the amino-terminal extracellular domain is required for polycystin-1
TRANSCRIPT
The amino-terminal extracellular domain is required for polycystin-1-
dependent channel activity
Victor Babich*,¶, Wei-Zhong Zeng*,¶, Byung-Il Yeh*, Oxana Ibraghimov-Beskrovnaya†,
Yiqiang Cai‡, Stefan Somlo‡, and Chou-Long Huang*
*Department of Internal Medicine (Division of Nephrology), The University of Texas
Southwestern Medical Center, Dallas, TX 75390;†Genomics and Genetics, Genzyme Corp.
Framingham, MA 01701; Departments of ‡Internal Medicine (Section of Nephrology), Yale
University School of Medicine, New Haven, CT 06519.
¶These authors contributed equally to this work and are listed according to alphabetic order.
Address correspondence to:
Chou-Long Huang, M.D., Ph.D.
Department of Medicine
UT Southwestern Medical Center
5323 Harry Hines Blvd
Dallas, TX 75390-8856
Tel: 214-648-8627; Fax: 214-648-2071
Email:[email protected]
JBC Papers in Press. Published on April 1, 2004 as Manuscript M402829200
Copyright 2004 by The American Society for Biochemistry and Molecular Biology, Inc.
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Running Title: Polycystin-1-dependent channel activity
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SUMMARY
Autosomal dominant polycystic kidney disease 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 amino-terminal extracellular region of
polycystin-1 contains potential motifs for protein and carbohydrate interaction. We now
report that expression of polycystin-1 alone in CHO cells and in PKD2 null cells can confer
Ca2+-permeable nonselective 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.
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INTRODUCTION
Autosomal dominant polycystic kidney disease (ADPKD)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 due to
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 4302 amino acids (4). The predicted structure
of polycystin-1 includes a large N-terminal extracellular region (~3109 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 leucine-rich
repeats (LRR), a C-type lectin domain, 16 copies of unique immunoglobulin (Ig)-like PKD
domains, a LDL-A related motif, and a region of homology with a sea urchin receptor for egg
jelly (suREJ) (5). The extracellular LRR, 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 transmembrane (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
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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 has shown 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 channels 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 C. elegans orthologues 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 orthologues (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.
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EXPERIMENTAL PROCEDURES
Cell Culture and Molecular Biology- CHO-K1 clone (from ATCC) were cultured in F12-
K medium (Gibco) containing 10% fetal calf serum. PKD2-/- cells, isolated as previously
described (23), were grown in a culture medium containing DMEM/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 -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- 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. Expression construct for N-terminal GFP-tagged polycystin-2 was generated by PCR-
based molecular cloning and confirmed by direct sequencing.
Electrophysiological Recording- Twenty-four hrs 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
mins. 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
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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 Na-
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 the equation PX+/PNa+
= exp(ErevF/RT)([Na+]i/[X+]o) and PCa2+/PNa+ = ([Na+]i/4[Ca2+]o)exp(ErevF/RT) {(1+exp(ErevF/RT)},
respectively (28). Data were shown as mean ± SEM. Statistical analysis was performed using un-
paired t-test.
Immunofluorescent Staining and Con-focal Immunofluorescent Imaging- Transiently
transfected cells (24-30 hrs later) were fixed (4% formalin in PBS for 10 mins), permeabilized
(0.1 % Triton-X 100 in PBS for 10 mins), blocked by 5% BSA in PBS at 37o C for 30 mins.
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 37o C for 1 hr, followed by Rhodamine Red-X conjugated anti-rabbit, 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 GST-fusion
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protein containing the C-terminus of human polycystin-2 (Cai and Somlo, unpublished results). 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 100 X
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 emission filter of 590 long-pass filter, 510-560 band-pass filter, and 670-810
band-pass filter, respectively. In Fig. 3, 4, and 6, image of CD4 was assigned pseudo-color of
either blue, red or green.
In Fig. 5A, PKD2-/- cells transfected with PKD1 were incubated with antibodies against Ig-
like PKD domains (25) at 4o C for 2 hrs (1:10 dilution in PBS containing 1 mM CaCl2 and 2 mM
MgCl2). After washing 3 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 hr.
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) TritonX-
100, and pre-made mixtures of protease inhibitors (Complete Mini protease inhibitor; Roche) and
incubated at 4o C for 1 hr. Lysates were centrifuged in a microfuge at 14,000g at 4o C for 30 mins.
Supernatants (800 µl) were incubated with 2 µg rabbit anti-GFP polyclonal antibodies (Santa
Cruz Biotechnology) or with rabbit anti-polycystin-2 polyclonal antibodies (29) (1:100 dilution)
at 4o C for 2 hr. After that, 30 µl protein-G agarose beads (1:1 suspension; Calbiochem) were
added and samples were further incubated overnight at 4o C. Immunoprecipitates were washed 5
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times with 1 ml of lysis buffer and resuspended in 40 µl SDS-gel loading buffer. Proteins were
separated by 7% SDS-PAGE and analyzed by western blotting using anti-polycystin-2 antibodies
and enhanced chemi-luminescence (ECL; Amersham).
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RESULTS
We measured currents using ruptured whole-cell patch-clamp recording in Chinese
hamster ovary (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 ± SEM currents at -100 mV and 100 mV were -410 ± 28 and
431 ± 35 pA, respectively, n = 156; p < 0.01 vs. control or PKD2 alone) as well as in cells co-
transfected with PKD1 and PKD2 (Fig. 1E; mean ± SEM currents at -100 mV and 100 mV were
-450 ± 35 and 476 ± 51 pA, respectively, n = 53; p < 0.01 vs control or PKD2 alone). Similar
currents were not detected in control untransfected cells (Fig. 1B; mean ± SEM 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 ± SEM currents at -100 mV and 100 mV were -35 ± 11 and 40 ± 8
pA, respectively, n = 108; not significant vs control). The molecular identity of currents in
PKD1-transfected cells is yet unclear (see “Discussion” below). Here, we refer these currents as
polycystin-1-dependent.
We further characterized the polycystin-1-dependent currents (Fig. 2). With 150 mM Na-
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 NMDG, measured with 150 mM Na+ in the pipette and
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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 mM 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+ vs 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 ± SEM) 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).
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 polycystin-1
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stainings 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) due to 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 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 immuno-staining 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 to 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 to 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
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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 due to 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. Co-localization was more
abundant in the peri-nuclear region but was also present near surface membrane. Such partial co-
localization 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 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 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
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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 green fluorescent protein
(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" vs “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 vs. 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,
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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 vs 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- ) with deletion of a.a. 290-2960 is localized to surface
membrane of Sf-21 cells ( 25). We found that in CHO cells Nhe- 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- 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 a.a. 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- with or without PKD2. No currents were detected in CHO cells
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transfected with Nhe- alone (Fig. 6C; 0 out of 57 recordings) or in cells transfected with Nhe- +
PKD2 (Fig. 6D; 0 out of 34 recordings). As shown in Fig. 1, cells transfected with PKD1 (29 out
of 51 recordings; not shown here) and co-transfected with PKD1 plus PKD2 (19 out of 32
recordings; not shown here) expressed currents under the same experimental conditions.
The region of polycystin-1 deleted in the Nhe- 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 solid line in Fig. 7 represents averaged current (at -100 mV) of such 5 to 6 recordings from
cells of the same transfection and treated without and with antibody, respectively. As shown, we
found in each of 5 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 ± SEM currents of 5 experiments: 397 ± 86 pA and 159 ± 34 pA without and with
antibodies, respectively; p<0.02, paired t-test). Incubation with control antibodies (anti-ROMK
antibodies, ref. 36) did not reduce polycystin-1-dependent currents (Fig. 7).
The region between 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 mutated Asp-4053 and Glu-4078 to glycine individually. Both D4053G and E4078G mutants,
however, were not targeted to cell surface (not shown). Neither mutant produced currents in CHO
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cells (0 out of 31 and 0 out of 32 for D4053G and E4078G, respectively, expressed current
density [at -100 mV] higher than -10 pA/pF).
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DISCUSSION
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, 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: 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 vs. 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- , 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- and
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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
hetero-multimeric 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 vs sub-
plasma 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
reported that expression of membrane-anchored C-terminal intracellular fragment of PKD1 in
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human embryonic kidney (HEK) cells activates endogenous cation currents with a single-channel
conductance of ~20 pS in Xenopus oocytes and in HEK cells (15). We found that Nhe- 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 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 subunits from
pertussis toxin-sensitive Gi/o-type G proteins (19). 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 homologues 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
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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.
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REFERENCES
1. Gabow, P. A. (1993) N. Eng. J. Med. 329: 332-342.
2. The European Polycystic Kidney Disease Consortium. (1994) Cell 77: 881-894.
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.
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.
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.
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.
7. Tsiokas, L., Arnould, T., Zhu, C., Kim, E., Walz, G., and Sukhatme, V. P. (1999) Proc.
Natl. Acad. Sci. USA 96, 3934-3939.
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. USA
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. Comm. 282, 341-350.
by guest on April 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
23
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.
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.
12. Pazour, G. J., San Agustin, J. T., Follit, J. A., Rosenbaum, J. L., and Witman, G. B. (2002)
Curr. Biol. 12, R378-R380.
13. Yoder, B. K., Hou, X., and Guay-Woodford, L. M. (2002) J. Am. Soc. Nephrol. 13, 2508-
2516.
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) Nature Genet. 33, 129-
137.
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.
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.
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.
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.
by guest on April 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
24
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.
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.
22. Barr, M. M., DeModena, J., Braun, D., Nguyen, C. Q., Hall, D. H., and Sternberg, P. W.
(2001) Curr. Biol. 11: 1341-1346.
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.
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.
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.
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.
27. Zeng, W. Z., Babich, V., Ortega, B., Quigley, R., White, S. J., Welling, P. A., Huang, C.
L. (2002) Am. J. Physiol. 283, F630-F639.
28. Hille, B. Ion channels of excitable membranes. (Sinauer Associates, Sunderland,
Massachusetts, 2001).
29. Foggensteiner, L., Bevan, A. P., Thomas, R., Coleman, N., Boulter, C., Bradley, J.,
by guest on April 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
25
Ibraghimov-Beskrovnaya, O., Klinger, K., and Sandford, R. (2000) J. Am. Soc. Nephrol.
11, 814-827.
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.
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.
32. Bukanov, N. O., Husson H, Dackowski WR, Lawrence BD, Clow PA, Roberts BL,
Klinger KW, Ibraghimov-Beskrovnaya O. (2002) Human Mol. Genet. 11, 923-936.
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.
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.
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. J. Am. Soc. Nephrol. 10, 2342-2351.
36. Huang, C. L., Feng, S., and Hilgemann, D. W. (1998). Nature 391, 803-806.
37. Qian, F., Germino, F. J., Cai, Y., Zhang, X., Somlo, S., and Germino, G.G.. (1997) Nat.
Genet. 16, 179-183.
38. van Adelsberg, J. (1999) Dev. Genet. 24, 299-308.
39. Praetorius, H.A., and Spring, K. R. (2003) Curr. Opin. Nephrol. Hypertens. 12, 517-520.
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.
by guest on April 4, 2018
http://ww
w.jbc.org/
Dow
nloaded from
26
41. Kim E, Arnould T, Sellin LK, Benzing T, Fan MJ, Gruning W, Sokol SY, Drummond I,
Walz G. (1999) J. Biol. Chem. 274, 4947-4953.
42. Lehtonen, S., Ora, A., Olkkonen, V. M., Geng, L., Zerial, M., Somlo, S., and Lehtonen, E.
(2000) J. Biol. Chem. 275, 32888-32893.
43. Geng, L., Burrow, C. R., Li, H. P., and Wilson, P. D. (2000) Biochim. Biophys. Acta. 535,
21-35.
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Footnotes
1The abbreviations used are: ADPKD, autosomal-dominant polycystic kidney disease; PKD1,
type-1 polycystic kidney disease; PKD2, type-2 polycystic kidney disease; TM, transmembrane;
REJ, receptor for egg jelly; LDL, low-density lipoprotein; CHO, Chinese hamster ovary; GFP,
green fluorescent protein; LRR, leucine-rich repeat; Ig, immunoglobulin; D511V, aspartate-511
to valine mutation; Erev, reversal potential.
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Acknowledgments
We thank Dr. Peter Igarashi for critical reading of an earlier version of the manuscript; Dr.
Moshe Levi for sharing the con-focal microscope; Xinji Li for assistance with cell culture. This
work was supported by grants from the National Institutes of Health, American Heart
Association, Polycystic Kidney Research Foundation, and Kidney Texas Foundation.
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FIGURE LEGENDS
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 “Methods” 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 to 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.
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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.
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). Immuno-
stainings were performed using respective antibodies (see METHODS). Images of CD4 was
assigned pseudo-color of either blue, red or green as indicated by the color of lettering in each
panel.
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
DNA of PKD1 plus 2 µg DNA of either pCDNA3, PKD2 or D511V were used. This is equivalent
to 1:2 molar ratio of DNA for PKD1:PKD2 (and for PKD1:D511V). cDNA for GFP (0.5 µg) was
included in each transfection.
Fig. 5. Whole-cell currents in PKD2 null cells transfected with PKD1. A, PKD2-/- cells were
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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 vs. GFP. Inset shows
surface membrane expression of PKD1 (red 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 “Methods”). No staining was observed in cells transfected with GFP alone
(not shown). B, I-V relationships for currents shown in (A).
Fig. 6. Subcellular distribution and whole-cell Na+ current of Nhe- with or without
polycystin-2. Cells were transfected with cDNAs for Nhe- + CD4 (A), for Nhe- + PKD2 + CD4
(B), for Nhe- + GFP (C), and for Nhe- + PKD2 + GFP (D).
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 4o C for 1 hr. Incubation at 4o C prevents endocytosis of antibodies.
Open square and closed square with error bar represent mean ± SEM of averaged current of 5
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.
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Cai, Stefan Somlo and Chou-Long HuangVictor Babich, Wei-Zhong Zeng, Byung-Il Yeh, Oxana Ibraghimov-Beskrovnaya, Yiqiang
channel activityThe amino-terminal extracellular domain is required for polycystin-1-dependent
published online April 1, 2004J. Biol. Chem.
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