From the Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center and the Departments of Medicine and Cell Biology, Harvard Medical School, Boston, Massachusetts 02215
Received for publication, July 14, 2000, and in revised form, October 3, 2000
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ABSTRACT |
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The cytoplasmic C-terminal portion of the
polycystin-1 polypeptide (PKD1(1-226)) regulates several
important cell signaling pathways, and its deletion suffices to cause
autosomal dominant polycystic kidney disease. However, a functional
link between PKD1 and the ion transport processes required to drive
renal cyst enlargement has remained elusive. We report here that
expression at the Xenopus oocyte surface of a transmembrane
fusion protein encoding the C-terminal portion of the PKD1 cytoplasmic
tail, PKD1(115-226), but not the N-terminal portion, induced a large, Ca2+-permeable cation current, which shifted oocyte
reversal potential (Erev) by +33 mV. Whole cell
currents were sensitive to inhibition by La3+,
Gd3+, and Zn2+, and partially inhibited by
SKF96365 and amiloride. Currents were not activated by bath
hypertonicity, but were inhibited by acid pH. Outside-out patches
pulled from PKD1(115-226)-expressing oocytes exhibited a 5.1-fold
increased NPo of endogenous 20-picosiemens cation channels of linear conductance. PKD1(115-226)-injected oocytes
also exhibited elevated NPo of unitary calcium
currents in outside-out and cell-attached patches, and elevated calcium permeability documented by fluorescence ratio and
45Ca2+ flux experiments. Both Ca2+
conductance and influx were inhibited by La3+. Mutation of
candidate phosphorylation sites within PKD1(115-226) abolished the
cation current. We conclude that the C-terminal cytoplasmic tail of
PKD1 up-regulates inward current that includes a major contribution
from Ca2+-permeable nonspecific cation channels.
Dysregulation of these or similar channels in autosomal dominant
polycystic kidney disease may contribute to cyst formation or expansion.
~85% of autosomal dominant polycystic kidney disease
(ADPKD)1 is secondary to
mutations in the polycystin-1 (PKD1) gene (1). The 4303-aa PKD1
polypeptide has a large N-terminal extracellular domain region
encompassing leucine-rich repeats, a C-type lectin domain, a low
density lipoprotein A-like domain (2), 16 PKD domain repeats, and a
single sperm receptor for egg jelly domain (3). Following a complex
polytopic transmembrane domain is a 226-aa C-terminal cytoplasmic tail.
The hallmark pathology of ADPKD is the two-hit generation and gradual
expansion of renal cysts. These cysts compress and, ultimately, render
nonfunctional adjacent, apparently normal renal tissue (4). Cyst
expansion is associated with the conversion of tubular epithelial cells
from the normal phenotype of net solute reabsorption to the cystic
phenotype of net secretion. This secretory phenotype may involve the
function of the apically localized chloride channel, CFTR (5), but a
direct link between the ADPKD disease genes for PKD1 and PKD2 and the
ion transport processes required to mediate cyst expansion has remained
elusive, despite the creation of mouse lines genetically deficient in
PKD1 (6) or PKD2 (7). PKD disease mutations have been found throughout
the PKD1 coding region (8-10), but absence of the C-terminal
cytoplamic tail suffices to cause ADPKD (11).
Multiple potential signaling and binding functions of the 226-aa PKD1
C-terminal cytoplasmic tail have been defined through study of
overexpressed transmembrane fusion proteins encompassing all or
portions of this C-terminal region (12-14). In this fusion protein
context, the PKD1 C-terminal cytoplasmic tail activates PKC Overexpression of the extreme C-terminal part of the PKD1 cytoplasmic
tail, PKD1(115-226), activates signaling by Wnt polypeptides via
frizzled family receptors to inhibit glycogen synthase kinase 3 The varied signaling and binding functions of the PKD1 C-terminal
cytoplasmic tail led us to hypothesize that it might regulate ion
transport processes that mediate or regulate cyst expansion. We now
report that the distal fragment of the cytoplasmic C-terminal tail of
PKD1 (PKD1(115-226)), expressed as a transmembrane fusion protein,
up-regulates inward current and a Ca2+-permeable
nonspecific cation channel of Xenopus oocytes.
CDNAs--
cDNAs encoded tripartite fusion proteins in
which the N-terminal extracellular domain of human CD16 was fused to
the human CD7 transmembrane region (27), whose cytoplasmic terminus was fused in turn to portions of the 226-amino acid (aa) C-terminal cytoplasmic domain of the 4303-aa human PKD1 polypeptide. These fusion
proteins were subcloned in the oocyte expression vector, pXT7. The
longest construct encoded the complete C-terminal cytoplasmic fragment
of PKD1 (PKD1 aa 4078-4303), and was termed "CD16.7-PKD1-(1-226)" (12, 13, 15). "CD16.7-PKD1(115-226)" encoded only the C-terminal 112 aa of the 226-aa PKD1 C-terminal tail (PKD1 aa 4192-4303). "CD16.7-PKD1(1-92)" encoded the N-terminal 92 aa of the PKD1
C-terminal tail (PKD1 aa
4078-4169).2 Other PKD1
constructs studied included "CD16.7-PKD1(1-155)" encoding PKD1 aa
4078-4232, "CD16.7-PKD1(115-189)" encoding PKD1 aa 4192-4266, and "CD16.7-PKD1(115-203)" encoding PKD1 aa 4192-4280. "CD16.7 control" encoded a cytoplasmic tail polypeptide of the same length as
PKD1(1-92), but of unrelated, novel
sequence.3
These and other CD16.7-PKD1 wild-type constructs have been described
(15). PKD1 C-terminal cytoplasmic domain fragments fused to the CD16.7
transmembrane anchor display intracellular protein binding, signaling,
and 293T cell surface expression properties indistinguishable from the
same PKD1 constructs linked to immunoglobulin ectodomain membrane
anchors and to various epitope tags (12-16). Point mutations in
CD16.7-PKD1(115-226) were constructed by four primer polymerase chain
reaction techniques. Primer sequences are available upon request.
Integrity of the polymerase chain reaction products and of ligation
junctions was confirmed by DNA sequencing.
Preparation of cRNA--
SalI- or
NotI-linearized recombinant pXT7 templates were transcribed
with T7 RNA polymerase. Xenopus oocyte isolation, culture, and microinjection were performed using standard techniques (28, 29).
Oocytes injected with 12-25 ng of cRNA in a volume of 50 nl maintained
integrity up to 18 days after injection. Generally, however, oocytes
were subjected to electrical recording studies 2-3 days after microinjection.
Immunocytochemistry--
Oocytes injected 3, 5, or 10 days
previously with cRNA were incubated with purified 3G8 mouse anti-human
CD16 monoclonal antibody (gift of O. Mandelstam, J. Strominger, and J. Unkeless; or purchased from Meditech) at a concentration of 2 µg/ml
in ND96 at room temperature for 3-4 h, then rinsed several times in
ND96. Oocytes were fixed either in absolute methanol or in 3%
paraformaldehyde in 140 mM NaCl, 20 mM sodium
phosphate, pH 7.4 (PBS). Paraformaldehyde-fixed oocytes were rinsed in
PBS, quenched with several washes in PBS plus 50 mM
glycine, then in PBS. Methanol-fixed oocytes were rehydrated into PBS.
Oocytes were then incubated in secondary Cy3-conjugated donkey
anti-mouse IgG (Jackson Immunoresearch Laboratories, West Grove, PA)
diluted 1/500 in PBS, rinsed several times in PBS, then dehydrated in
methanol, and cleared in benzyl benzoate/benzyl alcohol (2:1) prior to
imaging with a Bio-Rad MRC 1024 confocal microscope. Some oocytes were
incubated with both primary and secondary antibody prior to fixation,
with indistinguishable results.
Two-electrode Voltage Clamp--
Oocytes were placed in a 1-ml
chamber (model RC-11, Warner Instruments, Hamden, CT) on the stage of a
dissecting microscope and impaled with microelectrodes under direct
view. Electrodes were pulled from borosilicate glass with a Narashige
puller, filled with 3 M KCl, and had resistances of 2-3
megohms. Currents were measured with a Geneclamp 500 amplifier (Axon
Instruments, Burlingame, CA) interfaced to a Hewlett Packard computer
with a Digidata 1200 interface (Axon Instruments). Data acquisition and
analysis utilized pCLAMP 6.0.3 software (Axon Instruments). Voltage
pulse protocols consisting of 720-ms 20-mV steps between
Standard bath solution was 96 mM NaCl, 2 mM
KCl, 5 mM HEPES, 1.8 mM CaCl2, and
1 mM MgCl2, and holding potential was Patch Clamp Recording of Cation Currents--
Patch pipettes
were pulled to a resistance of 5-8 megohms, and oocytes were
devitellinized as described previously (29). Gigaseals were first
attained in the cell-attached mode. Outside-out patches were formed by
breaking the oocyte plasma membrane with a pulse and withdrawing the
pipette slowly. Currents were measured with an Axopatch 1D amplifier
(Axon Instruments) interfaced via a Digidata 1200 AD/DA board to a HP
Vectra computer. Data was acquired at 1 kHz and digitized at 5 kHz. For
outside-out experiments, pipette solutions contained 128 mM
cesium aspartate, 12 mM cesium EGTA, and 10 mM
HEPES and the bath solution was 140 mM sodium methanesulfonate, 10 mM HEDTA, 10 mM HEPES. In
cell-attached experiments, the pipette contained 100 mM
CaCl2, 10 mM HEPES, and 10 mM HEDTA.
Calcium Fluorescence Measurements--
Oocytes from both groups
were injected with 70-kDa Calcium Green dextran (Molecular Probes) to a
final concentration of 3.5-7 µM, then subjected to bath
[Ca2+] change from 0 to 10 mM. Relative
change in intracellular calcium concentration
([Ca2+]i) was determined by
exciting the fluorophore at 490 nm and imaging at 530 nm as described
(29). Additional oocytes were loaded for 45 min with 8 µM
Fura2-AM, then subjected to bath [Ca2+] changes from 1.8 mM to nominal 0 mM to 10 mM. In
these oocytes [Ca2+]i was
determined by alternately exciting the fluorophore at 340 and 380 nm.
510-nm emission images were acquired and analyzed as described (29),
using IMAGE1 and IMAGE1-FL software (Universal Imaging).
45Ca2+ Influx Studies--
The uptake
procedure was as reported (30), with modifications. Oocytes were
incubated 1 h in ND-96 modified to contain 0.13 mM
CaCl2 with 2.5 µCi/µl 45Ca2+,
washed in ND-96, then counted in a liquid scintillation counter (Packard 2200CA).
CD16.7-PKD1(115-226) Expression Induces a Large, Inward
Current--
Expression in Xenopus oocytes of
CD16.7-PKD1(115-226) was associated with an inward current not
observed in oocytes expressing CD16.7 control (Fig.
1, A and B). The
current measured at
Several subdomains of the PKD1 cytoplasmic C-terminal tail as
CD16.7-PKD1 fusion proteins were expressed in oocytes further to
delimit the regions required for activation of cation currents (Fig.
1D), which were sensitive to block by La3+
(inset). La3+-sensitive inward currents elicited
at
The increased inward current and positive voltage-shifted
Erev elicited by CD16.7-PKD1(115-226) but not
by CD16.7 control was not explained by differences in oocyte surface
expression of the different fusion proteins. As shown in Fig.
2, immunostaining of intact, unfixed
oocytes with anti-CD16 antibody revealed comparable intensities of CD16
signal at the oocyte surface whether the epitope was borne on
CD16.7-PKD1(115-226) or by CD16.7 control. Surface staining intensity
increased at longer times after cRNA injection and in oocytes
coexpressing both cRNAs (data not shown). The inactive CD16.7-PKD1(1-92) constructs were also expressed at the surface of
57% of injected oocytes at fluorescence intensities comparable to that
of the active construct CD16.7-PKD1(115-226). Surface staining was
always absent in water-injected oocytes (data not shown) and in oocytes
expressing CFTR, which lacks the CD16 epitope (Fig. 2d).
CD16.7-PKD1(115-226)-induced Current Exhibits Preferential
Specificity for Cations--
The ion selectivity of the
CD16.7-PKD1(115-226)-associated current was tested by NaCl bath
substitution with either sodium gluconate or
N-methyl-D-glucamine chloride. Bath change from
NaCl to sodium gluconate produced little reduction in current and no change in Erev (
The relative anion/cation permeability of oocytes expressing
CD16.7-PKD1(115-226) was further tested by a mannitol dilution test.
Shifting from 96 mM NaCl to a bath containing 9.6 mM NaCl, with mannitol added to maintain constant osmotic
strength, decreased inward current at
Monovalent cation selectivity of the induced current was tested by
equimolar substitution of bath NaCl with KCl, LiCl, and NH4Cl. Bath substitution with KCl or LiCl depolarized
Erev by +15 ± 3 mV and +7 ± 3 mV,
respectively (n = 5), in oocytes expressing CD16.7
control, as expected for the moderate K+ conductance and
small cation conductance native to oocytes (32). In contrast, these
bath changes did not alter Erev in oocytes expressing CD16.7-PKD1(115-226). Whereas bath substitution with NH4Cl depolarized Erev in CD16.7
control-expressing oocytes by +41 ± 4 mV and significantly
increased conductance (n = 5, data not shown) as in
native oocytes (32), CD16.7-PKD1(115-226)-expressing oocytes shifted
the (extrapolated) Erev only slightly, +6 ± 3 mV (n = 6; F = 5.7, p = 0.02, data not shown). Thus,
CD16.7-PKD1(115-226)-associated currents exhibited little specificity
among monovalent cations, and approximated in magnitude the native
NH4+ conductance of oocytes (33).
The CD16.7-PKD1(115-226)-induced Current Is Associated with
Elevated Ca2+ Permeability--
The hypothesis that the
PKD1(115-226)-associated current might conduct Ca2+ was
tested by subjecting oocytes to voltage ramps first in the absence and
then in the presence of 10 mM bath Ca2+ (Fig.
4A). The difference current at
45Ca2+ influx into oocytes expressing
PKD1(115-226) was 3.94 ± 0.14 pmol/oocyte·h (Fig.
4D, n = 24), and greatly exceeded the 0.85 ± 0.26 pmol/oocyte·h in oocytes expressing
CD16.7-PKD1(1-92) (n = 9, p < 0.00001). 1 mM La3+ inhibited
45Ca2+ influx into oocytes expressing
PKD1(115-226) by 69% to 1.24 ± 0.17 pmol/oocyte·h
(n = 26, p < 0.00001), whereas
La3+ was without effect on 45Ca2+
influx into oocytes expressing PKD1(1-92) (n = 4, Fig.
4D). Thus, 1 mM La3+ inhibited 88%
of the 45Ca2+ influx attributable to expression
of CD16.7-PKD1(115-226).
Calcium Green fluorescence estimates of oocyte [Ca2+]
indicated that the elevated Ca2+ influx into oocytes
expressing PKD1(115-226) led to elevated free
[Ca2+]i (Fig. 4E). Upon
bath shift from 0 to 10 mM Ca2+, global oocyte
[Ca2+]i in oocytes expressing
CD16.7-PKD1(115-226) exhibited a sustained increase of 38 ± 7 fluorescence intensity units, significantly higher than the 9.5 ± 1.4 units in oocytes expressing CD16.7 control (t = 4.0, p = 0.007, by unpaired t test,
n = 4). Since the nonratiometric Calcium Green did not
allow comparison of absolute
[Ca2+]i values among oocytes, we
also measured [Ca2+]i in
Fura2-AM-loaded oocytes. Basal
[Ca2+]i in oocytes expressing
CD16.7-PKD1(1-92) and CD16.7-PKD1(115-226) was indistinguishable,
87 ± 2 nM and 91 ± 6 nM,
respectively (n = 4, p > 0.1). These
values did not change detectably upon shift to nominally
Ca2+-free bath. However, subsequent bath shift to 10 mM Ca2+ increased
[Ca2+]i 34 ± 9 nM in PKD1(115-226)-expressing oocytes, but only 5 ± 1 nM in PKD1(1-92)-expressing oocytes (n = 4, p < 0.01). The elevated
[Ca2+]i persisted without decay
for at least 45 min.
Single-channel Properties of CD16.7-PKD1(115-226)-activated
Na+ Current--
Fig.
5A shows unitary currents
recorded at a holding potential of Single-channel Properties of CD16.7-PKD1(115-226)-activated
Ca2+ Currents--
Fig.
6A shows an outside-out patch
from an oocyte expressing CD16.7-PKD1(115-226). Ca2+
currents were recorded at
Induced Ca2+ channel activity was also detected in
cell-attached patches on oocytes expressing CD16.7-PKD1(115-226). Fig.
7A shows representative traces
from a cell-attached patch with 100 mM Ca2+ as
the main charge-carrying ion in the pipette. Oocytes were preinjected
with EGTA (estimated final concentration 5 mM) and then
preincubated 2 h in sodium methanesulfonate to deplete
intracellular chloride and minimize endogenous
Ca2+-activated chloride currents. Unitary current was again
1.1 pA at Pharmacology and Regulation of CD16.7-PKD1(115-226)-associated
Cation Currents--
Na+ current measured at
The effect of bath pH on the induced cation current was tested. Bath pH
change from 7.4 to 6.0 decreased inward current measured at Importance of Selected Serine and Tyrosine Residues for Cation
Current Activation by CD16.7-PKD1(115-226)--
Several amino acid
residues have been shown to be phosphorylated within PKD1(115-226)
fusion proteins in vitro by exogenously added kinases and in
transiently transfected cells (23). Within this region, serine 175 (Ser-4252 of holo-PKD1) is a preferred substrate of
cAMP-dependent protein kinase, and tyrosine 160 (Tyr-4237 of holo-PKD1) is a preferred substrate of c-Src. The adjacent serine
residues 174 and 175 (4251/4252) were mutated in tandem either to
alanine or to aspartate, since each is a candidate
cAMP-dependent protein kinase site. Each double mutation
abolished CD16.7-PKD1(115-226)-associated inward currents (Fig.
8A), despite unimpaired
expression of the doubly mutant polypeptides at the oocyte surface
(Fig. 8B). Similarly, mutation of tyrosine 160 to
phenylalanine (Y4237F) abolished CD16.7-PKD1(115-226)-associated current (Fig. 8A) despite unimpaired surface expression of
this mutant polypeptide (Fig. 8B).
We report that expression of the CD16.7-PKD1(115-226) fusion
protein, encoding the 112 C-terminal residues of the PKD1 cytoplasmic C-terminal tail fused to the CD16.7 transmembrane chimera, up-regulates a Ca2+-permeable cation conductance in Xenopus
oocytes. The cation conductance is nonselective among monovalent
cations, is inhibited by La3+, Gd3+, SKF96365,
amiloride, and Zn2+, but not by DIDS or niflumate, and is
not activated by hypertonicity. Expression of CD16.7 control, or of
CD16.7-PKD1(1-92), CD16.7-PKD1(1-155), or of CD16.7 without any
intracellular tail failed to activate this cation conductance, despite
normal surface expression of these inactive polypeptides. Activation of
whole cell currents was accompanied by increased open probability
(NPo) of cation channels of 20-22 pS linear
conductance, which likely mediated or substantially contributed to the
recorded whole cell currents.
The CD16.7-PKD1(115-226) fusion protein spans the lipid bilayer only
once. The transmembrane domain derived from CD7 within the CD16.7
fusion proteins is not expected itself to form an ion channel. Indeed,
similar cation channel activity appeared present at 5-8-fold lower
activity in oocytes previously injected with water or with cRNA
encoding either CD16.7 control or CD16.7-PKD1(1-92). Taken together,
the data suggest that the PKD1(115-226) domain activates a cation
channel native to oocytes. This hypothesis suggests that
CD16.7-PKD1(115-226) either triggers a signaling pathway that
tonically activates cation channel activity, or that the PKD1 fragment
can serve as a chaperonin or a functional subunit of an endogenous
pore-forming polypeptide or polypeptide complex.
Which signaling pathways might mediate cation channel activation by
CD16.7-PKD1(115-226)? This portion of the PKD1 C-terminal cytoplasmic
tail activates the siamois promoter via inhibition of
GKS-3 PKD1(115-226) also harbors a major in vitro
cAMP-dependent protein kinase phosphorylation site (23,
24). We tested by mutagenesis the functional importance of this site
for cation channel activation. Mutation of the tandem serine residues
4251/4252 to either alanine or to aspartate nearly abolished
CD16.7-PKD1(115-226)-associated cation currents, despite undiminished
expression of the mutant polypeptides at the oocyte surface. Mutation
of the putative tyrosine kinase site Tyr-4237 (23) to Phe also reduced
cation current without decreasing surface expression of polypeptide.
Therefore, these residues play important roles in cation channel
activation. Whether this occurs by direct phosphorylation or by another
mechanism remains to be determined.
With which cation channel might CD16.7-PKD1(115-226) directly or
indirectly interact? Qian et al. and Tsiokas et
al. showed that this region of PKD1 can complex with the
C-terminal tail of PKD2 (14, 17). Although PKD2 and PKD1 colocalization
within cells remains uncertain (19), PKD2 and PKD1 can be
coimmunoprecipitated (14, 17). The PKD2 homolog PKDL, although not a
cause of heritable human polycystic kidney disease (36), does
produce Ca2+-permeable cation channels in
Xenopus oocytes (30) different in character from those we
have measured in oocytes expressing CD16.7-PKD1(115-226).
Tsiokas et al. (18) showed that PKD2 through distinct
binding domains complexes with the Ca2+-permeable cation
channel hTRPC1, but not with hTRPC3. Akaike and colleagues (37) showed
that degenerate hTRP-1 oligonucleotides reduced thapsigargin-stimulated
currents in oocytes injected with a rat TRP4 homolog. More recently,
Berridge and colleagues have cloned Xtrp1 (38), but its function
remains unreported. Our preliminary experiments failed to demonstrate
reduction in CD16.7-PKD1(115-226)-associated cation channel activity
after coinjection of Xtrp1 antisense oligonucleotides (data not shown).
Xenopus oocytes exhibit an Icrac-like
store-operated Ca2+ current (39, 40). Preliminary
experiments indicate that thapsigargin, although reproducibly
activating a cation current in oocytes, did not activate current
attributable to CD16.7-PKD1-(115-226). CD16.7-PKD1(115-226)-elicited
cation current also differed from oocyte Icrac in its more
modest La3+ sensitivity in intact oocytes, its moderate
amiloride sensitivity, and its lack of inward rectification (40).
Nonselective cation channels and mechanosensitive cation channels have
been reported in Xenopus oocytes (reviewed in Ref. 41). The
cation conductances activated in oocytes by maitotoxin (42) and by
palytoxin (43) each differed from CD16.7-PKD1(115-226)-activated cation conductance. Unlike the widely distributed
hypertonicity-activated cation channel (33),
CD16.7-PKD1(115-226)-activated cation conductance was insensitive to
moderate increase in bath tonicity.
Unlike depolarization-activated hemi-gap junction-mediated cation
currents (44), CD16.7-PKD1(115-226)-activated cation conductance was
voltage-independent. Unlike the external divalent-blocked cation
channel (45, 46), CD16.7-PKD1(115-226)-activated cation conductance
was not blocked by DIDS or by niflumate. Endogenous oocyte cation
conductance has been activated by simple overexpression of heterologous
K+ channels (47), but, unlike these currents,
CD16.7-PKD1(115-226)-activated cation current was insensitive to DIDS
and not gated by hyperpolarization.
In summary, expression in Xenopus oocytes of a CD16.7
transmembrane fusion protein containing the PKD1 C-terminal cytoplasmic tail aa 115-226 activated Ca2+-permeable nonspecific
cation currents in Xenopus oocytes likely mediated by
channels of 20-pS linear conductance. Preliminary experiments (data not
shown) indicate that similar channels were activated in 293T cells
transiently transfected with CD16.7-PKD1(115-226). In oocytes, the
elevated cation conductance correlated with increased Ca2+
entry and elevation of [Ca2+]i.
These changes in ion channel activity are the first directly linked to
the expression of any portion of the PKD1 polypeptide. We propose that
changes in intracellular Ca2+ signaling resulting from
mutations in PKD1 contribute to the creation and/or maintenance of the
secretory and/or proliferative phenotype of renal cysts in ADPKD1, and
to the resultant cyst expansion that ultimately leads to renal failure.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and (in
a process requiring active Rac or Cdc-42) c-Jun N-terminal kinase. Both
pathways are required for downstream activation of AP-1-dependent transcriptional events (12, 13).
and
stabilize
-catenin, leading to activation of the TCF/LEF family of transcription factors (13). PKD1(115-226) also binds and
stabilizes the G protein regulator RGS7 (15) and, through RGS7, 14-3-3 (16). PKD1(115-226) also binds, stabilizes (14, 17), and may target to
the plasma membrane (18, 19) the ADPKD2 gene, PKD2, a structural
homolog of Ca2+ and cation channels (20). Overexpressed
PKD2 or its C-terminal cytoplasmic tail also up-regulate AP-1-mediated
transcription through pathways synergistic with those activated by the
PKD1 C-terminal cytoplasmic tail (21). The signaling activities
associated with overexpression of C-terminal fragments of PKD1 have
been noted independent of type of membrane-anchored fusion protein (12-16). Additional reported activities of the C-terminal cytoplasmic tail of PKD1 include activation of G
o and
G
i proteins in vitro (22), and
phosphorylation on serine and tyrosine residues in vitro and
in transfected cells (22-24). Endogenous PKD1 can be precipitated in a
complex with E-cadherin and the
/
/
-catenin complex and
colocalizes with these components in human fetal kidney (25, 26).
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 and +20
mV were generated by the Clampex subroutine. Bath resistance was
minimized by the use of agar bridges filled with 3 M KCl,
and a virtual ground circuit clamped bath potential to zero.
30 mV. All solutions were of pH 7.40. Cation/anion permeability determinations were by equimolar replacement of bath Na+ with
N-methyl-D-glucamine (NMDG), and of bath
Cl
with gluconate. Determination of relative monovalent
cation permeabilities was by equimolar substitution of bath
Na+ with K+, Li+, and
NH4
.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 mV in the CD16.7-PKD1(115-226) group was
910 ± 68 nA (n = 97 oocytes from 18 frogs)
compared with
131 ± 11 nA in the CD16.7 control group
(n = 79 oocytes from 15 frogs, t = 10.2, p < 0.0001 by unpaired two-tailed t
test, Fig. 1C). The average conductances of 2.1 and 8.7 µS
and the whole cell reversal potentials (Erev) of
31 mV and +2 mV in oocytes expressing CD16.7 control and
CD16.7-PKD1(115-226), respectively (Fig. 1C), each differed
significantly by unpaired t tests (p < 0.0001). The currents in CD16.7 control-expressing oocytes were
indistinguishable from those measured in oocytes previously injected
with CD16.7-PKD1(1-92) cRNA or with water (Fig. 1D).
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Fig. 1.
Expression in oocytes of
CD16.7-PKD1(115-226) induces a large current not observed in oocytes
expressing CD16.7 control or CD16.7-PKD1(1-92). A,
current traces in normal bath (ND96) generated in a two-microelectrode
voltage clamp experiment on a CD16.7 control-injected oocyte. The
voltage pulse protocol generated 20-mV voltage steps from 100 mV to
+20 mV, from a holding potential of
30 mV. B,
representative current traces from a PKD1(115-226)-injected oocyte.
C, composite current-voltage relationship of CD16.7
control-injected oocytes and CD16.7-PKD1(115-226)-injected oocytes.
Slope conductances were 2.1 and 8.7 µS, and reversal potentials were
31 and +2 mV in the control and PKD1(115-226) groups, respectively.
D, analysis of the indicated deletion constructs implicates
a region of the cytoplasmic tail encompassing residues 115-189 as
required for expression of increased current.
La3+-sensitive current (2 mM La3+)
was assayed at
100 mV during two microelectrode studies. *,
p < 0.05; **, p < 0.01, by post-test
versus control. Oocytes expressing CD16.7-PKD1(115-226)
(shaded bar) were studied in a separate
experiment (inset, I-V relationship of
La3+-sensitive difference current).
100 mV in oocytes expressing CD16.7-PKD1(115-203) or -(115-189)
were of magnitude comparable to those observed with
CD16.7-PKD1(115-226) (Fig. 1D and data not shown) with
CD16.7-PKD1(1-226). In contrast, expression of CD16.7 control, or of
CD16.7-PKD1(1-92), (), or () did not induce such currents.
Expression of the constructs shown in Fig. 1D was not
associated with evident oocyte morbidity over at least 4 days after
cRNA injection.
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Fig. 2.
Surface immunostaining of CD16 epitope in
unfixed oocytes injected 3 days (a-c) or 10 days
(d-f) previously with cRNA encoding CD16.7
(a; 2 of 3 unfixed oocytes were CD16-positive), CD16.7
control (b and e; 11 of 11 unfixed
oocytes were CD16-positive), CD16.7-PKD1(115-226) (c
and f; 40 of 43 unfixed oocytes were
CD16-positive), or CFTR (d; 0 of 4 unfixed oocytes
were CD16-positive). Unfixed oocytes were incubated anti-CD16
monoclonal antibody 3G8, rinsed, post-fixed with methanol
(a-c) or paraformaldehyde (d-f), then labeled
with Cy3-conjugated secondary anti-Ig, and again fixed. Relative
intensities in panels a-c are directly
comparable, as are panels d-f. Water-injected
oocytes (data not shown; 0 of 10 unfixed oocytes were CD16-positive)
were indistinguishable from those expressing CFTR (d). 13 of
23 unfixed oocytes expressing the inactive CD16.7-PKD1(1-92) were
detectably CD16-positive (data not shown). Each panel represents 1 mm2.
1 ± 1 mV,
n = 15, Fig.
3A). In contrast, bath change
from NaCl to N-methyl-D-glucamine chloride
significantly decreased inward current at
100 mV from
573 ± 58 nA to
327 ± 52 nA, p < 0.001), and
hyperpolarized Erev
19 ± 5 mV
(n = 15), each significantly greater than the
5 ± 1 mV hyperpolarization (p < 0.001, n = 5, unpaired t test), and
99 ± 13 nA
inward current at
100 mV (p = 0.0019, n = 5) observed in oocytes expressing CD16.7(control).
In the presence of the impermeant cation NMDG, the depolarized
Erev of CD16.7-PKD1(115-226)-expressing oocytes shifts toward the equilibrium potentials for Cl
and
K+. These results are most simply explained by preferential
activation of a cation conductance.
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Fig. 3.
CD16.7-PKD1(115-226)-stimulated currents
preferentially pass cations. A,
PKD1(115-226)-associated currents were significantly reduced and the
reversal potential was significantly hyperpolarized when bath sodium
was replaced by NMDG. Bath chloride subsitution with gluconate had no
effect on reversal potential. B, significant
La3+-sensitive currents persisted after 16 h chloride
depletion and subsequent recording in a sodium methanesulfonate bath
after injection of EGTA. Current at 100 mV was
421 ± 52 nA
and
270 ± 34 nA in control and La3+-containing
bath, respectively; t = 5.7, p = 0.0001, by paired t test.
100 mV from
634 ± 155 to
464 ± 130 nA (t = 5.5, p = 0.0009, by two-tailed t test), and
hyperpolarized Erev from
11 ± 4 mV to
19 ± 6 mV (t = 4.1, p = 0.005, by paired t test). The direction of this shift in
Erev indicates that
CD16.7-PKD1(115-226)-stimulated current was more selective for cations
than for anions. Oocytes were then ~90% depleted of intracellular
chloride by overnight incubation in a Cl-free sodium methanesulfonate
medium (31). Following subsequent acute injection with EGTA to minimize
residual Ca2+-activated Cl
currents, oocytes
expressing CD16.7-PKD1(115-226) continued to exhibit
La3+-sensitive current (Fig. 3B) significantly
greater than that observed in PKD1(1-92)fs-expressing oocytes.
100 mV attributable to Ca2+ was
436 ± 95 nA in
CD16.7-PKD1(115-226)-expressing oocytes compared with +21 ± 27 nA in oocytes previously injected with water (p = 0.004, two-tailed t test). Thus, the nonspecific cation
current activated in oocytes by expression of PKD1(115-226) also
exhibited substantial permeability to Ca2+. The time course
of holding currents was studied with calcium bath substitution (see
Fig. 4B). The holding potential chosen was
30 mV, close to
the chloride equilibrium potential, to minimize potential contribution
of endogenous chloride currents. The upper trace from an oocyte
expressing CD16.7-PKD1(115-226) reveals decreased inward current upon
bath Na+ substitution with NMDG, which increased upon
subsequent addition to the NMDG of 10 mM Ca2+
(representative of four experiments). The lower trace from an oocyte
expressing CD16.7 control shows that current remained unaltered during
the same bath change protocol (representative of four experiments), as
expected from the dominant endogenous chloride conductance. Fig.
4C shows current voltage relationships of oocytes subjected to the protocol of Fig. 4B. The calcium difference current
(solid line) was significant, and the
Erev was consistent with that expected of a
cation current.
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Fig. 4.
Oocytes expressing CD16.7-PKD1(115-226)
exhibit Ca2+ currents. A, difference
currents were constructed from 2600 msec voltage ramps ( 150 to +50
mV, upper panel), executed in baths containing
first 0 and then 10 mM CaCl2. The difference
currents (lower panel) in
PKD1(115-226)-expressing oocytes (black trace)
were significantly larger than those of water-injected oocytes
(gray trace). B, representative
experiment illustrating time course of holding currents during bath
change from ND-96 with NMDG (0 Ca2+) to NMDG with 10 Ca2+. For an oocyte expressing CD16.7-PKD1(115-226)
(upper panel), inward current was reduced at the
holding potential when NMDG (0 Ca2+) replaced the control
bath, and the inward current reappeared with the substitution of a
Ca2+-containing bath. In contrast, for an oocyte expressing
CD16.7 control (lower panel), inward current was
unchanged during these maneuvers. C, current-voltage
relation of CD16.7-PKD1(115-226)-expressing oocytes recorded in NMDG
baths containing first 0 and then 10 mM Ca2+.
The Ca2+ difference current exhibited a 6.8-µS slope
conductance and Erev of +15 mV
(r2 = 0.994). D,
45Ca2+ uptake into
CD16.7-PKD1(115-226)-expressing oocytes greatly exceeded uptake into
CD16.7-PKD1(1-92)-expressing oocytes, and was blocked by
La3+. E, Calcium Green fluorescence intensity
(495/530 nm) indicates a greater Ca2+ permeability in
oocytes expressing CD16.7-PKD1(115-226) than in those expressing
CD16.7 control. At the arrow, 10 mM
Ca2+ was added to the Ca2+-free NMDG bath. A
single oocyte trace is shown for each group. Similar results were
obtained with Fura-2 fluorescence ratio imaging.
50 mV from a representative
outside-out patch with cesium aspartate in the pipette and sodium
methanesulfonate in the bath. Addition to the bath of 1 mM
La3+ suppressed nearly completely the inward
Na+ currents. The unitary Na+ current exhibited
an amplitude of
1.1 pA at
50 mV (Fig. 5B), and a linear
conductance of 20 pS (r2 = 0.99) with
Erev ~0 mV
(PNa/PCs ~1; Fig.
5C). The mean NPo of 0.16 ± 0.04 was 5.1-fold higher than the NPo (0.03 ± 0.03) of indistinguishable channels detected in patches pulled from
oocytes expressing CD16.7 control (Fig. 5D,
n = 5, p = 0.03). Bath addition of 1 mM La3+ to patches pulled from oocytes
expressing CD16.7-PKD1(115-226) reduced mean
NPo by 96% (Fig. 5A), from 0.47 ± 0.19 to 0.02 ± 0.01 (n = 5, p = 0.04, one-tailed t test). Bath addition of 10 µM SKF96365 reduced NPo 82% to
0.03 ± 0.02 (n = 5, p = 0.03). Both inhibitors were considerably more potent as antagonists of outside-out patch currents than of whole oocyte currents (Table I).
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Fig. 5.
Outside-out patches from oocytes expressing
CD16.7-PKD1(115-226) display increased activity of
La3+-sensitive cation channels. A,
representative traces from an outside-out patch pulled from a
CD16.7-PKD1(115-226)-expressing oocyte into a 96 mM sodium
methanesulfonate bath with cesium aspartate in the pipette, before and
after addition of 1 mM La2+
(Vm was 50 mV). B, amplitude histogram
of single-channel events recorded from a representative experiment
during control conditions (Vm was
50 mV).
C, current-voltage relation of the cation channel, with
slope conductance of 20 pS. Erev near 0 mV
indicates PNa/PCs ~1.
D, NPo of single-channel events in
outside-out patches from CD16.7-PKD1(115-226)-expressing oocytes was
significantly higher than in CD16.7 control-expressing oocytes
(Vm =
50 mV, n = 5, p < 0.04).
Pharmacology of PKD1(115-226) currents (in Xenopus oocytes, measured
at 100 mV holding potential)
50 mV in a 100 mM
Ca2+ bath with EGTA in the pipette. Unitary
Ca2+ current in this patch was
1.1 pA at
50 mV (Fig.
6B). This current exhibited a linear I-V relationship (Fig.
6C, n = 10-12), and was undetectable in
oocytes expressing CD16.7 control (Fig. 6D). Unitary
Ca2+ current was inhibited 88% by addition of 1 mM La3+, reducing NPo
from 1.13 ± 0.39 to 0.14 ± 0.10 (n = 7, p < 0.02 by paired t test, Fig.
6E).
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Fig. 6.
Oocytes expressing CD16.7-PKD1(115-226)
exhibit increased La3+-sensitive Ca2+ currents
in outside-out patches. A, representative trace from
outside-out patch of a CD16.7-PKD1(115-226)-expressing oocyte,
recorded in a calcium bath with EGTA-buffered cesium aspartate in the
pipette (Vm = 50 mV). B, representative
amplitude histogram from recordings such as in panel
A (fitted by gaussian, Vm =
50 mV).
C, single-channel current-voltage relationship from
recordings such as in panel A, with slope
conductance of 11 pS (n = 7, gray
line). D, NPo of
single-channel currents from outside-out patches at
50 mV in
Ca2+ bath was significantly greater in oocytes expressing
CD16.7-PKD1(115-226) (0.775 ± 0.232) than in those expressing
CD16.7 control (0.006 ± 0.004; t = 2.23, p = 0.04, two-tailed, unpaired t test).
E, NPo of single-channel currents in
outside-out patches from oocytes expressing CD16.7-PKD1(115-226) is
inhibited 88% by 1 mM La3+.
50 mV (Fig. 7B), with a linear conductance of 18 pS (Fig. 7C). NPo in cell-attached
patches from oocytes expressing CD16.7-PKD1(115-226) was 0.53 ± 0.09, significantly higher than in oocytes expressing CD16.7 control
(0.01 ± 0.005, t = 3.5, p = 0.005, by unpaired t test, Fig. 7D). Thus, cation
channels activated by CD16.7-PKD1(115-226) were also permeable to
Ca2+ in outside-out and cell-attached patch
configurations.
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Fig. 7.
Oocytes expressing CD16.7-PKD1(115-226)
exhibit increased unitary Ca2+ currents in cell-attached
patches. A, representative traces from cell-attached patches
on CD16.7-PKD1(115-226)-expressing oocytes with CaCl2 in
the pipette. Oocytes were preincubated in sodium methanesulfonate and
preinjected with EGTA to reduce activity of endogenous
Ca2+-activated chloride currents. B, amplitude
histogram of 50 mV trace shown in A. C,
single-channel current-voltage relationship of experiment in
panel A with slope conductance 18 pS
(gray line). D,
NPo of cell-attached patches recorded on
CD16.7-PKD1(115-226)-expressing oocytes (0.525 ± 0.093)
significantly exceeded that in CD16.7 control-expressing oocytes
(0.01 ± 0.005; t = 3.5, p = 0.005, unpaired
two-tailed t test).
100 mV
holding potential in CD16.7-PKD1(115-226)-expressing oocytes was
reduced by bath addition of 2 mM LaCl3 from
694 ± 110 nA to
365 ± 72 nA (n = 10, p < 0.01, two-tailed t test).
Water-injected oocytes exhibited much lower currents at the same
holding potential (
112 ± 13 nA) that were reduced minimally by
La3+ to
89 ± 12 nA, n = 4, data not
shown). The current was insensitive to 500 µM DIDS and to
100 µM niflumic acid, but exhibited moderate block by 50 µM SKF96365 (13%, p = 0.001), 1 mM amiloride (13%, p = 0.02), and 10 µM Zn2+ (19%, p = 0.008),
with increased inhibition at higher Zn2+ concentrations.
The degree of inhibition by Gd3+ was similar to that of
La3+ (Table I). Because cation channels of comparable
unitary conductance are activated by hypertonicity (34), oocytes
expressing CD16.7-PKD1(115-226) or CD16.7 control were subjected to a
bath change from isotonic ND-96 (212 mosM) to ND-96
rendered hypertonic by addition of mannitol (final 280 mosM). Although this degree of hypertonicity
suffices to activate endogenous oocyte Na+/H+
exchange (35), currents recorded in the hypertonic and isotonic baths
did not differ in either group of oocytes (data not shown).
100 mV by
33%, from
1041 ± 71 nA to
697 ± 69 nA
(n = 5, p = 0.0002). In contrast, bath
pH change to 8.0 was without effect on inward current.
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Fig. 8.
PKD1(115-226)-associated
La3+-sensitive current is abolished by mutation of
Ser-174/175 (4251/4252 of holo-PKD1) to Ala or to Asp, and by mutation
of Tyr-160 (Tyr-4237 of holo-PKD1) to Phe. A, oocytes
injected 48 h earlier with cRNA encoding wild-type
CD16.7-PKD1(115-226) (n = 19), CD16.7-PKD1(115-226)
with double Ser mutations to Ala (n = 12) or Asp
(n = 11), or with a Tyr mutation to Phe
(n = 7) were analyzed for La3+-sensitive
current measured at 100 mV. B, CD16 immunolocalization in
unfixed oocytes expressing wild-type CD16.7-PKD1(115-226)
(left panel), or the double mutants S174A/S175A
(18 of 21 oocytes were CD16-positive) and S174D/S175D (32 of 33 oocytes
were CD16-positive; middle panels) and the mutant
Y160F (right panel, 9 of 10 oocytes were
CD16-positive).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
-catenin stabilization, and up-regulation of
TCF-mediated transcriptional signaling (13). Xwnt8-mediated
activation of the siamois promoter was also enhanced by
PKD1(115-226). Although itself incapable of activating transcriptional
signaling mediated by AP-1 elements, this PKD2-binding fragment of PKD1
enhanced activation by PKD2 of AP-1-mediated transcription (21).
PKD1(115-226) also dorsalized and prevented posterior development in
zebrafish embryos (13), and bound and stabilized RGS7 (15).
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ACKNOWLEDGEMENTS |
---|
We thank Leonidas Tsiokas and Joe Mindell for helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK51059, DK34854 (Harvard Digestive Diseases Center), and HL15157 (Boston Sickle Cell Center) (to S. L. A.) and DK52897 (to G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported by a Joseph E. Murray award from the Massachusetts/Rhode
Island affiliate of the National Kidney Foundation.
§ Current address: University Hospital Freiburg, 79106 Freiburg, Germany.
¶ To whom correspondence should be addressed: Molecular Medicine Unit, RW763, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston, MA 02215. Tel.: 617-667-2930; Fax: 617-667-2913; E-mail: salper@caregroup.harvard.edu.
Published, JBC Papers in Press, October 23, 2000, DOI 10.1074/jbc.M006252200
2 In most experiments, CD16.7-PKD1(1-92) was expressed as either of two variants which included C-terminal extensions beyond PKD1 cytoplasmic domain residue 92, originating from polylinker sequence. One form added a single C-terminal Ser residue. The other form added the 7-residue C-terminal sequence Ser-Ala-Ala-Ala-Arg-Glu-Ile. Both of these fusion proteins were expressed at the oocyte surface, but neither increased oocyte currents. The experiment shown in Fig. 6D utilized a form of CD16.7-PKD(1-92) devoid of any C-terminal extension.
3 The construct CD16.7 control provides a novel, PKD1(1-92)-unrelated cytoplasmic C-terminal amino acid sequence of the same length as PKD1(1-92). The sequence is: ESWHLSPLLCVGLWALRLWGALRLGAVILRWRYHALRGELYRPAWEPQDYEMVELFLRRLRLWMGLSKVKES. This sequence is encoded by a frameshifted PKD1(1-92) cDNA (due to a single nucleotide deletion, GenBankTM NM000296 nucleotide 12446).
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ABBREVIATIONS |
---|
The abbreviations used are: ADPKD, autosomal dominant polycystic kidney disease; PKD, polycystic kidney disease; DIDS, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; S, siemen(s); aa, amino acid(s); CFTR, cystic fibrosis transmembrane regulator; PBS, phosphate-buffered saline.
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REFERENCES |
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