P2U purinergic receptor
inhibits apical IsK/KvLQT1
channel via protein kinase C in vestibular dark cells
Daniel C.
Marcus1,
Hiroshi
Sunose1,
Jianzhong
Liu1,
Zhijun
Shen1, and
Margaret A.
Scofield2
1 Biophysics Laboratory, Boys
Town National Research Hospital, Omaha 68131; and
2 Molecular Pharmacology
Laboratory, Department of Pharmacology, Creighton University School
of Medicine, Omaha, Nebraska 68178
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ABSTRACT |
Vestibular dark
cells (VDC) are known to electrogenically secrete
K+ via slowly activating
K+
(IsK) channels, consisting of
IsK regulatory and KvLQT1 channel subunits, and the associated short-circuit current
(Isc) is
inhibited by agonists of the apical
P2U
(P2Y2) receptor (J. Liu, K. Kozakura, and D. C. Marcus. Audit.
Neurosci. 2: 331-340, 1995). Measurements of
relative K+ flux
(JK) with a
self-referencing K+-selective
probe demonstrated a decrease in
JK after apical
perfusion of 100 µM ATP. On-cell macropatch recordings from gerbil
VDC showed a decrease of the IsK
channel current
(IIsK) by 83 ± 7% during pipette perfusion of 10 µM ATP. The magnitude of the
decrease of Isc
by ATP was diminished in the presence of inhibitors of phospholipase C
(PLC) and protein kinase C (PKC), U-73122 and GF109203X. Activation of
PKC by phorbol 12-myristate 13-acetate (PMA, 20 nM) decreased
IIsK by 79 ± 3% in perforated-patch whole cell recordings, whereas the inactive
analog, 4
-PMA, had no effect. In contrast, elevation of cytosolic
Ca2+ concentration by A-23187
increased the whole cell
IIsK . The expression of the isk gene transcript was confirmed, and the
serine responsible for the species-specific response to PKC was found to be present in the gerbil IsK
sequence. These data provide evidence consistent with a direct effect
of the PKC branch of the PLC pathway on the
IsK channel of VDC in response to
activation of the apical P2U
receptor and predict that the secretion of endolymph in the human
vestibular system may be controlled by PKC in the same way as in our
animal model.
P2Y2 receptor; phospholipase
C; perforated-patch whole cell voltage clamp; minK channel; gerbil; self-referencing probe; slowly activating potassium channel
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INTRODUCTION |
VESTIBULAR DARK CELLS (VDC) secrete
K+ in the vestibular labyrinth to
a luminal concentration that is unusually high for a vertebrate
extracellular space (~140 mM) and the luminal
K+ is used to carry the
transduction current through the sensory hair cells. The constitutive
ion transport mechanisms employed by VDC to take
K+ across the basolateral membrane
and to secrete it across the apical membrane have been established (see
Ref. 15 for review). These include the
Na+-K+-ATPase,
Na+-Cl
-K+
cotransporter, and Cl
channels in the basolateral membrane and
K+-selective channels of the
slowly activating K+
(IsK, or minK) type in the apical
membrane. IsK channels have been
shown in other systems to consist of
IsK regulatory and
KvLQT1 channel subunits (2, 23).
The transepithelial short-circuit current
(Isc) has been
shown to be accounted for by electrogenic
K+ secretion (18, 19) and has
recently been found to be reduced in the presence of apically perfused
extracellular nucleotides (13). The mediator of this effect was
determined to be a purinergic receptor of the
P2U
(P2Y2) subtype in the apical
membrane. The present experiments were designed to test the hypothesis
that activation of the apical P2U
receptors results in inhibition of the
K+ secretory flux
(JK) by
inhibition of the current through the apical
IsK channels and to determine the
signal pathway between these two events. In other systems,
P2U receptors are coupled to
phospholipase C (PLC), which activates parallel signal pathways, resulting in elevation of cytosolic
Ca2+ via inositol
1,4,5-trisphosphate
(InsP3) and in
phosphorylation of effector proteins via protein kinase C (PKC) (4, 6). The present results are consistent with the phosphorylation of the
IsK channel by PKC as the signal
mechanism of the apical P2U receptor, rather than elevation of cytosolic
Ca2+.
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METHODS |
Tissue preparations.
Gerbils were anesthetized with pentobarbital sodium (50 mg/kg, ip) and
decapitated. The temporal bone was removed, and VDC epithelium was
dissected without enzymatic treatment at 4°C in solution 2 (Table
1) as described previously (31). The tissue was either transferred to a recording chamber continuously perfused at
37°C or frozen in liquid nitrogen within 10 min of death for reverse transcription-polymerase chain reaction (RT-PCR). The heart was
cut and frozen on dry ice or liquid nitrogen within 5 min of death.
Solutions and chemicals.
The compositions of solutions are listed in Table 1. Nystatin (200 µg/ml; Sigma, St. Louis, MO) was dissolved with sonication in the
pipette solution (solution 4, Table 1)
just before use. ATP (Sigma) was dissolved directly in pipette and bath
solution, whereas U-73122, U-73343 (BioMol, Plymouth Meeting, PA),
A-23187, phorbol 12-myristate 13-acetate (PMA),
4
-phorbol 12,13-didecanoate (4
-PMA) (Calbiochem, La Jolla, CA),
and GF109203X (Tocris Cookson, St. Louis, MO) were predissolved in
dimethyl sulfoxide (DMSO). The final concentration of DMSO was 0.1%.
All other chemicals for the electrophysiological experiments were
purchased from Sigma or Fluka (Ronkonkoma, NY).
Self-referencing
K+-selective
probe.
The self-referencing probe techniques used were nearly identical to
those previously described (14, 18). Signals were sampled from the
probe amplifier with a 16-bit analog-to-digital convertor
(CIO-DAS1602/16, ComputerBoards, Mansfield, MA), and the probe was
moved on manipulators (Applicable Electronics, Forest Dale, MA) by a
486-based computer and specialized probe software (ASET version 1.0, Science Wares, East Falmouth, MA). Tissues were mounted in the
micro-Ussing chamber, and the relative
JK was monitored
at 24°C with a K+-selective
microelectrode moved with an excursion of 25 µm perpendicular to the
plane of the tissue (10, 11). The electrode voltage was sampled at 200 Hz for 1 s at each position after a 1-s waiting period and without
automatic amplifier feedback. The microelectrodes were constructed from
borosilicate glass capillary (1.5 mm OD, 0.84 mm ID)
pulled to a tip of ~3 µm ID and silanized with
hexamethyldisilazane. The tips contained a column of
K+-selective ligand (no. 60398, Fluka Chemical) ~150 µm long, and the electrode was backfilled with
100 mM KCl and 0.5% agar. The reference was Ag/AgCl with a bridge of 3 M NaCl and 3% agar. Electrodes were only used if the slope was at
least 56 mV/decade in 1 and 10 mM KCl solutions. The probe was located
near the thin connective tissue overlying the basal membrane of the VDC
epithelium such that the signal under control conditions was >30
times the noise level. Data are expressed as voltage difference
detected by the K+-selective
electrode and summarized as percent of the reading under experimental
conditions compared with that under control conditions.
PCR amplification, subcloning, and sequencing.
Genomic DNA for the mouse and rat
IsK consists of a single exon
encoding the entire translated region (open-reading frame) and the
3'-untranslated region and two alternate exons, which are
generated from multiple transcription start sites and encode the
5'-untranslated region (12). A set of primers [sense:
5'-ACC CTG GGC ATC ATG CTG AG-3', anti-sense: 5'-GCC
GCC TGG TTT TCA ATG AC-3'] was designed (A. F. Ryan, Univ.
of California, La Jolla, CA) and synthesized on an Applied Biosystems
392 oligonucleotide synthesizer (Creighton Molecular Biology Core
Facility). The sequence of the primers was based on the known sequences
in the coding region of the rat and mouse and encompassed the consensus
sequence for phosphorylation by PKC (S/T X R/K). The primers were
expected to yield an RT-PCR product of 170 base pairs (bp).
Extraction of total RNA.
Total RNA was extracted from samples of gerbil heart, blood, and
vestibular labyrinth (mostly ampullae and utricle) by methods similar
to those described previously (24). Within 5 min of death, the hearts
were frozen in liquid nitrogen and pulverized in liquid nitrogen, and
100 mg of the powder were immediately transferred into 1 ml TRIzol
reagent (GIBCO BRL, Life Technologies), a monophasic solution of phenol
and guanidine isothiocyanate. The total RNA was then extracted using
TRIzol reagent according to the manufacturer's procedure. Total RNA
was precipitated by isopropanol and dissolved in ribonuclease
(RNase)-free water (diethyl pyrocarbonate-treated water). The nucleic
acid concentration was determined spectrophotometrically, the integrity
of the RNA was determined by the presence of 28S and 18S ribosomal RNA
bands by horizontal agarose (1%) gel electrophoresis, and the final nucleic acid concentration was adjusted to ~1-2 µg/µl. RNA
samples were stored at
70°C.
Vestibular labyrinth from gerbil was isolated by microdissection, and
tissues were frozen in liquid nitrogen within 12 min of death of each
animal. Tissues from eight ears were pooled in TRIzol reagent and were
triturated through a 25-gauge needle. Total RNA was then extracted
according to the manufacturer's procedure. Total RNA from 100 µl of
blood was also isolated using 1 ml of TRIzol reagent. The final
nucleotide concentration of the RNA samples from vestibular labyrinth
and blood was ~0.3 and 0.25 µg/µl, respectively. Directly before
the RT-PCR procedure, residual genomic DNA in RNA samples from heart,
vestibular labyrinth, and blood was removed by treatment with
amplification-grade RNase-free deoxyribonuclease I (GIBCO BRL, Life
Technologies) for 30 min at room temperature followed by heat
inactivation in the presence of EDTA, according to the protocol
specified by the manufacturer.
cDNA synthesis and PCR amplification.
Total RNA was reverse transcribed into cDNA in a 10-µl reaction. The
reaction contained 0.1-0.5 µg total RNA, 10 units RNasin (Promega), 1 mM dNTP (GIBCO BRL, Life Technologies), 25 units Moloney
murine leukemia virus (MMLV) reverse transcriptase (Perkin-Elmer), 2.5 mM MgCl2 (GIBCO BRL, Life
Technologies), 25 pmol oligo(dT), 20 mM
tris(hydroxymethyl)aminomethane (Tris) · HCl, and
50 mM KCl. Tris · HCl and KCl were added from a
10× PCR buffer (GIBCO BRL, Life Technologies). The RT reaction
was incubated at room temperature for 10 min, at 42°C for 50 min,
at 99°C for 5 min, and at 5°C for 5 min.
The 50-µl PCR mixture contained the 10 µl RT reaction mixture in
addition to 25 pmol each of antisense and sense primers for IsK and 1.25 units
Taq DNA polymerase (GIBCO BRL, Life
Technologies). The final concentrations of
MgCl2, KCl, and
Tris · HCl were adjusted to 2.5, 50, and 20 mM,
respectively. The PCR mixture was incubated as follows: 1 denaturation
cycle for 3 min at 95°C; 45 amplification cycles consisting of
denaturation for 1 min at 95°C, annealing for 1 min at 63°C,
and extension for 1 min at 72°C; and one extension cycle for 5 min
at 72°C in a Perkin-Elmer thermocycler 480. PCR products were
analyzed by horizontal electrophoresis in 2.5% agarose gels and
visualized by ethidium bromide.
Cloning and sequencing of amplified cDNA fragments.
Amplified cDNA fragments were extracted from the agarose gels using the
QIA quick gel extraction kit (Qiagen) and cloned into a pCR2.1 vector
with a TA cloning kit (Invitrogen). Recombinant plasmids were isolated
from the colonies using the standard alkaline lysis procedure, purified
by phenol/chloroform extraction, and precipitated and washed with
ethanol. Insertion of the PCR product into the plasmid was confirmed by
restriction endonuclease digestion with
EcoR I and subsequent horizontal gel
electrophoresis. The recombinant double-stranded plasmid served as a
template for cycle sequencing using M13 forward and reverse primers and
fluorescence-based dideoxy nucleotides (PRISM Ready Reaction dye deoxy
terminator cycle sequencing kit, Perkin Elmer). The sequence was then
determined using the ABI model 373 DNA sequencer (Applied Biosystems,
Creighton Molecular Biology Core Facility).
Micro-Ussing chamber.
The methods were described previously (16). Briefly, tissue was placed
in a micro-Ussing chamber, and the seal to the aperture (80 µm
diameter) between two hemichambers was made with the apical side of the
VDC epithelium. The apical and basolateral sides of the tissue were
perfused independently, and exchange of solution (24 or 37°C) on
each side was complete within 1 s. Transepithelial voltage
(Vt) was
measured between calomel electrodes connected to the hemichambers via
flowing 1 M KCl bridges. Transepithelial resistance
(Rt) was
obtained from the voltage change induced by current pulses (50 nA for
34 ms at 0.3 Hz). Sample and hold circuitry was used to obtain a signal
proportional to
Rt. The
equivalent Isc
was derived from
Vt and
Rt
(Isc = Vt/Rt).
Isc and
Rt were normalized for the area defined by the aperture of the micro-Ussing chamber.
Macropatch clamp.
The macropatch technique was described previously (17). Pipettes
(3-6 µm ID) were made from Corning 7052 glass capillary with a
two-stage puller and a microforge. Pipette tips were coated with a 2:1
mixture of
-tocopherol acetate and heavy mineral oil and filled with
NaCl pipette solution (solution 3,
Table 1). Tissues were folded into a loop with the apical membrane
facing outside and mounted in the recording chamber. High-resistance seals (2-8 G
) were made to the apical membrane of VDC, and
currents were recorded in the cell-attached configuration. The
recording technique was identical to that previously reported (17).
The patch-pipette perfusion technique was described previously (25). In
brief, an internal perfusion pipette was connected to either of two
solution vials via a valve and the internal pipette positioned within
the macropatch pipette. Positive pressure applied to the vials drove
the selected solution through the patch pipette. The arrival of new
solution was delayed by ~2 min due to diffusion over ~300 µm
between the outlet of the internal perfusion pipette and the membrane
patch.
Perforated-patch whole cell clamp.
The perforated-patch whole cell clamp technique was chosen over the
conventional whole cell technique to avoid rundown of channel activity
(17). Perforated-patch experiments were conducted in the absence of
Cl
to reduce the
contribution of the basolateral
Cl
conductance to the whole
cell conductance and thereby to increase the ratio between the cell
membrane resistance and the access resistance. This was a necessary
condition to effectively clamp the membrane potential in these cells.
An unusually high Cl
conductance in the basolateral membrane of these cells is responsible for an extremely low resistance of the cell membrane, typically <20
M
/cell in the presence of 150 mM
Cl
in the bathing medium
(32). K+ secretion by the apical
conductive pathway was supplied under whole cell conditions by the
pipette electrolyte diffusing through the nystatin perforations.
The tissues were continuously perfused with
Cl
-free bath solution
(solution 2, Table 1). The internal
diameter of the tip of the patch pipette was 1-2 µm with a
resistance of 8-12 M
in the
Cl
-free bath solution. The
tip of the patch pipette was backfilled with a 1-cm column of
K+-rich,
Cl
-free solution
(solution 4, Table 1) containing 200 µg/ml nystatin. The rest of the pipette was backfilled with 15 mM
Cl
solution
(solution 5) to stabilize the
Ag/AgCl junction. Gigaohm seals were made to the apical membrane, after
which the membrane patch was perforated by insertion of the nystatin.
After a stable whole cell configuration was established, the access
resistance (31 ± 1 M
, n = 23)
and the membrane capacitance (53 ± 23 pF, n = 23) were measured with the
circuitry in the patch amplifier (Dagan 3900 and Axon Instruments
200A). Capacitance was nearly fully compensated and resistance
compensated by typically 65%.
The seemingly large capacitance observed in the present study is
consistent with the large membrane area due to extensive basolateral
infoldings. Currents from individual VDC can be recorded in the native
epithelium, since no gap junctions have been found among them (9).
Voltage protocols.
Voltages are expressed with the usual convention of the intracellular
compartment with respect to the extracellular side. In on-cell
experiments, voltages are not corrected for the cell membrane
potential. The membrane potential is about
18 mV in vitro with
NaCl physiological saline bathing both sides of the epithelium (32).
The whole cell voltage protocol has been described in detail previously
(27), and the protocol was repeated every 30 s to record the time
course of changes during experimental treatments. For each instance of
the protocol, the cell was first held at 0 mV; a tail-current
I-V
plot was produced from four pulses of 10-20 ms each. The
IsK channel current
(IIsK) was then
deactivated by holding the cell at
50 mV for 4 s (17), and a
tail-current I-V
plot was produced of the "leak" current. The currents were sampled for the
I-V
plots after decay of capacitive transients, and the active currents
were corrected for the leak currents. The interval between protocols
was chosen to allow ample time for recovery of channel activity at the
activating holding potential (0 mV). Measurements of currents under
both control and experimental conditions in each cell allowed the
application of paired statistics.
The voltage protocol used for on-cell macropatch recordings was
similar, but repeated every 15 s. This interval was found to be
adequate for full recovery of activation under these conditions. The
deactivating voltage was
40 mV (the cell membrane voltage hyperpolarized the membrane an additional amount of ~18 mV). Currents at two voltages (
40 and 0 mV) were used to estimate the leak conductance. Parameters derived from these recordings include the
current and conductance of the apical
IsK channels
(IIsK and gIsK,
respectively), the reversal voltage without correction for leak
(Vr), and the
time constant of deactivation
(
off).
Data presentation and statistics.
Data are expressed as means ± SE
(n = number of cells). Student's
t-test of paired samples was applied,
and increases or decreases in parameters were considered significant at
a level of P < 0.05.
 |
RESULTS |
Inhibition of JK by
extracellular ATP.
Because it was necessary to measure
JK at 24°C,
we controlled for the temperature dependence of the response to
P2U receptor activation by first
comparing the response of
Isc at 24 and
37°C. Apical perfusion of ATP (100 µM) for 2 min at 24°C led
to a maximal decrease in
Isc by 40.8 ± 3.0% (n = 6) below the control value (Fig.
1A,
Table 2). This effect was
slower than that seen at 37°C (13) but similar in magnitude; it
occurred with a delay of 13 ± 1 s, reaching its peak value at 148 ± 9 s. Similarly, apical perfusion of ATP (100 µM) for 3 min at
24°C led to a maximal decrease in
JK by 40.9 ± 9.2% (n = 7) below the control value (Fig. 1B). The slower and delayed
response of JK
compared with that of
Isc was most
likely related to the lack of stirring in the basolateral compartment
required for the probe measurement. Similar differences in response
times between Isc
and JK were seen
for basolateral application of adenosine 3',5'-cyclic
monophosphate (cAMP) (27). The results clearly show, however, that the
response of VDC to apical ATP involves a decrease in transepithelial
JK.

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Fig. 1.
Summaries of effects of apical perfusion at 24°C of ATP (100 µM)
for 2 min on short-circuit current
(Isc,
A) and for 3 min on
K+ flux
(JK,
B) across vestibular dark cell
epithelium. Values are means ± SE
(n = 6 and 7 for
A and
B, respectively). Error bars are shown
only at intervals for clarity.
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Table 2.
Transepithelial voltage, transepithelial resistance, and short-circuit
current in the absence or presence of ATP or drug
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Inhibition of IIsK by
extracellular ATP.
On-cell macropatch-clamp recordings were made of
IIsK,
gIsK,
Vr, and
off from VDC as described in
METHODS. Data were obtained by
averaging the last three values of the control period and of the
experimental period for each experiment.
Perfusion of ATP (10 µM) for 4 min through the patch pipette led to a
decrease in IIsK
by 83.4 ± 7.4% and in
gIsK by 48.6 ± 9.3% and to a depolarization of
Vr by 24.6 ± 5.6 mV (n = 5; Fig.
2, Table 3).
The
off could not be measured
for the entire duration of inhibition by ATP but did not change
significantly during the first 90 s, when the current had
already decreased by 58 ± 18%.

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Fig. 2.
Summary of on-cell macropatch-clamp recordings of slowly activating
K+
(IsK) channel current
(IIsK),
IsK channel conductance
(gIsK), and reversal
voltage
(Vr)
during 5-min pipette perfusion of ATP. Values are means ± SE
(n = 5).
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Inhibition of PLC reduces effect of apical ATP.
Addition of U-73122 at 4 µM to the apical perfusate had no
significant effect on
Isc
(n = 5). However, apical ATP (1 µM)
caused less of a decrease of
Isc in the
presence of U-73122 than in its absence (Fig.
3A, Table
2). ATP decreased
Isc by 24.1 ± 3.9% in the presence of U-73122 and by 32.7 ± 4.1% in its absence
corresponding to a fractional response to ATP of 73 ± 4% in the
presence of U-73122 compared with that in its absence. Raising the
concentration of U-73122 to 10 µM resulted in a further reduction of
the effect of ATP [by 52.0 ± 8.0% without U-73122 vs. 30.0 ± 6.4% (n = 6) in the presence of
U-73122], although there was also a small decrease of
Isc from the
U-73122 itself at this concentration (17.6 ± 4.3%). The fractional
response to ATP in the presence of 10 µM U-73122, 56 ± 7% of
that in the absence of U-73122, was less than that at 4 µM U-73122.
The inactive analog, U-73343 (4 µM), had no effect on
Isc, and ATP (1 µM) caused the same magnitude decrease in the absence (30.3 ± 4.2%) and presence (32.0 ± 3.9%,
n = 6; Fig.
3B, Table 2) of U-73343.

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Fig. 3.
Summary of measurements of
Isc in vestibular
dark cells showing the effect of apical perfusion of ATP (1 µM) in
the presence and absence of an inhibitor of phospholipase C, U-73122 (4 µM, A), and an inactive analog,
U-73343 (4 µM, B). Values are
means ± SE (n = 5).
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Elevation of cytosolic
Ca2+ increases
IIsK.
The effect on the
IIsK of raising
cytosolic Ca2+ was tested in whole
cell patch-clamp recordings by addition of the
Ca2+ ionophore A-23187 (5 µM) to
the bathing solution. Data were obtained from the average of the last
two values of the control and experimental periods in each experiment.
Bath perfusion of VDC with A-23187 for 3 min led to an increase in
IIsK by 43 ± 11% of the original current (n = 11) and an increase in
gIsK by 34 ± 9% of the original conductance and to a hyperpolarization of
Vr by 4.8 ± 2.0 mV (Fig. 4, Table 3). The
off did not change
significantly.

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Fig. 4.
Summary of nystatin-perforated whole cell patch-clamp recordings of
IIsK,
gIsK, and
Vr of vestibular
dark cells during 3-min bath perfusion with the
Ca2+ ionophore A-23187 (5 µM).
Values are means ± SE (n = 11).
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Activation of PKC reduces
IIsK.
The effect of stimulating PKC on the
IIsK was tested
in whole cell patch-clamp recordings by addition of the phorbol ester PMA to the bathing solution. Data were obtained from the average of the
last 2 values of the control and experimental periods in each
experiment.
Bath perfusion of VDC with PMA (20 nM) for 3 min led to a decrease in
IIsK by 79.1 ± 2.8% of the original current (n = 6) and in gIsK
by 58.1 ± 3.5% of the original conductance and to a depolarization
of Vr by 25.5 ± 3.3 mV (Fig.
5A, Table
3). There was no significant change in
off. None of these parameters
changed significantly after 3-min perfusion of 20 nM 4
-PMA, an
inactive analog of PMA (n = 6; Fig.
5B, Table 3).

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Fig. 5.
A: summary of nystatin-perforated
whole cell patch-clamp recordings of
IIsK,
gIsK, and
Vr during 3-min
bath perfusion of an activator of protein kinase C, phorbol
12-myristate 13-acetate (PMA, 20 nM; means ± SE,
n = 6).
B: similar experiments with the
inactive analog, 4 -PMA, had no effect
(n = 6).
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Sequence of gerbil cDNA at putative PKC phosphorylation site.
RT-PCR of total RNA from a tissue known to express the isk
gene (heart) resulted in a product of the expected size based on the
gene-specific primers used during amplification (Fig.
6). Furthermore, an RT-PCR product of the
same expected size was also amplified from the vestibular labyrinth RNA
and thus indicates the presence of an isk gene transcript in
this tissue. The IsK identity of
the 170-bp DNA fragments from the heart and vestibular labyrinth was
confirmed by cloning and sequencing. There is no evidence of genomic
DNA contamination as seen by the absence of the RT-PCR product in RNA
samples subjected to only the PCR in the absence of the reverse
transcriptase (MMLV). In addition, the isk gene transcript
was not a result of blood contamination of the tissues, since the same
RNA isolation procedure and RT-PCR of the isolated RNA from blood did
not result in the amplification of a 170-bp product. The sequence is
shown in Fig. 7 (GenBank no. AF029765) and
is distinguished by its high homology to the same region of rat
IsK cDNA (89% sequence identity
between the primers) and predicted amino acid composition (91%). In
particular, the PKC consensus sequence in rat is preserved in the
gerbil. The serine at position 102 in the rat
IsK amino acid sequence, which
mediates the species-specific response to PKC, was found to be present
at the analogous position in the gerbil
IsK sequence.

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Fig. 6.
Gel electrophoresis of reverse transcription-polymerase chain reaction
(RT-PCR) products from reactions with 0.5, 0.15, and 0.125 µg total
RNA from gerbil heart (H), vestibular labyrinth (VL), and blood (B),
respectively. +, Reactions performed in the presence of reverse
transcriptase; , reactions performed in the absence of reverse
transcriptase. Position of the bands for the expected lengths of the
RT-PCR products [170 base pairs (bp)] is indicated, and a
100-bp ladder marker is shown (M). Image was digitally captured and
inverted.
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Fig. 7.
Base sequence for the 170-bp segment of gerbil
IsK RT-PCR products and the
corresponding amino acid sequence. A:
diagrammatic map of the location of 170-bp segments within the open
reading frame (TM, transmembrane segment; PKC, protein kinase C
consensus sequence). B: sequence of
bases and predicted amino acids from amino acid positions 58 to 114 compared between gerbil and rat. Bars indicate grouping of bases coding
for amino acids; large letters next to bars indicate the corresponding
amino acid; , positions where bases differ between
species. Amino acids for rat sequence are only indicated at positions
that include a mark. Sequences corresponding to the
sense and antisense primers are indicated by horizontal arrows.
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Inhibition of PKC reduces response to ATP.
It would be expected that if PKC is a mediating signal element in the
effect of extracellular ATP on the
IIsK then
interference with PKC activation would reduce the effect of ATP. We
tested this hypothesis by addition of the PKC inhibitor GF109203X to the apical perfusate.
Addition of GF109203X at 10 µM to the apical perfusate had no
significant effect on
Isc
(n = 7). However, apical ATP (1 µM) caused less of a decrease of
Isc in the
presence of GF109203X than in its absence (Fig.
8, Table 2). ATP decreased
Isc by 38.5 ± 3.2% in the absence of GF109203X and by 20.7 ± 1.6% in its
presence. The fractional response to ATP in the presence of 10 µM
GF109203X was 54 ± 2%.

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Fig. 8.
Summary of measurements of
Isc showing
effect of apical perfusion of ATP (1 µM) in the presence and absence
of an inhibitor of protein kinase C, GF109203X (10 µM), in vestibular
dark cells (values are means ± SE;
n = 7).
|
|
 |
DISCUSSION |
Purinergic (P2) receptors
responsive to UTP have been referred to as
P2U subtypes. More recently,
P2U receptors have been found to
represent a family of three subtypes responsive to uridine nucleotides,
which are designated P2Y2,
P2Y4, and
P2Y6 (22). The P2Y family of
purinergic receptors is coupled via heterotrimeric G proteins in the
plasma membrane to PLC (5), which leads via one branch of the pathway
to activation and translocation of PKC from the cytosol to the plasma
membrane (3) and via a second branch of the pathway to elevation of
cytosolic Ca2+ concentration
([Ca2+]c),
which occurs in response to production of
InsP3 (5). Most ion transport mechanisms that are influenced by
P2U receptors respond to changes
in
[Ca2+]c
(5). In contrast, we have presented evidence that activation of apical
P2U receptors in VDC leads to
inhibition of K+ secretion and
that this inhibition is mediated by the PKC branch of the PLC pathway.
The involvement of PLC in the response to apical ATP was shown by the
reduced effect of ATP in the presence of the PLC inhibitor, U-73122
(7). The involvement of PKC was demonstrated both by inhibition of the
IIsK in response
to activation of PKC with PMA and by the reduced effect of ATP in the
presence of the PKC inhibitor, GF109203X.
PKC most likely acts by direct phosphorylation of the
IsK protein. The
IsK protein in rat is known to
have a consensus sequence for phosphorylation by PKC, and this sequence
is altered in guinea pig. The difference in sequence is correlated with
different electrophysiological responses of the
IIsK to
stimulation of PKC (28). PKC activation in
Xenopus oocytes expressing rat
IsK leads to a decrease in
current, as found here for gerbil, whereas wild-type guinea pig
IsK (and rat
IsK with the consensus sequence
mutated to the corresponding sequence from guinea pig) are stimulated
by PKC. Those results led to the prediction that the consensus sequence
for phosphorylation by PKC in gerbil would be identical to that of rat.
The result of molecular cloning and sequencing of a segment of the
gerbil IsK cDNA from the
vestibular labyrinth confirms that the isk gene is expressed
in this tissue and that it contains the putative PKC phosphorylation
site, as predicted. On the basis of these results and the presence of
the same PKC phosphorylation site in human isk (21), it can
be expected that the secretion of endolymph in the human vestibular
system is controlled in the same way as in our animal model.
It is conceivable that extracellular nucleotides inhibit
K+ secretion indirectly via cAMP,
rather than via direct phosphorylation of the
IsK channel. In
DDT1MF-2 smooth muscle cells,
activation of the P2U receptor
activated PLC but also activated a distinct, pertussis toxin-sensitive
G protein pathway, which caused a large sustained decrease of the level
of cytosolic cAMP (26). We have recently shown that there is
constitutive production of cAMP and that elevation of cytosolic cAMP
stimulates the
IIsK in VDC (27), so it is likely that reduction of cAMP would lead to a decrease in
IIsK. If the
P2U receptors in the apical
membrane of VDC are coupled to Gi
as well as to PLC, it is conceivable that at least part of the effect
of apical perfusion of ATP was due to a reduced cAMP level, although no
such coupling has yet been demonstrated in VDC.
The increase of
IIsK observed in
VDC under conditions expected to elevate
[Ca2+]c
argues against a major role of the
InsP3 pathway,
with its subsequent increase of
[Ca2+]c,
in the response of K+ secretion to
activation of the apical P2U
receptor. Although Ca2+ is
classically known to increase PKC activity, isozymes have been
identified that are Ca2+
independent (PKC-
) (8). PKC-
has been identified in stria vascularis (1), a tissue in the cochlea that contains cells homologous
to VDC in many other respects (29). It is not yet clear what process is
directly affected by addition of A-23187, although it is most likely
due to an increase in cytosolic
Ca2+. Although A-23187 is a
divalent cation/H+ exchanger, it
is not likely that the effect was due to a change in cytosolic pH,
since an influx of Ca2+ would be
expected to lead to alkalinization and, by our current understanding, a
concomitant decrease in K+
secretion (30).
Although the sources of agonist and the physiological functions of the
apical purinergic receptors are not yet clear, several hypotheses can
be advanced. The constitutive level of ATP in the luminal fluid of the
cochlea has been estimated at 13 nM (20), a concentration that causes a
small but significant decrease in Isc of VDC (13).
If one assumes a similar level of ATP in the vestibular labyrinth, the
receptor would be poised to be either stimulated further by additional
agonists or inhibited by antagonists. Functionally, it may be extremely
important for these epithelial cells to receive signals from
neighboring VDC, since they are not coupled to each other by gap
junctions. In addition, there are many other epithelial cell types
lining the vestibular lumen that may communicate via apical release of
ATP. Conditions stimulating this hypothetical release of ATP remain to
be discovered.
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Institute on Deafness and Other
Communication Disorders research grant 5R01-DC-00212 to D. C. Marcus.
 |
FOOTNOTES |
Address for reprint requests: D. C. Marcus, Biophysics Laboratory, Boys
Town National Research Hospital, 555 No. 30th St., Omaha, NE 68131.
Received 21 January 1997; accepted in final form 20 August 1997.
 |
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