD1 + pCDNA3 empty vector, PKD1 + PKD2, or PKD1 + D511V. In each transfection condition, 2 µg of DNA of PKD1 plus 2 µg of DNA of either pCDNA3, PKD2, or D511V were used. This is equivalent to a 1:2 molar ratio of DNA for PKD1:PKD2 (and for PKD1:D511V). cDNA for GFP (0.5 µg) was included in each transfection.

 



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  		FIG. 6.
Subcellular distribution and whole cell Na+ current of Nhe-{delta} with or without polycystin-2. Cells were transfected with cDNAs for Nhe-{delta} + CD4 (A), for Nhe-{delta} + PKD2 + CD4 (B), for Nhe-{delta} + GFP (C), and for Nhe-{delta} + PKD2 + GFP (D).

 
In Fig. 5A, PKD2-/- cells transfected with PKD1 were incubated with antibodies against Ig-like PKD domains (25) at 4 °C for 2 h (1:10 dilution in PBS containing 1 mM CaCl2 and 2 mM MgCl2). After washing three times in the same buffer to remove unbound antibodies, cells were fixed in 4% formalin and incubated with Rhodamine Red-conjugated secondary antibodies at room temperature for 1 h.



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  		FIG. 5.
Whole cell currents in PKD2-null cells transfected with PKD1. A, PKD2-/- cells were transfected with GFP alone (left) or cDNAs for PKD1 plus GFP (right). Whole cell currents were recorded from cells expressing green fluorescence. Currents (at -100 mV) were normalized to cell capacitance and shown as current density (pA/pF). * indicates p < 0.01 versus GFP. Inset shows surface membrane expression of PKD1 (white labeling) in a cell transfected with GFP plus PKD1. The cell was stained with antibodies against Ig-like PKD domains (25) without permeabilization of cell membrane (see "Experimental Procedures"). No staining was observed in cells transfected with GFP alone (not shown). B, I-V relationships for currents shown in A.

 
Co-immunoprecipitation—Transfected cells were lysed in a lysis buffer containing (in mM) 20 sodium phosphate, pH 7.2, 150 NaCl, 1 EDTA, 10% (v/v) glycerol, 5% (v/v) Triton X-100, and pre-made mixtures of protease inhibitors (Complete Mini protease inhibitor; Roche Applied Science) and incubated at 4 °C for 1 h. Lysates were centrifuged in a microcentrifuge at 14,000 x g at 4 °C for 30 min. Supernatants (800 µl) were incubated with 2 µg of rabbit anti-GFP polyclonal antibodies (Santa Cruz Biotechnology) or with rabbit anti-polycystin-2 polyclonal antibodies (29) (1:100 dilution) at 4 °C for 2 h. After that, 30 µl of protein G-agarose beads (1:1 suspension; Calbiochem) were added, and samples were further incubated overnight at 4 °C. Immunoprecipitates were washed five times with 1 ml of lysis buffer and resuspended in 40 µl of SDS-gel loading buffer. Proteins were separated by 7% SDS-PAGE and analyzed by Western blotting using anti-polycystin-2 antibodies and enhanced chemiluminescence (ECL, Amersham Biosciences).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We measured currents using ruptured whole cell patch-clamp recording in CHO cells transfected with PKD1 and/or for PKD2 (Fig. 1). We detected La3+-sensitive cation currents with almost linear current-voltage (I-V) relationship in cells transfected with PKD1 alone (Fig. 1C; mean ± S.E. currents at -100 mV and 100 mV were -410 ± 28 and 431 ± 35 pA, respectively, n = 156; p < 0.01 versus control or PKD2 alone) as well as in cells co-transfected with PKD1 and PKD2 (Fig. 1E; mean ± S.E. currents at -100 mV and 100 mV were -450 ± 35 and 476 ± 51 pA, respectively, n = 53; p < 0.01 versus control or PKD2 alone). Similar currents were not detected in control untransfected cells (Fig. 1B, mean ± S.E. currents at -100 mV and 100 mV were -33 ± 8 and 38 ± 13 pA, respectively, n = 78) or cells transfected with PKD2 alone (Fig. 1D; mean ± S.E. currents at -100 mV and 100 mV were -35 ± 11 and 40 ± 8 pA, respectively, n = 108; not significant versus control). The molecular identity of currents in PKD1-transfected cells is yet unclear (see "Discussion" below). Here, we refer to these currents as polycystin-1-dependent.



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  		FIG. 1.
Channel activity in CHO cells expressing polycystin-1 and/or polycystin-2. A, left, voltage pulse protocol (-100 mV to +100 mV in 20 mV increments); right, ruptured whole cell configuration. See "Experimental Procedures" for details. B–E, representative whole cell current and I-V relationship from control untransfected cells (B), cells transfected with PKD1 (C), with PKD2 (D), and with PKD1 and PKD2 (E), respectively. Currents were recorded from cells co-expressing green fluorescent proteins. Where indicated, 1 mM LaCl3 (La3+) was added to the bath solution. I(pA) indicates currents (I) in picoamperes (pA). Membrane potential (Vm) is shown in millivolts (mV). None of control cells (n = 78) and PKD2-transfected cells (n = 108) expressed currents above background (<20 pA La3+-sensitive currents at -100 mV). On average, ~40–80% of cells from each independent transfection with PKD1 (71 cells with currents out of 156 recordings in total 23 transfections combined) or with PKD1 plus PKD2 (22 cells with current out of 53 in total 8 transfections combined) expressed currents (>50 pA La3+-sensitive currents at -100 mV). When controlled for the amount of DNA used in transfection, the overall frequency of currents between PKD1-transfected and PKD1/PKD2-transfected cells were not significantly different (see Fig. 4C). Presumably due to difficult expression of large protein such as polycystin-1, we found that all green fluorescent cells do not express polycystin-1 in immunofluorescent staining (not shown). The percentage of cells that expressed currents roughly correlated with the percentage of cells that showed PKD1 immunoreactivity in the immunostaining experiments.

 
We further characterized the polycystin-1-dependent currents (Fig. 2). With 150 mM sodium aspartate (NaAsp) in bath and 135 mM NaAsp plus 15 mM NaCl in the pipette, the reversal potential (Erev) for polycystin-1-dependent currents was -1.4 ± 0.4 mV (n = 59). Substituting this bath solution with a solution containing 150 mM NaCl did not cause a shift in Erev (Erev, -2.3 ± 1.2 mV in 150 mM NaCl), indicating that currents were carried by cations. Consistent with this idea, lowering bath Na+ from 150 to 15 mM caused a shift in Erev by -53 ± 4 mV (n = 18) (Fig. 2A). The permeability ratio for Na+,K+,Cs+, and N-methyl-D-glucamine, measured with 150 mM Na+ in the pipette and 150 mM of individual monovalent ion in the bath, was 1: 0.98: 0.95: 0.52 (Fig. 2B). The channels in PKD1-transfected cells were also permeable to Ca2+ ions. Reducing extracellular Ca2+ concentration from 50 to 5 mM caused a shift in Erev by -27 ± 5 mV (n = 9) (Fig. 2C). This shift in Erev is consistent with that predicted from the Nernst equation for Ca2+-permeable channels. The permeability ratio for Ca2+ versus Na+ (PCa2+/PNa+) measured with 5 mM Ca2+ in the bath and 150 mM Na+ in the pipette was 3.8:1. Currents in PKD1-transfected cells were inhibited by extracellular Ca2+ (Fig. 2D). Na+ currents (mean ± S.E.) at 100 mV and -100 mV were 405 ± 30 pA and -305 ± 35 pA, respectively, with 1 mM Ca2+ in the bath and 530 ± 51 pA and -420 ± 45 pA, respectively, with 1 mM EGTA and nominal Ca2+-free solution in the bath. These characteristics of whole cell currents in PKD1-transfected cells are essentially indistinguishable from the currents observed in cells co-transfected with PKD1 and PKD2 in our studies (not shown).



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  		FIG. 2.
Characterization of currents in PKD1-transfected cells. A, shift in Erev by lowering bath NaAsp from 150 to 15 mM. B, permeability ratio of currents for different monovalent cations. C, permeability of currents for Ca2+. D, effect of bath Ca2+ on Na+ currents. Open and closed circles indicate without Ca2+ and with 1 mM Ca2+ in the bath, respectively.

 
We examined subcellular localization of polycystin-1 and/or polycystin-2 expressed in CHO cells by double or triple labeling immunofluorescent staining and imaging by a laser-scanning confocal microscope (27). Cells were co-transfected with CD4 expression plasmid to monitor distribution of expressed proteins in plasma membrane. In cells transfected with PKD1, polycystin-1 was distributed in punctate pattern intracellularly as well as in the plasma membrane (Fig. 3a, left panel). CD4 was distributed mostly in plasma membrane and to a lesser extent in intracellular location (see Fig. 3b, middle panel). The intracellular staining of CD4 likely represents proteins in the biosynthetic and/or forward trafficking pathway. Localization of polycystin-1 to the plasma membrane was confirmed by co-localization with CD4 in merged image (Fig. 3a, middle panel). As shown in the magnified image (4x), some of the polycystin-1 staining clearly reached the outermost margin of cell surface (Fig. 3a, right panel, arrowheads). The punctate distribution of polycystin-1 in cell surface and intracellular membranes is similar to several other studies in expression system and in cells expressing native proteins (30–33). The punctate distribution of native polycystin-1 may be (at least partly) because of its association with desmosomes (30–32). As reported previously for cells transfected with PKD2 (26), polycystin-2 was distributed in punctate/reticular pattern intracellularly (Fig. 3b). Polycystin-2 was not detected in the surface membrane of these cells (Fig. 3b).

In cells co-transfected with PKD1, PKD2, and CD4, polycystin-1 was also present in cell surface and intracellular membranes (Fig. 3c, panels i, iv, and vii). Though it is difficult to analyze the immunostaining results quantitatively, in multiple experiments it appeared that the abundance of polycystin-1 in the surface membrane was increased by co-expression with polycystin-2 (compare images in panels i, iv, and vii of Fig. 3c with images in the middle and right panels of Fig. 3a). Localization of polycystin-1 to surface membrane was apparent by the finding of overlapping signals of polycystin-1 and CD4 reaching all the way to the outermost margin of cell surface (Fig. 3c, panels iv and vii; also see Fig. 4A, panels i and iv).

The distribution of polycystin-2 was also altered by co-expression with polycystin-1. Compared with cells without co-transfection of PKD1 (Fig. 3b, right panel), there appeared to be more polycystin-2 distribution toward surface membrane in cells co-transfected with PKD1 (Fig. 3c, panels ii, v, and viii). The extent of polycystin-2 signals reaching the margin of cell surface is much less as compared with polycystin-1 and varies considerably between multiple experiments. As shown, polycystin-2 overlapped with CD4 (Fig. 3c, panels v and viii, yellow color) to a significantly lesser extent than polycystin-1 overlapped with CD4 (panel iv). Moreover, except for a few scattered areas in occasional images, virtually all of the overlapping signals of polycystin-2 and CD4 did not include overlapping CD4 signals in the outer rim (Fig. 3c, panels v and viii; also see Fig. 4A, panel v and Fig. 6B, panel v). This may be because of the fact that CD4 was more abundantly expressed in the plasma membrane (and thus the signal could be detected over a broader window) than polycystin-2. Alternatively, the majority of polycystin-2 staining that overlaps with CD4 might not be localized to the plasma membrane, but rather was localized to structure(s) that are right underneath and closely apposed to the plasma membrane. Also shown in panels vi and ix of Fig. 3c, polycystin-1 and -2 partially co-localized. Colocalization was more abundant in the perinuclear region but was also present near surface membrane. Such partial colocalization of polycystin-1 and -2 was also reported by other investigators (33, 34).

We investigated whether expression of polycystin-1-dependent currents in our experimental system depends on functional channel activity of the endogenous polycystin-2 in CHO cells. Mutation of Asp-511 to valine (D511V) in the third putative TM domain of polycystin-2 causes type 2 polycystic kidney disease (35). In the LLC-PK1 cell culture model, D511V mutant distributes similarly as the wild-type polycystin-2 but does not confer the vasopressin-induced increase in intracellular Ca2+ (10). We found that the D511V mutant showed similar distribution to wild-type polycystin-2 when co-expressed with polycystin-1 (compare Fig. 4A, panel vi with Fig. 3c, panel vi). D511V also enhanced surface expression of polycystin-1 (compare Fig. 4A, panel iv with Fig. 3a, middle panel). Similar to wild-type polycystin-2, overlap of D511V with CD4 (Fig. 4A, panel v) did not reach outermost margin of cell surface and was significantly less than overlap of polycystin-1 with CD4 (Fig. 4A, panel iv). D511V partially co-localized with polycystin-1 as well (Fig. 4A, panel vi). Together, these results indicate that D511V behaves similarly to wild-type polycystin-2.

To see whether D511V interacts with wild type polycystin-2, we carried out co-immunoprecipitation study in cells transfected with D511V and/or GFP-tagged wild-type PKD2 (GFP-PKD2). In cells co-transfected with D511V and GFP-PKD2, anti-GFP antibody co-precipitated D511V mutant with GFP-PKD2 (Fig. 4B, lane 3 from left). In the negative control, anti-GFP antibody did not co-precipitate D511V with GFP (lane 2). The identity of GFP-PKD2 (~140 kDa) was verified by immunoprecipitation with anti-GFP antibody from cells transfected with GFP-PKD2 alone (lane 4). The identity of D511V (~110 kDa) was verified by its immunoprecipitation with anti-PKD2 antibody from cells transfected with D511V alone (lane 1).

We next compared currents in cells co-transfected with either PKD1 plus pCDNA3 empty vector, PKD1 plus PKD2, or PKD1 plus D511V (Fig. 4C). I-V relationships of currents were not different among these cells (not shown). The average whole cell La3+-sensitive inward Na+ currents (at -100 mV) were 211 ± 25 pA, 351 ± 45 pA, 360 ± 50 pA, respectively. Assuming that endogenous polycystins in CHO cells interact in the same manner as the expressed human polycystins, lack of inhibition of currents by D511V (PKD1+PKD2 versus PKD1+D511V, not significant) suggests that functional channel activity of the endogenous polycystin-2 is not necessary for polycystin-1-dependent currents. Interestingly, co-expression of PKD2 (or D511V) with PKD1 increased currents (p = 0.05, PKD1 + pCDNA3 versus PKD1 + PKD2).

To confirm that polycystin-1 can produce currents independent of polycystin-2, we recorded currents from PKD2-null cells (23) transfected with PKD1. In the total 39 cells transfected with GFP, the mean whole cell current density at -100 mV was -5 ± 1 pA/pF (Fig. 5A, left column). None of the GFP-transfected cells had current density (at -100 mV) higher than -15 pA/pF, the maximal current density observed in the control, untransfected cells (not shown). Not every green fluorescence-positive cell co-transfected with GFP plus PKD1 expressed currents. Out of the total 41 green fluorescence-positive cells co-transfected with GFP plus PKD1, 10 expressed current density (at -100 mV) higher than -20 pA/pF. This frequency of expression of PKD1 in PKD2-null cells (~24%) is much lower than that in CHO cells (see legend to Fig. 1). For the 10 current-positive cells, the mean current density (at -100 mV) was -136 ± 26 pA/pF (p < 0.01 versus GFP-transfected control cells; Fig. 5A, right column). Cell surface expression of polycystin-1 in PKD1-transfected cells was confirmed by immunofluorescent staining of non-permeabilized cells using antibodies against the extracellular Ig-like PKD domains (Fig. 5A, Inset). I-V relationships (Fig. 5B) and ion selectivity (not shown) for these PKD1-mediated currents in PKD2-null cells were similar to those in CHO cells (Fig. 2).

A polycystin-1 mutant (Nhe-{delta}) with deletion of amino acids 290–2960 is localized to surface membrane of Sf-21 cells (25). We found that in CHO cells Nhe-{delta}) protein was targeted to plasma membrane when expressed alone (Fig. 6A, middle and right panels) or with PKD2 (Fig. 6B, panel iv). Similar to wild-type polycystin-1, Nhe-{delta} protein partially co-localized with polycystin-2 (Fig. 6B, panel vi) and increased distribution of polycystin-2 toward surface membrane (compare Fig. 6B, panel v with Fig. 3b, right panel). Thus, deletion of amino acids 290–2960 of the extracellular region of polycystin-1 did not affect its ability to interact with polycystin-2.

We used the mutant to examine the role of the extracellular domain of polycystin-1 in the regulation of polycystin-1-dependent currents. Whole cell cation currents were recorded from cells transfected with Nhe-{delta} with or without PKD2. No currents were detected in CHO cells transfected with Nhe-{delta} alone (Fig. 6C, 0 of 57 recordings) or in cells transfected with Nhe-{delta} + PKD2 (Fig. 6D, 0 out of 34 recordings). As shown in Fig. 1, cells transfected with PKD1 (29 of 51 recordings; not shown here) and co-transfected with PKD1 plus PKD2 (19 of 32 recordings, not shown here) expressed currents under the same experimental conditions.

The region of polycystin-1 deleted in the Nhe-{delta} mutant includes C-type lectin domain, LDL-A-related motif, Ig-like PKD domains, and a large part of the REJ domain (5). We further examined the role of the Ig-like PKD domains for polycystin-1-mediated currents using antibodies raised against repeats II-XVI of the Ig-like PKD domains of polycystin-1 (25). In each single experiment, CHO cells expressing polycystin-1 from the same transfection were divided into 2 groups and incubated with or without antibodies. Whole cell cation currents from 5 to 6 cells of each group were recorded and averaged. Each pair of open and closed circles connected with a solid line in Fig. 7 represents the averaged current (at -100 mV) of such 5–6 recordings from cells of the same transfection and treated without and with antibody, respectively. As shown, we found in each of five such experiments the averaged polycystin-1-dependent current was lower for cells incubated with the antibodies against Ig-like PKD domains than for cells without antibodies (Fig. 7; mean ± S.E. currents of five experiments: 397 ± 86 pA and 159 ± 34 pA without and with antibodies, respectively; p < 0.02, paired Student's t test). Incubation with control antibodies (anti-ROMK antibodies, Ref. 36) did not reduce polycystin-1-dependent currents (Fig. 7).



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  		FIG. 7.
Inhibition of polycystin-1-dependent currents by antibodies against Ig-like PKD domains. Cells were transfected with cDNA for PKD1. Left panel, cells were either incubated with antibodies against Ig-like PKD domains of polycystin-1 (25) (1:10 dilution; labeled Anti-Ig Ab) or without antibodies at 4 °C for 1 h. Incubation at 4 °C prevents endocytosis of antibodies. Open squares and closed squares with error bar represent mean ± S.E. of averaged current of five independent experiments without and with antibodies, respectively. Right panel, experimental paradigm as in the left panel, except that antibodies against ROMK channel (36) (labeled Control Ab) were used.

 
The region between the 10th and 11th TM domains of polycystin-1 corresponds to the putative pore region of polycystin-2. To examine the role of this region of polycystin-1 in channel activity, we individually mutated Asp-4053 and Glu-4078 to glycine. Both D4053G and E4078G mutants, however, were not targeted to the cell surface (not shown). Neither mutant produced currents in CHO cells (0 of 31 and 0 of 32 for D4053G and E4078G, respectively; expressed current density (at -100 mV) higher than -10 pA/pF).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The role of polycystin-1 in mediating channel activity was suggested by its homology with polycystin-2 in the transmembrane region and the presence of a REJ domain in the extracellular region. One such function of polycystin-1 is to interact with polycystin-2 to form channels (11). In the present study, we report an additional mechanism for polycystin-1 regulation of ion currents. Expression of polycystin-1 can regulate channel activity independent of the channel activity of polycystin-2 and that the extracellular region of polycystin-1 is necessary for this channel activity.

Our conclusion that polycystin-1 can regulate channel activity independent of the channel activity of polycystin-2 is based on the following findings. First, the I-V relationship of whole cell currents we detect in both PKD1-transfected and PKD1/PKD2-transfected cells is almost linear. González-Perrett et al. (8) and Koulen et al. (10) reported that the activity of polycystin-2 channels reconstituted in planar lipid bilayers is strongly voltage-dependent. The opening probability decreases sharply in positive membrane potentials. These bilayer studies predict that, if polycystin-2 solely contributes to the whole cell currents, I-V relationship of the currents would be inwardly rectifying. Alternatively, differences in lipid composition in planar lipid bilayer versus plasma membrane may give rise to different biophysical properties. Second, we found that co-expression of D511V mutant of polycystin-2 did not reduce polycystin-1-dependent currents, suggesting that functional channel activity of polycystin-2 is not necessary for the currents. Third, we found that a mutant of polycystin-1, Nhe-{delta}, was targeted to plasma membrane and partially co-localized with polycystin-2 in a manner similar to the full-length polycystin-1. Co-expression of Nhe-{delta} and polycystin-2, however, did not produce currents. Finally, similar PKD1-dependent currents were observed in PKD2-null cells.

Interestingly, we found that co-expression with polycystin-2 increased surface expression of polycystin-1 and polycystin-1-dependent currents. Polycystin-1 and -2 interact through their C termini (7, 37). One possibility for increase of polycystin-1-dependent currents by polycystin-2 in our experiments is that polycystin-1 and -2 both contribute to channel pore via formation of heteromultimeric channels. The lack of reduction of currents by D511V in our experiments argues against this possibility. Alternatively, polycystin-2 may increase surface expression and currents of polycystin-1 by stabilizing polycystin-1 on the cell surface or functioning as a chaperone for polycystin-1. This can occur irrespective of localization (plasma membrane versus subplasma membranous structures) and channel activity of polycystin-2. Our finding that both wild-type polycystin-2 and D511V mutant increase polycystin-1-dependent currents favors this possibility. If this is indeed the mechanism for polycystin-2 enhancing polycystin-1-dependent currents, it is possible that in some experimental systems the endogenous polycystin-2 in CHO cells was low so that surface polycystin-1-dependent channel activity could only be seen when co-expressed with polycystin-2 (11).

The identity of protein(s) responsible for the currents in PKD1-transfected cells remains elusive. Polycystin-1 may form ion channel pore by itself or activate endogenous channels with or without direct contribution to formation of the channel pore. Our attempts to examine whether polycystin-1 is a pore-forming protein were hampered by the fact that mutations of putative pore residues of polycystin-1 prevented cell surface expression (see "Results"). Vandorpe et al. (15) reported that expression of membrane-anchored C-terminal intracellular fragment of PKD1 in human embryonic kidney (HEK) cells activates endogenous cation currents with a single-channel conductance of ~20 picosiemens in Xenopus oocytes and in HEK cells. We found that Nhe-{delta} mutant containing the complete C-terminal fragment of polycystin-1, despite its targeting to surface membrane, did not produce currents in CHO cells. Delmas et al. (19) reported that expression of full-length mouse polycystin-1 activates endogenous voltage-activated Ca2+ channels and G protein-activated inward rectifying K+ channels in sympathetic neurons via release of {beta}{gamma} subunits from pertussis toxin-sensitive Gi/o-type G proteins. The polycystin-1-dependent currents in our study are distinct from the above Ca2+ and K+ currents and are not sensitive to pertussis toxin (not shown). Further studies are required to identify protein(s) responsible for the PKD1-dependent currents.

Several recent findings provide strong evidence that cilia are an important site of polycystin function. The C. elegans homologs of polycystin-1 and -2 are localized to the sensory cilia of nematodes (22). Polycystin-1 and -2 have also been localized to the primary cilia of kidney epithelial cells (12, 13). Bending of the cilia in cultured epithelial cells by flow causes calcium influx (39). Nauli et al. (14) recently reported that the calcium response to bending is abolished in cells lacking cilia, in cells lacking PKD1, or in cells treated with a blocking antibody to PKD2. These experiments provide strong support that PKD1 and PKD2 are involved in calcium influx activated by flow-induced bending of the apical cilium. Defects in fluid flow sensation by cilia and Ca2+ influx likely play pivotal roles in cyst formation in kidney and other organs in polycystic kidney diseases.

What is the potential role of polycystin-1-dependent channel activity independent of polycystin-2? Polycystin-1 is also expressed in the basolateral membrane of epithelial cells and likely play important roles in cell-cell and cell-matrix interactions (32). We found that the extracellular region of polycystin-1 is critical for its function of regulating ion currents across the plasma membrane. This region of polycystin-1 contains many potential domains for cell-cell and cell-matrix interactions. Among these are the 16 repeats of Ig-like PKD domains. Small peptides from PKD domains interfere with branching morphogenesis of the ureteric bud (38). Repeats II-XVI of PKD domains of polycystin-1 are capable of forming strong homophilic interactions, possibly mediating homodimerization and/or heterodimerization between two molecules from contacting cells (25). Extracellular application of antibodies against these repeats of PKD domains of polycystin-1 perturbs cell-cell adhesion between MDCK cells (25). Our results that application of anti-PKD domain antibodies reduces polycystin-1-dependent currents support the idea that polycystin-1 is involved in cell-cell interactions and that alteration of ion currents is one of the downstream signals for cell-cell interactions. It is well accepted that polycystin-2 is present on the surface membrane of cilia and likely mediates Ca2+ entry at this site. However, whether polycystin-2 is present on the surface of the basolateral membrane remains debatable. If polycystin-2 is indeed not present on the surface of basolateral membrane, we suggest that the polycystin-2-independent channel activity associated with polycystin-1 may play an important role at this site. Thus, extracellular domain of polycystin-1 may be involved in cell interactions with neighboring cells and basement membrane. These interactions may cause alterations of intracellular ion concentration and/or currents. Changes in intracellular ion concentration and/or currents, likely acting in concert with many other signaling pathways activated by the intracellular domain of polycystin-1 (17, 18, 40–43), control renal epithelial cell growth, and promote normal tubulogenesis.


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health, American Heart Association, Polycystic Kidney Research Foundation, and Kidney Texas Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ These authors contributed equally to this work and are listed in alphabetic order. Back

** To whom correspondence should be addressed: Dept. of Medicine, UT Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-8856. Tel.: 214-648-8627; Fax: 214-648-2071; E-mail: chou-long.huang{at}UTSouthwestern.edu.

1 The abbreviations used are: PKD, polycystic kidney disease; PKD1, type-1 PKD; PKD2, type-2 PKD; TM, transmembrane; REJ, receptor for egg jelly; LDL, low density lipoprotein; CHO, Chinese hamster ovary; GFP, green fluorescent protein; Erev, reversal potential; PBS, phosphate-buffered saline; pF, picoFarad. Back

2 Y. Cai and S. Somlo, unpublished results. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Peter Igarashi for critical reading of an earlier version of the manuscript, Dr. Moshe Levi for sharing the confocal microscope, and Xinji Li for assistance with cell culture.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Gabow, P. A. (1993) N. Eng. J. Med. 329, 332-342[Free Full Text]
  2. The European Polycystic Kidney Disease Consortium (1994) Cell 77, 881-894[CrossRef][Medline] [Order article via Infotrieve]
  3. Mochizuki, T., Wu, G., Hayashi, T., Xenophontos, S. L., Veldhuisen, B., Saris, J. J., Reynolds, D. M., Cai, Y., Gabow, P. A., Pierides, A., Kimberling, W. J., Breuning, M. H., Deltas, C. C., Peters, D. J., and Somlo, S. (1996) Science 272, 1339-1342[Abstract]
  4. Hughes, J., Ward, C. J., Peral, B., Aspinwall, R., Clark, K., San Millan, J. L., Gamble, V., and Harris, P. C. (1995) Nat. Genet. 10, 151-160[Medline] [Order article via Infotrieve]
  5. Sandford, R., Sgotto, B., Aparicio, S., Brenner, S., Vaudin, M., Wilson, R. K., Chissoe, S., Pepin, K., Bateman, A., Chothia, C., Hughes, J., and Harris, P. (1997) Human Mol. Genet. 6, 1483-1489[Abstract/Free Full Text]
  6. Moy, G. W., Mendoza, L. M., Schulz, J. R., Swanson, W. J., Glabe, C. G., and Vacquier, V. D. (1996) J. Cell Biol. 133, 809-817[Abstract/Free Full Text]
  7. Tsiokas, L., Arnould, T., Zhu, C., Kim, E., Walz, G., and Sukhatme, V. P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 3934-3939[Abstract/Free Full Text]
  8. Gonzalez-Perrett, S., Kim, K., Ibarra, C., Damiano, A. E., Zotta, E., Batelli, M., Harris, P. C., Reisin, I. L., Arnaout, M. A., and Cantiello, H. F. (2000) Proc. Natl. Acad. Sci. U. S. A. 98, 1182-1187
  9. Vassilev, P. M., Guo, L., Chen, X. Z., Segal, Y., Peng, J. B., Basora, N., Babakhanlou, H., Cruger, G., Kanazirska, M., Ye, C. P., Brown, E. M., Hediger, M. A., and Zhou, J. (2001) Biochem. Biophys. Res. Commun. 282, 341-350[CrossRef][Medline] [Order article via Infotrieve]
  10. Koulen, P., Cai, Y., Geng, L., Maeda, Y., Nishimura, S., Witzgall, R., Ehrlich, B. E., and Somlo, S. (2002) Nat. Cell Biol. 4, 191-197[CrossRef][Medline] [Order article via Infotrieve]
  11. Hanaoka, K., Qian, F., Boletta, A., Bhunia, A. K., Piontek, K., Tsiokas, L., Sukhatme, V. P., Guggino, W. B., and Germino, G. G. (2000) Nature 408, 990-994[CrossRef][Medline] [Order article via Infotrieve]
  12. Pazour, G. J., San Agustin, J. T., Follit, J. A., Rosenbaum, J. L., and Witman, G. B. (2002) Curr. Biol. 12, R378-R380[CrossRef][Medline] [Order article via Infotrieve]
  13. Yoder, B. K., Hou, X., and Guay-Woodford, L. M. (2002) J. Am. Soc. Nephrol. 13, 2508-2516[Abstract/Free Full Text]
  14. Nauli, S. M., Alenghat, F. J., Luo, Y., Williams, E., Vassilev, P., Li, X., Elia, A. E., Lu, W., Brown, E. M., Quinn, S. J., Ingber, D. E., and Zhou, J. (2003) Nat. Genet. 33, 129-137[CrossRef][Medline] [Order article via Infotrieve]
  15. Vandorpe, D. H., Chernova, M. N., Jiang, L., Sellin, L. K., Wilhelm, S., Stuart-Tilley, A. K., Walz, G., and Alper, S. L. (2001) J. Biol. Chem. 276, 4093-4101[Abstract/Free Full Text]
  16. Boletta, A., Qian, F., Onuchic, L. F., Bhunia, A. K., Phakdeekitcharoen, B., Hanaoka, K., Guggino, W. B., Monaco, L., and Germino, G. G. (2001) Mol. Cell 6, 267-273
  17. Bhunia, A. K., Piontek, K., Boletta, A., Liu, L., Qian, F., Xu, P. N., Germino, F. J., and Germino, G. G. (2002) Cell 109, 157-168[CrossRef][Medline] [Order article via Infotrieve]
  18. Parnell, S. C., Magenheimer, B. S., Maser, R. L., Zien, C. A., Frischauf, A. M., and Calvet, J. P. (2002) J. Biol. Chem. 277, 19566-19572[Abstract/Free Full Text]
  19. Delmas, P., Nomura, H., Li, X., Lakkis, M., Luo, Y., Segal, Y., Fernandez-Fernandez, J. M., Harris, P., Frischauf, A. M., Brown, D. A., and Zhou, J. (2002) J. Biol. Chem. 277, 11276-11283[Abstract/Free Full Text]
  20. Lu, W., Peissel, B., Babakhanlou, H., Pavlova, A., Geng, L., Fan, X., Larson, C., Brent, G., and Zhou, J. (1997) Nat. Genet. 17, 179-181[CrossRef][Medline] [Order article via Infotrieve]
  21. Wu, G., Markowitz, G. S., Li, L., D'Agati, V. D., Factor, S. M., Geng, L., Tibara, S., Tuchman, J., Cai, Y., Park, J. H., van Adelsberg, J., Hou, H. Jr., Kucherlapati, R., Edelmann, W., and Somlo, S. (2000) Nat. Genet. 24, 75-78[CrossRef][Medline] [Order article via Infotrieve]
  22. Barr, M. M., DeModena, J., Braun, D., Nguyen, C. Q., Hall, D. H., and Sternberg, P. W. (2001) Curr. Biol. 11, 1341-1346[CrossRef][Medline] [Order article via Infotrieve]
  23. Grimm, D. H., Cai, Y., Chauvet, V., Rajendran, V., Zeltner, R., Geng, L., Avenor, E. D., Sweeney, W., Somlo, S., and Caplan, M. J. (2003) J. Biol. Chem. 278, 36786-36793[Abstract/Free Full Text]
  24. Ibraghimov-Beskrovnaya, O., Dackowski, W. R., Foggensteiner, L., Coleman, N., Thiru, S., Petry, L. R., Burn, T. C., Connors, T. D., Van Raay, T., Bradley, J., Qian, F., Onuchic, L. F., Watnick, T. J., Piontek, K., Hakim, R. M., Landes, G. M., Germino, G. G., Sandford, R., and Klinger, K. W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6397-6402[Abstract/Free Full Text]
  25. Ibraghimov-Beskrovnaya, O., Bukanov, N. O., Donohue, L. C., Dackowski, W. R., Klinger, K. W., and Landes, G. M. (2000) Human Mol. Genet. 9, 1641-1649[Abstract/Free Full Text]
  26. Cai, Y., Maeda, Y., Cedzich, A., Torres, V. E., Wu, G., Hayashi, T., Mochizuki, T., Park, J. H., Witzgall, R., and Somlo, S. (1999) J. Biol. Chem. 274, 28557-28565[Abstract/Free Full Text]
  27. Zeng, W. Z., Babich, V., Ortega, B., Quigley, R., White, S. J., Welling, P. A., and Huang, C. L. (2002) Am. J. Physiol. 283, F630-F639
  28. Hille, B. (2001) Ion Channels of Excitable Membranes, Sinauer Associates, Sunderland, Massachusetts
  29. Foggensteiner, L., Bevan, A. P., Thomas, R., Coleman, N., Boulter, C., Bradley, J., Ibraghimov-Beskrovnaya, O., Klinger, K., and Sandford, R. (2000) J. Am. Soc. Nephrol. 11, 814-827[Abstract/Free Full Text]
  30. Scheffers, M. S., van der Bent, P., Prins, F., Spruit, L., Breuning, M. H., Litvinov, S. V., de Heer, E., and Peters, D. J. (2000) Human Mol. Genet. 9, 2743-2750[Abstract/Free Full Text]
  31. Xu, G. M., Sikaneta, T., Sullivan, B. M., Zhang, Q., Andreucci, M., Stehle, T., Drummond, I., and Arnaout, M. A. (2001) J. Biol. Chem. 276, 46544-46552[Abstract/Free Full Text]
  32. Bukanov, N. O., Husson, H., Dackowski, W. R., Lawrence, B. D., Clow, P. A., Roberts, B. L., Klinger, K. W., and Ibraghimov-Beskrovnaya, O. (2002) Human Mol. Genet. 11, 923-936[Abstract/Free Full Text]
  33. Newby, L. J., Streets, A. J., Zhao, Y., Harris, P. C., Ward, C. J., and Ong, A. C. (2002) J. Biol. Chem. 277, 20763-20773[Abstract/Free Full Text]
  34. Scheffers, M. S., Le, H., van der Bent, P., Leonhard, W., Prins, F., Spruit, L., Breuning, M. H., de Heer, E., and Peters, D. J. (2002) Human Mol. Genet. 11, 59-67[Abstract/Free Full Text]
  35. Reynolds, D. M., Hayashi, T., Cai, Y., Veldhuisen, B., Watnick, T. J., Lens, X. M., Mochizuki, T., Qian, F., Maeda, Y., Li, L., Fossdal, R., Coto, E., Wu, G., Breuning, M. H., Germino, G. G., Peters, D. J., and Somlo, S. (1999) J. Am. Soc. Nephrol. 10, 2342-2351[Abstract/Free Full Text]
  36. Huang, C. L., Feng, S., and Hilgemann, D. W. (1998) Nature 391, 803-806[CrossRef][Medline] [Order article via Infotrieve]
  37. Qian, F., Germino, F. J., Cai, Y., Zhang, X., Somlo, S., and Germino, G. G. (1997) Nat. Genet. 16, 179-183[CrossRef][Medline] [Order article via Infotrieve]
  38. van Adelsberg, J. (1999) Dev. Genet. 24, 299-308[CrossRef][Medline] [Order article via Infotrieve]
  39. Praetorius, H. A., and Spring, K. R. (2003) Curr. Opin. Nephrol. Hypertens. 12, 517-520[Medline] [Order article via Infotrieve]
  40. Kim, E., Arnould, T., Sellin, L., Benzing, T., Comella, N., Kocher, O., Tsiokas, L., Sukhatme, V. P., and Walz, G. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 6371-6376[Abstract/Free Full Text]
  41. Kim, E., Arnould, T., Sellin, L. K., Benzing, T., Fan, M. J., Gruning, W., Sokol, S. Y., Drummond, I., and Walz, G. (1999) J. Biol. Chem. 274, 4947-4953[Abstract/Free Full Text]
  42. Lehtonen, S., Ora, A., Olkkonen, V. M., Geng, L., Zerial, M., Somlo, S., and Lehtonen, E. (2000) J. Biol. Chem. 275, 32888-32893[Abstract/Free Full Text]
  43. Geng, L., Burrow, C. R., Li, H. P., and Wilson, P. D. (2000) Biochim. Biophys. Acta 535, 21-35

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