From the Renal Unit, Department of Medicine,
Massachusetts General Hospital and Harvard Medical School, Charlestown,
Massachussetts 02129, the ¶ Laboratorio de Canales Iónicos,
Departamento de Fisicoquímica y Química
Analítica, Facultad de Farmacia y Bioquímica, Buenos
Aires, Argentina 1113, and the
Departamento de
Fisiología, Facultad de Medicina,
Buenos Aires, Argentina 1121
Received for publication, September 30, 2002, and in revised form, October 20, 2002
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ABSTRACT |
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Autosomal dominant polycystic kidney disease
(ADPKD) is a prevalent genetic disorder largely caused by mutations in
the PKD1 and PKD2 genes that encode the
transmembrane proteins polycystin-1 and -2, respectively. Both proteins
appear to be involved in the regulation of cell growth and maturation,
but the precise mechanisms are not yet well defined. Polycystin-2 has
recently been shown to function as a Ca2+-permeable,
non-selective cation channel. Polycystin-2 interacts through its
cytoplasmic carboxyl-terminal region with a coiled-coil motif in the
cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of
this interaction on its channel activity, however, are unknown. In this
report, we show that P1CC enhanced the channel activity of
polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to
P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type
polycystin-2 channel activity. The modulation of polycystin-2 channel
activity by polycystin-1 may be important for the various biological
processes mediated by this molecular complex.
ADPKD1 is a
common genetic disorder caused by mutations in either one of the two
genes, PKD1 and PKD2, that encode polycystin-1 and -2, respectively (1). Polycystin-1 is an 11-membrane-spanning desmosome-associated protein (2, 3) that may be involved in the
regulation of cell growth (4). Polycystin-2 is a six-span membrane
protein with homology to voltage-dependent (5) and transient receptor potential (TRP) channel proteins (6).
Recently, we and others demonstrated that polycystin-2 indeed functions as a Ca2+-permeable nonselective cation channel
(7-9).
Mutations in either PKD1 or PKD2 cause nearly
identical clinical manifestations, suggesting that these two proteins
either interact directly or are components of a common signaling
pathway (reviewed in Ref. 1). Polycystin-1 and -2 interact with each other through their carboxyl-terminal cytoplasmic tails both in vitro (10, 11) and in vivo (12). This interaction has
been implicated in various cellular processes, including the activation of Jak and the consequent regulation of cell growth (4), the activation
of whole-cell cation-permeable currents (13), and the regulation of
G-protein signaling (14). The functional consequences of polycystin-1
interaction on polycystin-2 channel activity, however, have not been
determined. Here, we demonstrate that binding of polycystin-2 to the
coiled-coil-containing segment of the polycystin-1 carboxyl-tail (P1CC)
increased and stabilized polycystin-2 channel function. In contrast, a
single point mutation in P1CC, Q4215P, abolished the regulatory role on
wild-type polycystin-2 channel function. Furthermore, the polycystin-2
truncation mutant R742X, an active channel (15) missing most
of its cytoplasmic tail (including the polycystin-1 binding segment),
was not regulated by P1CC.
Plasmid Constructs--
The PKD2 baculovirus expression
construct pVL1393-PKD2 has been described (7). To make a mammalian
expression construct, the PKD2-encoding fragment between
PstI (blunted) and XbaI was cut out and ligated
into vector pCI (Promega, Madison, WI) between EcoRI
(blunted) and NheI. The PKD2R742X truncation mutant in pCI was constructed by PCR and tagged at the COOH terminus by a FLAG epitope. To obtain the GST fusion constructs, DNA fragments
corresponding to various murine polycystin-1 carboxyl tail (P1CT)
regions were PCR-amplified with Vent DNA polymerase (New England
Biolabs, Beverly, MA) by introducing a BamHI site at the
5'-end and a translation stop codon at the 3'-end. The PCR fragments
were digested with BamHI and phosphorylated by T4
polynucleotide kinase at the 3'-end. The resulting fragments were
ligated into pGEX-2T (Amersham Biosciences) between the
BamHI and SmaI sites. The
GST·P1CC·Q4215Pconstruct was made by two steps of PCR. In
the first step, two separate PCR reactions were performed on the
GST·P1CC template to obtain two overlapping PCR fragments where the
mutation was introduced in the overlapping region. The two PCR
fragments were then used together as a template for the second step of
PCR using two end primers that flank the subcloning sites in
vector pGEX-2T. The oligonucleotides for the two end primers are
5'-CAGCAAGTATATAGCATGGC-3' (sense) and 5'-CAAGCTGTGACCGTCTCC-3'
(antisense). The two overlapping mutant primers are
5'-GGTCAAACGGGACAAGCAGACTTTC-3' (antisense) and
5'-GCTTGTCCCGTTTGACCGACT-3' (sense). The final PCR product was
digested with BamHI and EcoRI and ligated into
pGEX-2T between the same sites. The sequences of all constructs were
confirmed by the DNA sequencing facility at Massachusetts General Hospital.
Protein Expression and Isolation--
Plasmids for GST fusion
constructs were transformed into the bacterial strain BL21(DE3)
(Novagen, Milwaukee, WI) for protein expression. The bacteria were
grown in LB broth to an optical density of about 0.3 before
isopropyl-1-thio- In Vitro Transcription-Translation of FLAG-tagged
Polycystin-2--
In vitro transcription-translation
reactions were carried out on pCI-PKD2 and pCI-PKD2R742X for the
wild-type and R742X polycystin-2, respectively, using a
TNT-T7 coupled reticulocyte lysate system (Promega). To
determine the presence of the in vitro translation products,
one set of reactions was performed using [35S]methionine
(Amersham Biosciences) as a protein label. The reaction products were
diluted with 400 µl of phosphate-buffered saline containing 1%
Triton X-100 and 1× protease inhibitor mixture (Roche Diagnostics).
Anti-FLAG M2 antibody-bound protein G beads (10 µl, Sigma) were added
to the mixture to immunoprecipitate the translated products. The
proteins were released from the beads in boiling Laemmli sample buffer
containing 5% 2-mercaptoethanol, resolved on 4-12% SDS-PAGE, and
detected by autoradiography.
Ion Channel Reconstitution--
Lipid bilayers were formed with
a mixture of synthetic phospholipids (Avanti Polar Lipids, Birmingham,
AL) in n-decane as recently reported (7). The lipid mixture
was made of 1-palmitoyl-2-oleoyl phosphatydil-choline and
phosphatydil-ethanolamine in a 7:3 ratio. The lipid solution (~20-25
mg/ml) in n-decane was spread with a glass rod over the
250-µm diameter aperture of a polystyrene cuvette (CP13-150) of a
bilayer chamber (model BCH-13, Warner Instruments Corp.). Both sides of
the lipid bilayer were bathed with a solution containing 10 mM MOPS-KOH and 10 mM MES-KOH, pH 7.40, and
10-15 µM Ca2+. The final K+
concentration in the solution was ~15 mM. KCl was further
added to the cis compartment where membrane vesicles were
added so that final concentrations of 150 mM
K+, and 135 mM Cl Data Acquisition and Analysis--
Holding potentials were
applied from the trans chamber with either a DC voltage
source or a wave function generator with the opposite, cis
side, defined as virtual ground. Unless otherwise stated, a
cis minus trans voltage convention was utilized
throughout the study. Bilayer formation was monitored by applying a 2.5 mV peak-to-peak, 20 Hz triangular wave with a typical membrane
capacitance of 100-200 picofarads. All the experiments were performed
at room temperature (20-25 °C). Electrical signals were recorded
using a current-to-voltage converter with a 10 Gohm feedback resistor. Output (voltage) signals were low-pass filtered at 700 Hz ( Protein Preparations and in Vitro Translation Products--
To
study modulation of the channel properties of polycystin-2 by P1CT,
different portions of the murine polycystin-1 cytoplasmic region were
expressed as GST fusion proteins and purified for functional evaluation
(Fig. 1, A-C). These fusion
constructs included GST·P1CN, harboring the 84 N-terminal amino acids
of the polycystin-1 carboxyl-tail, and GST·P1CC, containing the 77 amino acids encompassing the coiled-coil motif known to interact with
polycystin-2. GST·P1CC·Q4215P, which harbors the mutation
equivalent to an ADPKD-causing mutation in human polycystin-1, Q4224P
(19), was also constructed. The Gln to Pro mutation in the
second seven-amino acid repeat of the coiled-coil motif is predicted to
disrupt the continuity of the helix. Full-length wild-type polycystin-2
and its ADPKD-causing truncation mutant R742X were in vitro
translated (Fig. 1D) and reconstituted in a lipid bilayer
system as reported recently (7, 17).
Single Channel Currents of Wild-type and R742X
Polycystin-2--
Wild-type and R742X-polycystin-2 ion channel
activity was assessed in the presence of a chemical gradient with 150 mM KCl in the cis side and 15 mM KCl
in the trans side of a reconstitution chamber (7). Wild-type
polycystin-2 showed single-channel activity as previously reported (7,
17) (Fig. 2A). The
R742X mutant also exhibited spontaneous channel activity in
agreement with a recent report (15). Similar to the wild-type,
R742X showed two most common single conductance substates of
77.2 ± 4.32 pS (n = 15) and 25.2 ± 1.19 pS
(n = 36) (Fig. 2B). However, the high conductance state, occasionally seen in the in vitro
translated wild-type polycystin-2 and most frequently in the endogenous
channel of human syncytiotrophoblast (hST) (7), was not observed in R742X. R742X polycystin-2 showed a smaller
subconductance state of 16.5 ± 0.87 pS (n = 24, Fig. 2B), which was not obvious in the wild-type channel.
R742X also tended to close more frequently than the
wild-type channel.
All in vitro translated wild-type polycystin-2 spontaneously
inactivated, in contrast to the stable channel function observed with
the human syncytiotrophoblast native protein (7). Spontaneous ion
channel inactivation of wild-type polycystin-2 occurred in 151 ± 29 s (n = 15) with varying times ranging from 15 to 440 s. Interestingly, ion channel inactivation could be
induced, in most cases, by switching the holding potential to negative
values. Voltage-induced inactivation occurred in 27.4 ± 5.54 s (n = 8). The time needed to elicit voltage
inactivation was statistically shorter (p < 0.05) than
that for spontaneous inactivation. In the only two cases in which
channel activity was not completely inactivated by voltage, channels
later inactivated spontaneously. Inactivated channels (by either
method) failed to restore ion channel activity within 30 min after
returning to positive holding potentials. A similar pattern of
inactivation was observed in R742X polycystin-2 (data not shown).
Modulation of Polycystin-2 Channel Activity by P1CC--
To
examine the regulatory role of the polycystin-1 carboxyl-tail on the
channel function of polycystin-2, the following experimental protocol
was adopted. Single channel currents of wild-type polycystin-2 in a
lipid bilayer membrane were first examined at the beginning of the
experiment. Membranes showing spontaneous channel activity were
inactivated either by voltage switch or spontaneously and then tested
for response to various P1CT fusion proteins. During this period, a
GST·P1CC fusion protein was added to the cis (but not
trans) side of the reconstitution chamber. Proteins were
added either to the bulk of the solution or, occasionally, directly to
the surroundings of the chamber orifice holding the lipid
bilayer membranes ("painting"). Fig.
3 shows the restoration of wild-type polycystin-2 ion channel activity by painting GST·P1CC from the cis side of the chamber. The addition of the
GST·P1CC·Q4215P to inactivated wild-type polycystin-2 did not
reactivate channel activity, thus establishing the specificity of the
stimulatory effect by wild-type P1CC (Fig.
4).
The addition of GST·P1CC induced a 5,070 ± 2,110%
(n = 5, p < 0.001, Fig.
5) increase in the mean currents compared
with those of the inactivated membranes. In contrast, the addition of
either GST·P1CTN, GST·P1CC·Q4215P, or GST alone had no effect.
The painting of either GST or GST·P1CC to membranes lacking
polycystin-2 did not produce any channel activity (data not shown).
Wild-type polycystin-2 channel activity was restored by P1CC in
22.3 ± 8.34 s (mean ± S.E., n = 5, Figs. 3 and 5), except for two instances where channel activity was
restored in 225 and 350 s, respectively. Polycystin-2 channel
activity restored by GST·P1CC did not inactivate for up to 15 min,
suggesting that the peptide prevented the spontaneous inactivation of
polycystin-2 channel function (not shown).
To further confirm the stimulatory effect of P1CC on wild-type
polycystin-2, a different experimental protocol was carried out. Equal
volumes of a polycystin-2 in vitro translation product were
incubated with equimolar amounts of the different P1CT fusion proteins.
Polycystin-2 ion channel activity was then measured as the mean current
per membrane integrated over the initial 12.5 s after membrane
reconstitution (Fig. 6). Typically, most
reconstituted complexes showed no channel activity (Fig. 6,
A and B). The polycystin-2·GST·P1CN complex
had a small increase of 206 ± 46.3% over the GST (control) complexes (mean ± S.E., n = 52, p < 0.05). The lipid bilayers reconstituted with the
polycystin-2·GST·P1CC mixture, however had a 2,730 ± 730%
(n = 30, p < 0.02) increase in channel
activity compared with that reconstituted with the mixture containing
GST alone. Complexes containing P1CC Q4215P were without effect (Fig. 6). The data indicate that the interaction between polycystin-1 and -2 facilitates the formation of active polycystin-2 channels.
Modulation of Polycystin-2 Channel Activity by Polycystin-1
Requires the Respective Interacting Segments in Both Proteins--
To
further substantiate the importance of polycystin-1/-2 interaction on
the channel activity of the latter, R742X polycystin-2 was tested for
its response to P1CC after channel inactivation. Although displaying
spontaneous channel activity, R742X failed to reactivate
following the addition of GST·P1CC after spontaneous inactivation
(Fig. 7, A and B),
indicating that the cytoplasmic tail of polycystin-2 mediates the
activation and stabilization by P1CC.
The present findings indicate that an interaction with
polycystin-1 activates and stabilizes the channel activity of wild-type polycystin-2 when reconstituted in a lipid bilayer system. In contrast
to the stable channel function of the native protein in the human
syncytiotrophoblast (7), the in vitro translated protein
spontaneously inactivates, a phenomenon that is also induced by
negative potentials. The polycystin-1/-2 interaction reversed both
spontaneous and voltage-induced inactivation of polycystin-2. This
regulation is largely achieved by the membrane-distal segment of
polycystin-1, which contains the polycystin-2-interacting region. The
proximal segment had little effect, and the disease-causing Q4215P
mutant completely lacked this effect. The polycystin-2 R742X
mutant, lacking most of the cytoplasmic tail, displayed spontaneous
channel activity as reported previously (15) but failed to reactivate
after the addition of the wild-type polycystin-1 carboxyl tail.
A number of functional consequences of polycystin-1/-2 interaction have
been suggested. It has been reported that polycystin-1 may be required
for the trafficking of polycystin-2 to the cell surface of Chinese
hamster ovary cells overexpressing both proteins (13). Recently, an
interaction between the two proteins was linked to the Jak-2-mediated
anti-proliferative activity of polycystin-1 (4), and the interaction
between polycystin-1 and -2 was found to block G protein signaling by
polycystin-1 heterologously expressed in neurons (14). The data in this
report present the first direct demonstration of the
electrophysiological significance of this interaction, namely the
activation and stabilization of polycystin-2 channel activity by
polycystin-1, which may underlie some of the above effects. The
resulting increase in channel activity by the polycystin-2/-1 complex
may be critical for the activation of cation-dependent
signaling pathway(s) normally associated with various cell functions
including cell cycle, vesicle trafficking, and ion transport.
Functional interruption of this interaction may account for
abnormalities in protein targeting, cell growth, and ion transport,
characteristic of ADPKD.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-D-galactopyranoside (IPTG) was added
to a final concentration of 0.2 mM. Induction was allowed
for 3-4 h at 37 °C, followed by centrifugation to collect the
bacteria. To isolate the proteins, bacteria were lysed by sonication in
10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1.5% Sarkosyl plus protease
inhibitors (Roche Diagnostics). Cell debris was removed by a 30 min
centrifugation at 10,000 rpm in a Sorval SLA-600TC rotor. The
supernatant was then incubated with glutathione beads (Amersham
Biosciences) for 30 min at 4 °C. The beads were washed three times
with 10 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, and three times with the same
buffer containing no Triton X-100. Proteins were eluted from the beads
in a solution containing 50 mM Tris-HCl, pH 8.0, and 20 mM reduced glutathione (Sigma). The protein eluates were
dialyzed against 20 mM Tris-HCl, pH 8.0, 1 mM
EDTA, and 1 mM dithiothreitol overnight with three changes
of the buffer. The final protein preparation was mixed with an equal
volume of 50% glycerol and either used freshly or stored at
80 °C. Protein concentration was determined by the Bradford method
(16). The expression level of GST·P1CTN was low.
were
achieved in this side of the chamber.
3 dB) with
an eight pole, Bessel-type filter (Frequency Devices, Haverhill, MA).
Signals were displayed on an oscilloscope, and channel recordings were
simultaneously digitized with a pulse code modulator (Sony PCM-501 ES),
and stored in videotapes with a video cassette recorder (Toshiba HQ).
Data were later transferred for subsequent analysis at 4 kHz to a
personal computer. Single channel current tracings were further
filtered (see "Results") for display purposes only. Unless
otherwise stated, pCLAMP Version 5.5.1 (Axon Instruments, Foster City,
CA) was used for data analysis, and Sigmaplot Version 2.0 (Jandel
Scientific, Corte Madera, CA) was used for statistical analysis and
graphics. Single channel conductances (
) under asymmetrical
conditions were calculated by the best fitting of current-to-voltage
experimental data to the Goldman-Hodgkin-Katz (GHK) equation, such that
= I/(Vh
Er) was obtained from Equation 1,
(Eq. 1)
where i (species in trans compartment) and
j (species in cis compartment) represent the
cation species (K+, Na+) on either side of the
membrane; Vh is the holding electrical potential
in mV; zi and zj, the
charge for species i and j, respectively; Ctrans and Ccis are the
trans and cis concentrations of i and j, respectively, and
= RTVh/ziF, and
= RTVh/zjF.
Pi and Pj represent the
permeability coefficient for either species i or j, respectively. Whenever a single salt was present in the
preparation (i.e. KCl), i and j
correspond to the cation and anion, respectively. F,
R, and T have their usual meanings. Each
reconstituted lipid-protein membrane preparation contained at least
three variables, namely, the number of active ion channels, different
single channel currents (due to multiple substates (7), and distinct
open probabilities under each condition (17). Thus, the data were
analyzed as follows; the mean membrane current for each membrane
preparation was determined prior to averaging data for each condition
separately. The averaged data represented I = Nipo, encompassing N, the total
number of active channels in the preparation, i, the average
single channel current for the channel species, and
po, the open probability of the open channel at
a given holding potential. Unless otherwise stated, data were obtained
at a holding potential of 40 mV. Whenever indicated, statistical
significance was obtained by unpaired t test comparison of
sample groups of similar size (18). Average data values were expressed
as the mean ± S.E. (n) under each condition, where
n represents the total number of experiments analyzed.
Statistical significance was accepted at p < 0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Polycystin-1 tail constructs and
in vitro translation products.
A, amino acid sequence of the cytoplasmic tail of murine
polycystin-1 from amino acids 4096 to 4293. The coiled-coil motif is
underlined, and Gln-4215 is marked by an
asterisk. B, schematic representation of the
glutathione-S-transferase (not shown) fusion constructs of
P1CT. PICN encompasses amino acids from 4098 to 4181, and P1CC from
4173 to 4249. The P1CC construct contains the coiled-coil motif from
4205 to 4239. P1CC·Q4215P carries the Gln to Pro mutation at amino
acid 4215, equivalent to the ADPKD causing mutation Q4224P in human
polycystin-1. C, two representative preparations of GST
fusion proteins stained by Coomassie Blue G-250. More than three
independent protein preparations were tested for each construct. The
numbers indicate molecular mass in kDa. The full-length P1CT
fusion protein was subject to degradation and not tested. D,
[35S]methionine-labeled in vitro translation
products of the wild-type and R742X mutant
polycystin-2.
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Fig. 2.
Single channel currents of the wild-type and
R742X mutant polycystin-2. A, single
channel currents of the wild-type polycystin-2 (top) and
R742X mutant (bottom). In vitro
translated wild-type and R742X mutant polycystin-2 were
reconstituted in a lipid bilayer system. Data were obtained in the
presence of asymmetrical KCl (150 mM) in the cis
side and 15 mM K+ in the trans side
and are representative of 24 and 36 experiments, respectively.
Dashed lines in between asterisks
indicate substates. B, I-V relation for the wild-type and
R742X mutant polycystin-2. Current-to-voltage relationships
were obtained from single channel tracings at different holding
potentials and fitted to the Goldman-Hodgkin-Katz equation as indicated
under "Materials and Methods." The two main single channel
conductance substates observed in R742X polycystin-2 were
77.2 pS, and 25.2 pS (thin lines), comparable to
the conductance levels observed in the wild-type polycystin-2
(dashed lines). A third, smaller conductance
state of 16.5 pS was unique to the mutant.
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Fig. 3.
Effect of P1CC fusion protein on the
wild-type polycystin-2 channel activity. Top
panel shows the strategy for testing the effect of P1CC on
polycystin-2 channel function. Polycystin-2 single channel activity was
observed at the beginning of the experiment (top
tracing, left). Inactivation occurred either
spontaneously (shown at the beginning of the top
right tracing) or after holding the membrane to
negative potentials. Addition of GST·P1CC restored polycystin-2
channel activity (top tracing, right).
The middle panel shows representative expanded
tracings for each condition, including spontaneous channel activity
(a), spontaneous inactivation (b), and two
subsequent channel reactivation levels after the addition of P1CC
(c and d). Dashed lines
indicate substates. The bottom panel shows the
corresponding all-point histograms from tracings a through
d in the middle panel. Data are
representative of seven experiments.
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Fig. 4.
Effect of P1CC·Q4215P fusion protein on the
wild-type polycystin-2 channel activity. Top, wild-type
polycystin-2 single channel activity was inactivated by shifting the
voltage to 20 mV from a holding potential of 40 mV, followed by
returning to positive potential. The addition of P1CC·Q4215P failed
to restore channel activity. Bottom, expanded tracings from
spontaneous channel activity (a), immediately after voltage
inactivation (b), and after the addition of P1CC·Q4215P
(c). Data are representative of three experiments.
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Fig. 5.
Effect of P1CT-derived fusion proteins on the
wild-type polycystin-2 channel activity. Mean currents per
membrane of the wild-type polycystin-2 at the beginning of successful
channel reconstitution (CTR), after inactivation
(Inact), and after the addition of P1CT fusion protein
GST·P1CC (P1CC) or GST·P1CC·Q4215P
(Q4215P). Data represent mean ± S.E. of seven
experiments except for Q4215P (three experiments). No statistically
significant difference was found between CTR and P1CC.
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Fig. 6.
Effects of P1CT fusion proteins on the
wild-type polycystin-2 channel activity. Wild-type polycystin-2
was premixed with P1CT fusion proteins and reconstituted into a lipid
bilayer. Spontaneous channel activity was measured for the first
12.5 s after reconstitution in the presence of asymmetrical KCl
(150 mM) in the cis side, and 15 mM
K+ in the trans side. Typically, most
reconstituted membranes showed no channel activity. A,
representative tracings for polycystin-2 premixed with GST, GST·P1CN
(P1CN), GST·P1CC (P1CC), or GST·P1CC·Q4215P
(Q4215P). B, mean currents per membrane of the
polycystin-2 premixed with different P1CT fusion proteins. Data are the
mean ± S.E. of 30, 52, 16, and 86 experiments, for GST, P1CN,
P1CC, and Q4125P, respectively. * and ** indicate significance at
p < 0.5 and p < 0.01, respectively,
compared with GST alone.
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Fig. 7.
Effects of P1CC on R742X
polycystin-2. A, single channel currents observed
at positive holding potentials (+30 mV, top
tracing); the expanded section (bold
line) of the tracing shows three channels. Middle
tracing shows the spontaneous inactivation of the
R742X channels. The addition of GST·P1CC (P1CC)
did not reactivate R742X polycystin-2 (bottom
tracing). B, mean currents per membrane of spontaneous
channel activity of the R742X mutant at the beginning
(CTR), after inactivation (Inact), and after the
addition of P1CC. Data represent the mean ± S.E. of seven paired
experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* These studies were supported in part by Program Project Grant DK54711 (to G. M. X., M. E., and M. A. A.).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.
§ These authors contributed equally to this work.
** To whom correspondence may be addressed: Renal Unit, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. Tel.: 617-726-5663; Fax: 617-726-5671; E-mail: arnaout@receptor. mgh.harvard.edu.
To whom correspondence may be addressed: Renal Unit,
Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129. Tel.: 617-726-5640; Fax: 617-726-5669; E-mail: cantiello@helix. mgh.harvard.edu.
Published, JBC Papers in Press, October 28, 2002, DOI 10.1074/jbc.M209996200
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ABBREVIATIONS |
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The abbreviations used are: ADPKD, autosoma dominant polycystic kidney disease; GST, glutathione S-transferase; P1CT, polycystin-1 carboxyl tail; P1CC, coiled-coil containing segment of P1CT; PICN, polycystin-1 that harbors 84 N-terminal amino acids; MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid.
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