ATP-dependent regulation of inwardly rectifying
K+ current in bovine retinal
pigment epithelial cells
Bret A.
Hughes and
Masayuki
Takahira
Departments of Ophthalmology and Physiology, University of
Michigan, Ann Arbor, Michigan 48105
 |
ABSTRACT |
Inwardly
rectifying K+ current
(IKir) in
freshly isolated bovine retinal pigment epithelial (RPE) cells was
studied in the whole cell recording configuration of the patch-clamp
technique. When cells were dialyzed with pipette solution containing no
ATP, IKir ran
down completely in <10 min [half time
(t1/2) = 1.9 min]. In contrast, dialysis with 2 mM ATP sustained
IKir for 10 min or more. Rundown was also prevented with 4 mM GTP or ADP. When 0.5 mM
ATP was used,
IKir ran down by
~71%. Mg2+ was a critical
cofactor because rundown occurred when the pipette solution contained 4 mM ATP but no Mg2+
(t1/2 = 1.8 min).
IKir also ran
down when the pipette solution contained 4 mM
Mg2+ + 4 mM
5'-adenylylimidodiphosphate
(t1/2 = 2.7 min)
or 4 mM adenosine 5'-O-(3-thiotriphosphate)
(t1/2 = 1.9 min),
nonhydrolyzable and poorly hydrolyzable ATP analogs, respectively. We
conclude that the sustained activity of
IKir
in bovine RPE requires intracellular MgATP and that the underlying
mechanism may involve ATP hydrolysis.
patch clamp; potassium channels; adenosine triphosphate
 |
INTRODUCTION |
POSITIONED BETWEEN the distal retina and the choroidal
blood supply, the retinal pigment epithelium (RPE) carries out a wide range of functions that are critical to the health and integrity of the
adjacent photoreceptor cells. One of these functions is control of the
composition and volume of the extracellular fluid surrounding the
photoreceptor outer segments through the transport of fluid, ions, and
metabolites. K+ channels in the
RPE apical and basolateral membranes play a central role in RPE
physiology. They not only participate directly in net
K+ transport (25) but they also
influence the active transport of
HCO
3 (14) and
Cl
(2, 9) by virtue of
their impact on resting membrane potentials (22, 26). Patch-clamp
studies on freshly isolated amphibian (31) and human (18) RPE cells
have shown that the predominant conductance in the physiological
voltage range is an inwardly rectifying
K+ conductance. The inward
rectification of this K+
conductance is relatively weak, such that it supports substantial outward K+ current at potentials
positive to the K+ equilibrium potential
(EK). Pharmacological studies on
the intact toad RPE-choroid preparation indicate that this channel is a
major component of the K+
conductance in the apical membrane (15), where it can support Na+-K+
pump activity by recycling K+.
In other cell types, a wide variety of inwardly rectifying
K+ channels have been identified
(27), some of which are regulated by intracellular ATP levels.
KATP channels, for example, have a
relatively high open probability when cell ATP levels are low but
become inactivated when the intracellular ATP levels are high (1).
Other inwardly rectifying K+
channels, such as the cloned channel Kir2.1 (ROMK1; Ref. 23), exhibit
rundown in the absence of cytoplasmic ATP as a result of the net
dephosphorylation of the channel. In the present study, we show
that inwardly rectifying K+
conductance in freshly isolated bovine RPE cells requires relatively high concentrations of intracellular ATP for sustained activity and,
furthermore, that this requirement may be specific for hydrolyzable nucleotide triphosphate analogs. A preliminary account of this work has
been presented previously in abstract form (17).
 |
METHODS |
Solutions. The standard bath solution
consisted of (in mM) 135 NaCl, 5 KCl, 10 HEPES, 10 glucose, 1.8 CaCl2, and 1.0 MgCl2 and was titrated to pH 7.4 with NaOH. In experiments where the concentrations of
K+,
Cs+, or
Ba2+ were varied, NaCl was
replaced by an equimolar amount of the appropriate
Cl
salt. Quinidine was
added to Ringer solution as a concentrated DMSO solution; the final
concentration of DMSO was <0.1%. Glybenclamide was added directly to
the Ringer solution. In some experiments, we added 100 µM
Gd3+ to the Ringer solutions to
block a noisy, nonselective cation current that was activated in some
cells by membrane hyperpolarization. Gd3+ had no obvious effects on
either the kinetics or voltage dependence of the inwardly rectifying
K+ currents
(IKir). The
osmolality of all external solutions was 288 ± 5 mosmol/kgH2O.
The standard pipette solution used in these experiments consisted of
(in mM) 30 KCl, 83 potassium gluconate, 10 HEPES, 5.5 EGTA-KOH, 0.5 CaCl2 (free
Ca2+ concentration
20 nM),
and 2.0 or 4 MgCl2, titrated to pH
7.2 with KOH. Addition of ATP (K+
salt), ADP, adenosine
5'-O-(3-thiotriphosphate)
(ATP
S), 5'-adenylylimidodiphosphate (AMP-PNP;
Li+ salt), and GTP
(Na+ or Tris salt) caused
acidification of the pipette solution, requiring further titration to
pH 7.2. Mg2+-free internal
solution was made by eliminating
MgCl2 and substituting 5.5 mM EDTA
for EGTA. The concentrations of MgATP, free
Ca2+, and free
Mg2+ were calculated using a Basic
program Calcium (4). In some early experiments, we used an internal
solution containing 100 mM potassium gluconate, which in some cells led
to the development of a swelling-activated, outwardly rectifying
Cl
current (3).
Orthovanadate stock solution, prepared by adjusting the pH of a 100 mM
NaVO4 solution to pH 10, was
stored at 4°C and boiled for 15 min before an aliquot was added to
the internal solution. The pH of the diluted orthovanadate solution was
then adjusted to pH 7.2 with HCl. All internal solutions had an
osmolality of 244 ± 5 mosmol/kgH2O except for the 100 mM
potassium gluconate solution, which had an osmolality of 278 ± 5 mosmol/kgH2O.
The cell isolation medium contained (in mM) 135 N-methyl-D-glucamine
(NMDG) chloride, 5 KCl, 10 HEPES, 3 EDTA-KOH, 10 glucose, and 3 cysteine, as well as 0.2 mg/ml papain (type III), and was titrated to
pH 7.4 with NMDG free base.
All chemicals were of reagent grade and were obtained from Sigma
Chemical (St. Louis, MO) except for
GdCl3, which was obtained from
Aldrich Chemical (Milwaukee, WI).
Cell isolation. Adult bovine eyes were
obtained from a local abattoir. Eyes were enucleated within 30 min of
death and placed on ice. Cells were isolated by enzymatic dispersion as
described previously for human RPE (20). Briefly, 5-mm-square pieces of RPE-choroid were dissected from the inferior or superior pigmented region of bovine eyecups and incubated for 5-10 min in the cell isolation medium at room temperature. The tissue was then incubated in
normal Ringer solution containing 0.1% BSA for 3 min and finally in
normal Ringer solution for another 10 min. This series of incubations was repeated three or four times before dissociating cells by vortexing
the tissue in 2-3 ml of Ringer solution. Isolated cells were
stored in normal Ringer solution at 4°C for up to 24 h before use.
Electrophysiological methods. Isolated
RPE cells were transferred to a continuously perfused Lucite recording
chamber (31). Cells selected for recording had a bright appearance
under phase-contrast microscopy. All experiments were conducted at room
temperature (23-25°C).
Membrane currents were recorded using the conventional whole cell
recording technique (12) or the amphotericin B perforated-patch method
(30). Pipettes were pulled from 7052 glass tubing (Garner Glass,
Claremont, CA) with the use of a multistage programmable puller (Sutter
Instruments, San Rafael, CA) and coated with Sylgard (Dow Corning,
Midland, MI). Before use, pipette tips were fire polished to
resistances in the range 1-5 M
. For perforated-patch recording,
patch pipettes were front filled with a 200- to 300-µm column of
internal solution and then backfilled with internal solution containing
amphotericin B. The amphotericin B solution was made just before use by
adding 10 µl of a freshly made stock solution (1.5 mg amphotericin
B/50 µl DMSO) to 2.5 ml of internal solution to give a final
concentration of 120 µg/ml. Series resistance commonly decreased to
25 M
or less within 5-15 min after gigaohm seal formation on
the basolateral membrane.
Currents were recorded with Axopatch 1D or Axopatch 200 amplifiers
(Axon Instruments, Foster City, CA) with the built-in low-pass filter
set to 1 kHz unless noted otherwise. Recordings were referenced to a
Ag-AgCl electrode separated from the bath by a short column of 150 mM
KCl set in 4% agar. Command potentials were generated by software
control (pCLAMP, Axon Instruments, Foster City, CA). Signals were
digitized on line every 1.2-2.5 ms and stored on the hard drive on
a 486 microcomputer for subsequent analysis. Series resistance
(Rs) and
membrane capacitance were calculated from uncompensated capacitative
transients (low-pass filter 5 kHz) as described previously (31).
Rs averaged 13 ± 6 (SD) M
(n = 91) for whole
cell recordings and 13 ± 11 (n = 6) for perforated-patch recordings and was commonly compensated 50% by
amplifier circuitry. Built-in circuits also compensated for pipette and
membrane capacitances. The pipette tip potential (
10 mV) was
measured against a flowing saturated KCl bridge and was used to correct
the apparent membrane potential as described previously (16). Liquid
junction potentials at interface between the agar bridge and bath were
<3 mV and were ignored.
Virtually every cell exhibited inwardly rectifying and delayed
rectifier K+ currents; in
addition, ~24% of the cells also expressed M-type K+ currents (32). The delayed,
outwardly rectifying current was routinely inactivated by holding the
membrane potential at
10 mV between voltage pulses and ramps.
Cells containing M-type K+
currents were excluded. In most experiments, we quantified the IKir by measuring
the component of whole cell current that was blocked by 20 mM external
Cs+, a near-saturating
concentration (see RESULTS).
Dose-response relationships. The
blocker sensitivity of the
IKir was
determined by measuring currents at selected voltages in the presence
of various concentrations of blocking ions and fitting the results to
the first-order equation
|
(1)
|
where
IK is the
component of whole cell current blocked by a particular concentration
of blocker ([B]),
Imax is the
component of current blocked by a saturating concentration of blocker,
and KD is the
concentration at which the block is half maximal. Data for each cell
were fitted using curve-fitting algorithms in SigmaPlot (SPSS, Chicago,
IL) to obtain the parameters
Imax and
KD.
Time course experiments. After the
membrane patch beneath the recording pipette was ruptured, the
steady-state current-voltage (I-V)
relationship of the cell was measured every 15 s by ramping the
membrane voltage from +40 to
160 mV (in some cells, to
110 mV) at a rate of 50 mV/s, with the voltage held at
10
mV between ramps. This voltage-clamp protocol inactivated the delayed
rectifier K+ current and elicited
currents through inwardly rectifying
K+ channels as well as
Cl
and
Na+-selective channels. The time
course of IKir
and residual current (Na+ + Cl
currents) was
constructed from this I-V data by
sampling currents at specific voltages. For
IKir, we first
determined the reversal potential of the residual current by measuring
the zero-current potential of the I-V
curve obtained in the presence of 20 mM external Cs+, a concentration that blocks
nearly all of
IKir. For the
residual current, we sampled the current at the reversal potential of
the Cs+-sensitive current, which
generally was within a few millivolts of
EK (
83
mV).
Statistics. Values are means ± SE,
except where noted. Statistical significance was determined using
Student's two-tailed t-test.
P
0.05 was considered significant.
 |
RESULTS |
Membrane parameters. Freshly isolated
bovine RPE cells had a "figure-eight" shape with distinct apical
and basolateral domains, as reported previously for frog (16) and human
(20) RPE. Cell size varied considerably, with the diameter of the
basolateral hemisphere ranging from 15 to 30 µm. The apical
hemisphere was generally two to four times smaller and had numerous
processes projecting from its surface. To ensure effective space clamp, we selectively recorded from the smallest cells in the recording chamber; these cells had an average membrane capacitance of 82 ± 26 (SD) pF (n = 118). The zero-current
potential (V0)
of bovine RPE cells, measured in the zero-current clamp mode
immediately after rupturing the membrane patch, averaged
64 ± 11 (SD) mV (n = 118). This value
compares favorably with the membrane potentials measured at 35°C in
the intact bovine RPE-choroid preparation with intracellular
microelectrodes (22).
Properties of IKir.
To characterize the properties of
IKir, we carried
out a series of experiments on bovine RPE cells dialyzed with 4 mM ATP; under this condition,
IKir was
sustained for 2 h or more. Figure 1 shows
representative recordings of whole cell currents in a cell that had
been dialyzed with 4 mM ATP (3.36 mM MgATP, 0.64 mM free ATP) for ~5
min. Currents were essentially time independent over the voltage range
examined, but their amplitude was substantially larger at
hyperpolarized voltages (Fig. 1A,
top). The steady-state I-V relationship (Fig.
1B, control) shows that the whole cell current reversed at
75 mV
(V0) and
exhibited mild inward rectification, with the slope conductance
decreasing gradually with membrane depolarization. Superfusing the cell
with 20 mM Cs+, an effective
blocker of IKir
in amphibian (31) and human (18) RPE cells, substantially reduced the
amplitude of the whole cell currents (Fig.
1A,
middle), depolarized
V0 to
31
mV (Fig. 1B), and markedly reduced
inward and outward slope conductances (Fig. 1B). The
Cs+-sensitive current, obtained by
taking the difference between the family of currents recorded in the
absence and presence of Cs+, is
shown in Fig. 1A,
bottom. The current activated rapidly
in response to hyperpolarizing voltage steps and did not exhibit inactivation. The I-V relationship of
the difference current (Fig. 1C)
shows that the underlying conductance was mildly voltage dependent and
that a substantial portion was activated in the physiological voltage
range, which lies between
70 and
50 mV (22). The reversal of the difference current at
82 mV near the predicted
K+ equilibrium potential
(
83 mV) confirms that the
Cs+-sensitive, inwardly rectifying
current is carried by K+.

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Fig. 1.
Properties of inwardly rectifying
K+ current
(IKir).
A,
top: family of currents recorded in a
bovine retinal pigment epithelium cell bathed with control Ringer
solution. Currents were evoked by 1-s voltage pulses to potentials
ranging from 140 to +40 mV from a holding potential of 10
mV. Zero-current potential is indicated by horizontal line to left of
current records. Pipette solution contained 4 mM ATP and bath
contained 100 µM Gd3+ to block
nonselective cation currents. Middle:
family of currents recorded in same cell bathed with 20 mM
Cs+ Ringer.
Bottom: isolated
IKir
(Cs+-sensitive current).
B: steady-state current-voltage
(I-V) relationships obtained in
control Ringer and 20 mM Cs+
Ringer. C:
I-V relationship of
IKir
(Cs+-sensitive current), obtained
by taking the difference between the
I-V curves in
B.
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|
Similar results were obtained in 10 other cells dialyzed with 4 mM ATP
for 5-25 min. Extracellular
Cs+ (10-50 mM) depolarized
V0 from a mean
value of
69.0 ± 1.3 mV to
46.1 ± 2.5 mV
(P < 10
5), decreased the
inward slope conductance measured between
120 and
110 mV
from 10.47 ± 1.26 to 2.35 ± 0.30 nS
(P < 10
5), and decreased the
outward slope conductance measured between
60 and
50 mV
from 5.95 ± 1.21 to 2.28 ± 0.37 nS
(P = 0.001). These results indicate
that the inwardly rectifying K+
conductance accounts for ~78% of the inward slope conductance and
62% of the outward slope conductance of the bovine RPE cell membrane.
To assess blocker sensitivity, we estimated the apparent dissociation
constants (KD)
of the Cs+- and
Ba2+-induced blocks of the
inwardly rectifying K+ conductance
by measuring IKir
in the presence of various blocker concentrations. Figure
2A shows
the dose-response relationship of the
Cs+-induced block of
IKir measured at
35 and
110 mV. Symbols represent the mean values of
normalized K+ current measured in
the presence of various Cs+
concentrations (n = 5), and the smooth
curves are the best fits of the data to Eq. 1 (see METHODS). The
Cs+-induced block was mildly
voltage dependent and had a mean
KD of 2.01 ± 0.32 mM at
35 mV and a
KD of 0.73 ± 0.08 mM at
110 mV (P = 0.005). At none of the concentrations tested did
Cs+ exhibit a time-dependent
effect on IKir
(not shown). These results indicate that 20 mM
Cs+ blocks >90% of
IKir, thus
validating the use of this blocker to isolate
IKir from other
currents.

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Fig. 2.
Dose-response relationships of
Cs+- and
Ba2+-induced blocks of
IKir.
A: concentration dependence of
Cs+-induced block. Symbols
represent mean values of fraction of
IKir present at
various Cs+ concentrations, and
vertical lines indicate means ± SE
(n = 5). Smooth curves are
least-squares fits of mean values to Eq. 1 with
KD values of 1.82 and 0.75 mM for measurements at 35 and 110 mV,
respectively. B: concentration
dependence of Ba2+-induced block.
Smooth curves are least-squares fits of mean values
(n = 4-6) to Eq. 1 with
KD values of 148 and 61 µM for measurements at 35 and 110 mV,
respectively.
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Figure 2B shows that external
Ba2+ also blocked
IKir in a weakly
voltage-dependent manner; the apparent
KD of the
Ba2+-induced block averaged 219 ± 68 µM at
35 mV and 85 ± 22 µM at
110 mV
(P = 0.02;
n = 6). In contrast to
Cs+, the
Ba2+-induced block was time
dependent at voltages negative to
EK (not shown).
Quinidine, a blocker of several types of
K+ channel (7), blocked ~50% of
the inwardly rectifying K+
conductance at a concentration of 20 µM
(n = 2), and at a concentration of 100 µM the block was essentially complete
(n = 4; not shown). Glybenclamide, a
potent inhibitor of ATP-sensitive
K+ channels (1), had no
significant effect on K+ currents
in bovine RPE cells at a concentration of 100 µM
(n = 5).
Dependence on intracellular ATP. To
investigate the possible dependence of
IKir on
intracellular ATP, we dialyzed cells with ATP-free pipette solution and
monitored the time course of changes in current at specific voltages
(see METHODS). The results of a
representative experiment are shown in Fig.
3. Figure
3A plots the time course of currents
measured at
30 and
82 mV, the reversal potentials of the
Cs+-insensitive (residual) and
Cs+-sensitive components of the
whole cell current, respectively (see Fig.
3B). At
30 mV, the sum of all
currents excluding
IKir was zero,
and hence the current at this voltage represents outward K+ movement through the inwardly
rectifying K+ conductance.
Likewise, at
82 mV
(
EK), there
was no current through the inwardly rectifying
K+ conductance, and hence the
current at this voltage represents the residual current, consisting
largely of Cl
and
Na+ currents. As shown in Fig.
3A,
IKir ran down
over a period of 8 min, while the residual current remained essentially
unchanged. A decrease in
IKir is also
evident from the data depicted in Fig. 3B, which compares
I-V curves measured after 30 s of
whole cell recording (a) and again
after 8 min of dialysis (b).
Dialysis with the ATP-free solution led to a large decrease in inwardly rectifying current, resulting in a shallow
I-V relationship. The difference
between the initial and steady-state
I-V curves (Fig. 3C, a
b) exhibits mild inward
rectification and a reversal potential of
82 mV, confirming that
the component of whole cell current that ran down was mainly
IKir. This
conclusion was corroborated by the finding that 20 mM
Cs+ had no effect on the
I-V relationship at 9 min, when the
current at
30 mV had reached zero (Fig.
3A). Similar results were obtained in nine other cells dialyzed with ATP-free pipette solution, with the
half time for
IKir rundown
averaging 1.9 ± 0.3 min (Fig. 3D and Table 1). The time course
of rundown was approximately five times slower than the rate expected
for simple diffusion of MgATP from the cytoplasm to the
pipette,1
suggesting that cellular ATP may be compartmentalized.

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Fig. 3.
Effects of internal dialysis with ATP-free pipette solution.
A: time course of changes in current
in cell dialyzed with pipette solution containing 0 ATP (1.63 mM free
Mg2+). Currents measured at
30 mV [I( 30
mV)] and 82 mV
[I( 82
mV)] represent
IKir and residual
current, respectively. , Sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
B:
I-V curves obtained at times indicated
by in A.
C: difference curves obtained from
data in B, showing
I-V relationship of rundown current
(a b) and the lack of a
Cs+-sensitive current
(b c) after 8 min of dialysis.
D: averaged time course of
IKir rundown in
cells dialyzed with ATP-free pipette solution
(n = 10).
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As already mentioned,
IKir was
sustained when bovine RPE cells were recorded with pipette solution
containing 4 mM ATP. Figure 4 shows the
results of dialyzing a cell with the pipette solution containing 2 mM
ATP (1.90 mM MgATP, 0.10 mM free ATP). During the first minute of
dialysis, IKir
(measured at
36 mV) increased slightly (Fig.
4A), possibly due to an increase in
intracellular K+ concentration.
Thereafter, IKir
was stable until the cell was superfused with 20 mM
Cs+ (Fig.
4A, open box), which
produced a reversible inhibition. Figure 4,
B and
C, shows that the
I-V relationship changed little after
6 min of dialysis and that the cell retained a large inwardly rectifying K+ conductance. The
results obtained in this and nine other cells dialyzed with 2 mM ATP
are summarized in Fig. 4D, which shows the time course of normalized
IKir.

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Fig. 4.
Effects of internal dialysis with 2 mM ATP.
A: time course of changes in current
during internal dialysis with the pipette solution containing 2 mM ATP.
Currents measured at 36 mV
[I( 36
mV)] and 82 mV
[I( 82
mV)] represent
IKir and residual
current, respectively. , sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
B:
I-V curves obtained at times indicated
by in A.
C: difference curves obtained from
data in B, showing that dialysis with
2 mM ATP produced little change in I-V
relationship (b a) and that the cell had a
relatively large
IKir
(b c).
D: averaged time course of
IKir in cells
dialyzed with 2 ATP (n = 10).
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We further tested the dependence of
IKir on
intracellular ATP by first depleting cells of ATP by metabolic
inhibition and then dialyzing them with ATP. We found that the exposure
of bovine RPE cells to 250 ng/ml oligomycin plus 10 mM deoxyglucose
(substituted for glucose) caused nearly complete rundown of the
inwardly rectifying K+ conductance
(not shown). In six cells recorded in the amphotericin B
perforated-patch configuration, the combined metabolic inhibitors reduced the inwardly rectifying K+
chord conductance (gKir)
at
110 mV from an average of 4.54 ± 0.74 to 0.34 ± 0.13 nS (P = 0.007). Figure
5 shows the results of an experiment in
which a cell preexposed to oligomycin and deoxyglucose for ~30 min
was recorded in the whole cell configuration with a pipette solution
containing 4 mM ATP (3.36 mM MgATP, 0.64 mM free ATP). Figure
5A plots the time course of currents
measured at
38 and
83 mV, which represent
IKir and the
residual current, respectively. At the first measurement after rupture
of the membrane patch,
IKir was near
zero, but it increased rapidly until it reached a steady level after
~3 min of dialysis. Inspection of
I-V relationships corresponding to
these time points (Fig. 5, B and
C) reveals that dialysis led to a
dramatic increase in both inward and outward conductances and that
these conductance increases were completely blocked by 20 mM
extracellular Cs+. The data
indicate that the inwardly rectifying
K+ conductance was small or absent
before dialysis with ATP began and that the change in whole cell
current can be attributed solely to an increase in inwardly rectifying
K+ conductance. Similar results
were obtained in two other cells that were first metabolically
inhibited and then dialyzed with 4 mM ATP. For these three cells,
IKir rose with an
average half time of ~1.8 min (Fig.
5D), and the steady-state
gKir at
110 mV averaged 6.69 ± 0.49 nS. These results provide strong
support for the idea that the magnitude of
IKir is
determined by intracellular ATP concentration.

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Fig. 5.
Effect of internal dialysis with 4 mM ATP in metabolically inhibited
cells. A: time course of changes in
current during internal dialysis. Currents measured at 38 mV
[I( 38
mV)] and 83 mV
[I( 83
mV)] represent
IKir and residual
current, respectively. Before whole cell recording, the cell was
exposed to 250 ng/ml oligomycin + 10 mM deoxyglucose for >30 min.
Pipette solution contained 4 mM ATP. , Sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
B:
I-V curves obtained at times indicated
by in A. Note the similarity in
the I-V relationships obtained at the
start of dialysis (a) and in
presence of Cs+ after
IKir became
activated (c).
C: difference curves derived from data
in B, showing activation of
IKir by dialysis
with ATP (b a) and its inhibition by
Cs+
(b c).
D: averaged time course of
IKir activation
in metabolically inhibited cells dialyzed with 4 ATP
(n = 3).
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To specify the ATP concentration range over which
IKir is
modulated, we measured
gKir at
110 mV in cells dialyzed with various amounts of ATP for
10-15 min, when diffusional exchange between the pipette and
cytoplasm was likely complete. The results of these experiments are
summarized in Fig. 6, which depicts average conductances for cells dialyzed with 0 ATP, 0.5 mM ATP (0.46 mM MgATP,
0.04 mM free ATP), 2 mM ATP (1.90 mM MgATP, 0.10 mM free ATP), and 4 mM
ATP (3.36 mM MgATP, 0.64 mM free ATP). Compared with the conductance
present in cells dialyzed with 4 mM ATP, cells dialyzed with
2 mM, 0.5 mM, and 0 ATP had conductances that were 28%
(P = 0.09), 71%
(P = 0.0004), and 95% smaller
(P < 10
7), respectively.
Together, these results suggest that the concentration of intracellular
ATP necessary to support a half-maximal
gKir probably
lies in the range 0.5-2 mM. It is worthwhile noting that the local
concentration of ATP in the vicinity of the
K+ channels or the regulatory
protein that modulates them may differ from the concentration of ATP in
the pipette because of diffusion limitations and endogenous ATP
production and consumption. Nevertheless, the data are consistent with
the idea that relatively high ATP concentrations, in the millimolar
range, are required to sustain maximal activity of
IKir.

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Fig. 6.
Relationship between ATP concentration and inwardly rectifying
K+ conductance
(gKir). Bars, average
values of gKir;
vertical lines, means ± SE. Each cell was dialyzed for 10-15
min and then exposed to 20 mM Cs+;
gKir was then
calculated from Cs+-sensitive
current at 110 mV using the chord conductance equation.
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We also tested whether other nucleoside triphosphates could sustain
IKir. As shown in
Fig. 7, internal dialysis with 4 mM GTP
(3.4 mM MgGTP, 0.6 mM free GTP) was effective in preventing the
rundown. During the initial 8 min of dialysis, there was a transient
increase in IKir,
but comparison of initial and steady-state I-V relationships showed little net
change (Fig. 7B,
a and
b, and Fig.
7C, a
b). Exposure of the cell to
20 mM Cs+ after 8 min of dialysis
blocked a major fraction of whole cell current, demonstrating the
presence of a large inwardly rectifying K+ conductance (Fig. 7,
B and
C). Although results were somewhat variable, there was no evidence of
IKir rundown in
cells dialyzed with 4 mM GTP (Fig.
7D). After 10-20 min of
dialysis, gKir at
110 mV averaged 5.47 ± 1.09 nS
(n = 10), which is not
significantly different from the value obtained in cells dialyzed with
4 mM ATP (P = 0.14). These results
suggest that the mechanism responsible for sustaining
IKir can utilize
GTP as well as ATP.

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Fig. 7.
Effect of internal dialysis with 4 mM GTP.
A: time course of changes in current
during internal dialysis with the pipette solution containing 4 mM GTP.
Currents measured at 50 mV
[I( 50
mV)] and 84 mV
[I( 84
mV)] represent
IKir and residual
current, respectively. , Sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
B:
I-V curves obtained at times indicated
by in A.
C: difference curves of data in
B, showing that dialysis with 4 mM GTP
produced little change in I-V
relationship (a b) and that the cell had a
relatively large
IKir
(b c).
D: averaged time course of
IKir in cells
dialyzed with 4 mM GTP (n = 5).
|
|
Effect of ATP analogs. If inwardly
rectifying K+ channel activity
were regulated by nonhydrolytic ATP binding, then
IKir should be
sustained by the nonhydrolyzable ATP analog, AMP-PNP. Figure 8 shows, however, that dialysis with 4 mM
AMP-PNP plus 4 mM Mg2+ led to
IKir rundown.
This effect cannot be attributed to the small amount of
Li+ that was liberated from
AMP-PNP, because
IKir was stable
in two cells dialyzed with 4 mM ATP plus 4 mM
Li+ (not shown). In six cells
dialyzed with 4 mM AMP-PNP,
IKir declined to
near zero with an average half time of 2.7 ± 0.6 min (Fig. 8D and Table 1); after 5-10 min
of dialysis, the magnitude of gKir at
110 mV averaged 0.13 ± 0.07 nS.

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Fig. 8.
Effect of internal dialysis with the nonhydrolyzable ATP analog,
5'-adenylylimidodiphosphate (AMP-PNP).
A: time course of current changes in a
cell dialyzed with pipette solution containing 4 mM AMP-PNP. Currents
measured at 20 mV
[I( 20
mV)] and 80 mV
[I( 80
mV)] represent
IKir and residual
current, respectively. , Sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
C: difference curves derived from data
in B.
D: averaged time course of
IKir rundown in
cells dialyzed with 4 mM AMP-PNP (n = 6).
|
|
These results suggest that simple nonhydrolytic ATP binding is not
sufficient to sustain the activity of
IKir. An
alternative explanation, however, is that AMP-PNP does not have the
appropriate three-dimensional structure to interact with the
nucleotide-binding site of the channel or regulatory protein. To
examine this possibility, we tested whether an excess of AMP-PNP could
prevent ATP from sustaining
IKir. In eight
cells, dialysis with 3.5 mM AMP-PNP plus 0.5 mM ATP led to a partial
rundown of IKir,
with the steady-state gKir at
110 mV averaging 3.5 ± 0.72 nS. This value is somewhat greater but not significantly different from the average steady-state conductance measured in cells dialyzed with 0.5 mM ATP alone
(P = 0.21), indicating that AMP-PNP
does not bind to the ATP regulatory site or else it does so with a much
lower affinity than does ATP. Hence the results with AMP-PNP do not
allow us to rule out the possibility that the mechanism responsible for
the sustained activity of
IKir involves
nonhydrolytic binding of ATP.
The poorly hydrolyzable ATP analog ATP
S can also substitute for ATP
in some mechanisms involving nonhydrolytic binding. In addition, it can
serve as a substrate for a number of protein kinases, whereas most
ATPases do not accept it (8). Figure 9
shows, however, that dialysis of a bovine RPE cell with 4 mM ATP
S
plus 4 mM Mg2+ did not prevent
rundown of IKir.
The change in current at
84 mV (Fig.
9A) indicates that the conductance
for an ion other than K+ also
decreased, but the fact that the rundown current reversed near
EK (
75.8 ± 2.8 mV, n = 4) leads to the
conclusion that the main effect of ATP
S was a decrease in
K+ conductance. In four cells
dialyzed with 4 mM ATP
S,
IKir rapidly declined with an average half time of 1.8 ± 0.2 min (Fig.
9D), and the steady-state
gKir measured at
110 mV averaged 0.26 ± 0.16 nS. In contrast to AMP-PNP,
dialysis with ATP
S together with ATP caused
IKir to run down
to a greater extent than did ATP alone. In eight cells dialyzed with
3.5 mM ATP
S plus 0.5 mM ATP, the steady-state
gKir at
110 mV averaged 0.30 ± 0.13 nS, which is significantly
smaller than the average steady-state conductance measured in cells
dialyzed with 0.5 mM ATP (P = 0.03). These results suggest that ATP
S can bind to the same regulatory site
as does ATP but is incapable of maintaining
IKir activity. Hence we can exclude nonhydrolytic ATP binding as the mechanism underlying regulation of the channel.

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Fig. 9.
Effect of internal dialysis with the poorly hydrolyzable ATP analog,
adenosine
5'-O-(3-thiotriphosphate)
(ATP S). A: time course of current
changes in cell dialyzed with pipette solution containing 4 mM ATP S.
Currents measured at 36 mV
[I( 36
mV)] and 84 mV
[I( 84
mV)] represent
IKir and residual
current, respectively. , Sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
C: difference curves derived from data
in B.
D: averaged time course of
IKir rundown in
cells dialyzed with 4 mM ATP S (n = 4).
|
|
The failure of ATP
S to prevent or slow
IKir rundown also
suggests that ATP dependence for sustained activity of
IKir does not
involve a phosphorylation mechanism. To test this possibility further,
we carried out a series of experiments in which cells were
dialyzed with an ATP-free pipette solution containing various protein
phosphatase inhibitors (Table 1). The inclusion of calyculin, a
specific inhibitor of protein phosphatases 1 and 2A, in the pipette
solution did not significantly affect the rate of rundown at
concentrations of either 10 nM or 1 µM. Likewise, rundown was not
affected by the nonspecific protein phosphatase inhibitor orthovanadate. Rundown was somewhat faster in cells dialyzed with pipette solution containing the serine-threonine protein phosphatase inhibitor okadaic acid, but this could have arisen from a smaller average cell volume for this experimental group. Although we cannot exclude the possibility that the inwardly rectifying
K+ channel is regulated by a
protein kinase at some level, the data suggest that the rundown of
IKir observed in
cells dialyzed with ATP-free solution is not the result of net
dephosphorylation by a protein phosphatase. Rather, it seems likely
that some other ATP-dependent mechanism is required to maintain
sustained channel activity.
ATP metabolites. It is possible that
the inwardly rectifying K+
conductance is regulated by ATP breakdown products rather than by ATP
itself. To test this possibility, we dialyzed cells with ADP or AMP. A
representative example of an experiment involving dialysis with 4 mM
ADP plus 4 mM Mg2+ is shown in
Fig. 10. ADP did not sustain the
IKir at its
initial level, but the rundown that did occur was relatively slow and incomplete. IKir
also exhibited modest rundown in three other cells dialyzed with ADP,
but in two cells no significant rundown occurred. For all cells, the
steady-state gKir
at
110 mV averaged 4.54 ± 1.45 nS, which is significantly
lower than that obtained with 4 mM ATP
(P = 0.03, one-tail
t-test). It is possible that the
partial preservation of the
IKir by ADP
resulted from the phosphorylation of ADP to ATP by endogenous adenylate
kinases. In contrast, cells dialyzed with 4 mM AMP consistently
exhibited a complete rundown of
IKir, with an
average half time of 1.9 ± 1.6 min
(n = 4). These results provide
additional support for the hypothesis that the sustained activity of
IKir requires
ATP.

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Fig. 10.
Effect of internal dialysis with 4 mM ADP.
A: time course of changes in cell
dialyzed with pipette solution containing 4 mM ADP. Currents measured
at 40 mV
[I( 40
mV)] and 84 mV
[I( 84
mV)] represent
IKir and residual
current, respectively. , Sampling times for
I-V curves shown in
B; open box indicates exposure of cell
to 20 mM external Cs+.
C: difference curves of data in
B. D:
averaged time course of
IKir in cells
dialyzed with 4 mM ADP (n = 6). Note
the partial rundown.
|
|
Mg2+
dependence.
For a number of enzymatic processes that involve ATP hydrolysis,
Mg2+ is a requisite cofactor. To
test whether MgATP is required to maintain
IKir, we dialyzed
four cells with a pipette solution containing 4 mM
K2ATP and 0 Mg2+. In each cell, the current
measured at the reversal potential of the residual
(Cs+-insensitive) current rapidly
declined to near zero within 5 min (half time = 1.8 ± 0.2 min),
with little change in the current measured at
EK, indicating
rundown of IKir
(not shown). This conclusion is supported by the findings that the
reversal potential of the rundown current was close to
EK (
75.6 ± 6.8 vs.
83 mV) and that 20 mM external
Cs+ had no significant effect on
the whole cell current after rundown was complete. These results
indicate that MgATP, rather than free ATP, is the molecule required for
sustained activation of the IKir.
 |
DISCUSSION |
The present study on freshly isolated bovine RPE cells demonstrates
that the inwardly rectifying K+
conductance, which is the predominant ion conductance at physiological voltages, requires intracellular ATP for sustained activity. The results suggest the possible involvement of a hydrolytic mechanism rather than simple nonhydrolytic ATP binding.
ATP dependence of IKir.
The sustained activity of the bovine RPE
IKir was found to
depend on the concentration of intracellular ATP. Internal dialysis with an ATP-free pipette solution resulted in the rapid rundown (half
time = 1.9 min) of
IKir. Rundown was
prevented, however, when the pipette solution contained 2 mM ATP. ATP
prevented rundown in a dose-dependent manner: at a concentration of 0.5 mM ATP, partial rundown occurred, the inwardly rectifying
K+ conductance declining ~71%.
ADP was partially effective in preventing the complete rundown of
IKir, but this
may have been due to the local generation of ATP from ADP by an
adenylate kinase. The conductance appears to be regulated by ATP itself
and not by its metabolites, because rundown occurred when AMP was used
in place of ATP.
There are several possible mechanisms that could underlie this ATP
regulation of
IKir. These
include simple ATP binding to a nucleotide-binding site,
energy-consuming processes that require ATP hydrolysis, and
phosphorylation. Our data suggest that hydrolysis of nucleotide
triphosphates may be required for the activation of the inwardly
rectifying K+ conductance. ATP and
GTP were both effective in preventing rundown, but
Mg2+, a cofactor required for ATP
hydrolysis reactions, was also required for the sustained activity of
IKir. The
possibility that nonhydrolytic binding of ATP to a nucleotide-binding
site regulates channel activity seems less likely, since high
concentrations of the nonhydrolyzable ATP analogs AMP-PNP and ATP
S
did not prevent rundown. It is possible, however, that these analogs do
not have the appropriate three-dimensional structure to interact with
the nucleotide-binding site of the channel (or regulatory protein).
Consistent with this idea, an excess of AMP-PNP did not interfere with
the regulatory effects of ATP. In contrast, similar experiments with
ATP
S yielded results indicating competitive inhibition. Hence we
conclude that the mechanism by which ATP regulates the inwardly
rectifying K+ channel does not
involve simple nonhydrolytic binding. ATP
S can also serve as a
cofactor for protein kinase phosphorylation (5, 8), and its failure to
support IKir
suggests that phosphorylation of the channel alone is not sufficient to
open the channel. This conclusion is supported by the finding that the
inhibition of protein phosphatase activity by calyculin, okadaic acid,
or orthovanadate did not slow
IKir rundown in
cells dialyzed with 0 ATP. Thus we conclude that RPE inwardly
rectifying K+ channel activity is
sustained by an ATP-dependent process that may involve ATP hydrolysis.
It is conceivable that the rundown of
IKir was an
indirect result of inhibition of an ATPase, such as the plasma membrane or endoplasmic reticulum Ca2+
pump. For example, inhibition of a
Ca2+-ATPase could lead to an
increase in intracellular Ca2+,
which leads to the inhibition of
IKir in both toad
(19) and bovine (unpublished observations) RPE cells. There are two
arguments, however, against this hypothesis. First, the pipette
solutions used in present experiments contained 5.5 mM EGTA, which
would be expected to buffer the free
Ca2+ concentration to
~10
8 M. Still, the
Ca2+-buffering capacity may have
been exceeded in the proximity of the plasma membrane if the rate of
Ca2+ influx where high. Second,
the dose dependency of the ATP regulation of
IKir is
inconsistent with the dependency of most ATPases on ATP. Our data
suggest that the
KD of the
ATP-dependent regulation of
IKir is >500
µM. This value is much higher than that obtained for
Na+-K+-ATPase
and Ca2+-ATPase
[KD,ATP = 0.2-2 µM (34)]. Other results suggest that IKir rundown does
not result from inhibition of the
Na+-K+
pump. In preliminary experiments on bovine RPE cells recorded in the
amphotericin B perforated-patch configuration, we found that inhibition
of the
Na+-K+-ATPase
with ouabain had no inhibitory effect on the inwardly rectifying
K+ conductance
(n = 5).
ATP-dependent regulation has been described in reports on various
members of the inwardly rectifying
K+ (Kir) channel family. For
example, the functional activity of a cloned inward rectifier
K+ channel, Kir2.1 (IRK1),
requires ATP for both phosphorylation and a separate mechanism
apparently involving ATP hydrolysis (10). On the other hand, ATP has a
dual effect on native ATP-sensitive K+ channels
(KATP). When present at low
concentrations on the cytoplasmic face of excised membrane patches, it
sustains channel activity through a phosphorylation mechanism (11, 35),
but at high concentrations it inhibits activity through nonhydrolytic
binding (6, 11, 28).
In a recent report, Ishii et al. (21) presented molecular biochemical
and immunocytochemical evidence that rat and rabbit RPE cells express
Kir4.1 (KAB-2). This ATP-regulated
K+ channel contains a Walker
type-A domain, which has been proposed to serve as a hydrolytic ATP
binding site that confers ATP dependence (33). This idea is somewhat
controversial, however, because recent studies on Kir1 (ROMK1), another
cloned inward rectifier K+ channel
containing a Walker type-A domain (13), suggest that nonhydrolytic
interactions between MgATP and the Walker A site are involved in
channel inhibition (24). The expression pattern and ATP dependence of
Kir4.1 suggest that this channel subtype may be the molecular basis for
the RPE inwardly rectifying K+
conductance, but several properties of the native RPE conductance differ from those of Kir4.1 expressed in
Xenopus oocytes; these include weaker
inward rectification and an anomalous decrease in slope conductance
when extracellular K+
concentration is increased (18, 31). Clearly, additional experiments are needed to determine the relationship between the RPE
inwardly rectifying K+ channel and
Kir4.1, as well as the mechanism underlying its ATP-dependent regulation.
Physiological significance. There is a
wide body of evidence indicating that the inwardly rectifying
K+ conductance in the RPE apical
membrane is an important control point for the transport of
K+ and other ions between the
subretinal space and the choroid. For instance, studies in intact
bovine RPE have shown that the large apical membrane
K+ conductance mediates the
recycling of most of the K+ that
enters the cell via the apical
Na+-K+
pump and that a decrease in its magnitude, produced by extracellular Ba2+, for example, stimulates net
K+ absorption (25). In addition,
the apical K+ conductance provides
a crucial link in the communication between photoreceptors and the RPE.
At light onset, the closure of cGMP-gated channels in the plasma
membrane of photoreceptor outer segments produces a hyperpolarization,
leading to a redistribution of K+
from the extracellular space into the photoreceptor cytoplasm. This
light-evoked decrease in subretinal
K+ concentration hyperpolarizes
the apical membrane of the RPE by virtue of its inwardly rectifying
K+ conductance (15), and this
triggers changes in several RPE transport mechanisms (9, 14).
The requirement of the inwardly rectifying
K+ conductance for relatively high
intracellular ATP levels has important implications with regard to RPE
function. An increase in glycolytic metabolism, for example, may
elevate the intracellular ATP concentration, causing an increase in
apical K+ conductance; this
conductance increase would lead to a greater degree of recycling of the
K+ that enters the cell through
the apical
Na+-K+
pump and a decrease in net K+
absorption. Circumstances that would tend to decrease intracellular ATP
levels, such as an increase in
Na+-K+-ATPase
activity secondary to an increase in
Na+-coupled ion and nonelectrolyte
transport, would have the opposite effect, decreasing the apical
K+ conductance and increasing net
K+ reabsorption (25). This
decrease in the apical K+
conductance would also diminish the responsiveness of the RPE to
photoreceptor-induced changes in subretinal
K+ concentration, which normally
leads to, among other things, volume changes of the RPE cell and
subretinal space (2). It is presently not known whether these
aforementioned mechanisms produce changes in intracellular ATP
concentration in the RPE under physiological conditions. Changes in the
metabolic capacity of the RPE resulting from disease processes or aging
could also lead to a decrease in the intracellular concentration of
ATP, resulting in a diminishment of the apical
K+ conductance, and abnormal
transport function.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Eye Institute Grant
EY-O8850, Core Grant EY-O7703, a Retinitis Pigmentosa
Research Center Grant, and a Research to Prevent Blindness Career
Development Award to B. A. Hughes.
 |
FOOTNOTES |
Present Address of M. Takahira: Dept. of Ophthalmology,
Kanazawa Univ. School of Medicine, Kanazawa, Ishikawa, Japan.
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. §1734 solely to indicate this fact.
1
The time course of dialysis between the patch
pipette and the cell interior is a complex function of cell volume and
geometry, access resistance between the pipette and the cell, and the
molecular weight of the test molecule (29). To quantify the rate of
diffusional equilibrium in bovine RPE cells, we carried out whole cell
recordings with K+-free pipette
solution (NMDG substitute) and measured the delayed rectifier current
at +40 mV every 5 s. In eight cells, the outward K+ current declined exponentially,
with an average half time of 10.2 ± 0.9 s. Assuming that the rate
of current decline is solely a function of diffusion of
K+ out of the cell and that the
diffusion rate between cytoplasm and pipette is proportional to the
inverse cube root of the molecular weight, then the half time for MgATP
diffusion can be estimated to be ~24 s. This value is about five
times faster than the observed half time for
IKir rundown in
cells dialyzed with ATP-free solution (112 s).
Address for reprint requests: B. A. Hughes, Dept. of Ophthalmology,
Univ. of Michigan Medical School, 1000 Wall St., Ann Arbor, MI 48105.
Received 21 April 1998; accepted in final form 20 August 1998.
 |
REFERENCES |
1.
Ashcroft, S. J.,
and
F. M. Ashcroft.
Properties and functions of ATP-sensitive K-channels.
Cell. Signal.
2:
197-214,
1990[Medline].
2.
Bialek, S.,
and
S. S. Miller.
K+ and Cl
transport mechanisms in bovine pigment epithelium that could modulate subretinal space volume and composition.
J. Physiol. (Lond.)
475:
401-417,
1994[Abstract].
3.
Botchkin, L. M.,
and
G. Matthews.
Chloride current activated by swelling in retinal pigment epithelium cells.
Am. J. Physiol.
265 (Cell Physiol. 34):
C1037-C1045,
1993[Abstract/Free Full Text].
4.
Chang, D. P.,
S. Hsieh,
and
D. C. Dawson.
Calcium: a program in Basic for calculating the concentration of calcium, magnesium, and other divalent cations.
Comput. Biol. Med.
18:
351-366,
1988[Medline].
5.
Chung, S. K.,
P. H. Reinhart,
B. L. Martin,
D. Brautigan,
and
I. B. Levitan.
Protein kinase activity closely associated with a reconstituted calcium-activated potassium channel.
Science
253:
560-562,
1991[Medline].
6.
Cook, D. L.,
and
C. N. Hales.
Intracellular ATP directly blocks K+ channels in pancreatic B-cells.
Nature
311:
271-273,
1984[Medline].
7.
Cook, N. S.,
and
U. Quast.
Potassium Channels: Structure, Classification, Function, and Therapeutic Potential, edited by N. S. Cook. Chichester, UK: Horwood, 1990, p. 181-255.
8.
Eckstein, F.
Nucleoside triphosphates.
Annu. Rev. Biochem.
54:
367-402,
1985[Medline].
9.
Edelman, J. L.,
H. Lin,
and
S. S. Miller.
Potassium-induced chloride secretion across the frog retinal pigment epithelium.
Am. J. Physiol.
266 (Cell Physiol. 35):
C957-C966,
1994[Abstract/Free Full Text].
10.
Fakler, B.,
U. Brandle,
E. Glowatzki,
H. P. Zenner,
and
J. P. Ruppersberg.
Kir2.1 inward rectifier K+ channels are regulated independently by protein kinases and ATP hydrolysis.
Neuron
13:
1413-1420,
1994[Medline].
11.
Findlay, I.,
and
M. J. Dunne.
ATP maintains ATP-inhibited K+ channels in an operational state.
Pflügers Arch.
407:
238-240,
1986[Medline].
12.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
13.
Ho, K.,
C. G. Nichols,
W. J. Lederer,
J. Lytton,
P. M. Vassilev,
M. V. Kanazirska,
and
S. C. Hebert.
Cloning and expression of an inwardly rectifying ATP-regulated potassium channel.
Nature
362:
31-38,
1993[Medline].
14.
Hughes, B. A.,
J. S. Adorante,
S. S. Miller,
and
H. Lin.
Apical electrogenic NaHCO3 cotransport. A mechanism for HCO3 absorption across the retinal pigment epithelium.
J. Gen. Physiol.
94:
125-150,
1989[Abstract].
15.
Hughes, B. A.,
A. Shaikh,
and
A. Ahmad.
Effects of Ba2+ and Cs+ on apical membrane K+ conductance in toad retinal pigment epithelium.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1164-C1172,
1995[Abstract/Free Full Text].
16.
Hughes, B. A.,
and
R. H. Steinberg.
Voltage-dependent currents in isolated cells of the frog retinal pigment epithelium.
J. Physiol. (Lond.)
428:
273-297,
1990[Abstract].
17.
Hughes, B. A.,
and
M. Takahira.
ATP-dependent regulation of the inwardly rectifying K+ current in isolated bovine retinal pigment epithelial cells (Abstract).
Invest. Ophthalmol. Vis. Sci.
37:
S229,
1996.
18.
Hughes, B. A.,
and
M. Takahira.
Inwardly rectifying K+ currents in isolated human retinal pigment epithelial cells.
Invest. Ophthalmol. Vis. Sci.
37:
1125-1139,
1996[Abstract].
19.
Hughes, B. A.,
M. Takahira,
and
Y. Segawa.
Calcium-dependent regulation of the inward-rectifying K+ conductance in toad retinal pigment epithelium (Abstract).
Invest. Ophthalmol. Vis. Sci.
35:
1759,
1994.
20.
Hughes, B. A.,
M. Takahira,
and
Y. Segawa.
An outwardly rectifying K+ current active near resting potential in human retinal pigment epithelial cells.
Am. J. Physiol.
269 (Cell Physiol. 38):
C179-C187,
1995[Abstract/Free Full Text].
21.
Ishii, M.,
Y. Horio,
Y. Tada,
H. Hibino,
A. Inanobe,
M. Ito,
M. Yamada,
T. Gotow,
Y. Uchiyama,
and
Y. Kurachi.
Expression and clustered distribution of an inwardly rectifying potassium channel, KAB-2/Kir4.1, on mammalian retinal Muller cell membrane: their regulation by insulin and laminin signals.
J. Neurosci.
17:
7725-7735,
1997[Abstract/Free Full Text].
22.
Joseph, D. P.,
and
S. S. Miller.
Apical and basal membrane ion transport mechanisms in bovine retinal pigment epithelium.
J. Physiol. (Lond.)
435:
439-463,
1991[Abstract].
23.
McNicholas, C. M.,
W. Wang,
K. Ho,
S. C. Hebert,
and
G. Giebisch.
Regulation of ROMK1 K+ channel activity involves phosphorylation processes.
Proc. Natl. Acad. Sci. USA
91:
8077-8081,
1994[Abstract].
24.
McNicholas, C. M.,
Y. Yang,
G. Giebisch,
and
S. C. Hebert.
Molecular site for nucleotide binding on an ATP-sensitive renal K+ channel (ROMK2).
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F275-F285,
1996[Abstract/Free Full Text].
25.
Miller, S. S.,
and
J. L. Edelman.
Active ion transport pathways in the bovine retinal pigment epithelium.
J. Physiol. (Lond.)
424:
283-300,
1990[Abstract].
26.
Miller, S. S.,
and
R. H. Steinberg.
Passive ionic properties of frog retinal pigment epithelium.
J. Membr. Biol.
36:
337-372,
1977[Medline].
27.
Nichols, C. G.,
and
A. N. Lopatin.
Inward rectifier potassium channels.
Annu. Rev. Physiol.
59:
171-191,
1997[Medline].
28.
Noma, A.
ATP-regulated K+ channels in cardiac muscle.
Nature
305:
147-148,
1983[Medline].
29.
Pusch, M.,
and
E. Neher.
Rates of diffusional exchange between small cells and a measuring pipette.
Pflügers Arch.
411:
204-211,
1988[Medline].
30.
Rae, J.,
K. Cooper,
P. Gates,
and
M. Watsky.
Low access resistance perforated patch recordings using amphotericin B.
J. Neurosci. Methods
37:
15-26,
1991[Medline].
31.
Segawa, Y.,
and
B. A. Hughes.
Properties of the inwardly rectifying K+ conductance in the toad retinal pigment epithelium.
J. Physiol. (Lond.)
476:
41-53,
1994[Abstract].
32.
Takahira, M.,
and
B. A. Hughes.
Isolated bovine retinal pigment epithelial cells express delayed rectifier type and M-type K+ currents.
Am. J. Physiol.
273 (Cell Physiol. 42):
C790-C803,
1997[Abstract/Free Full Text].
33.
Takumi, T.,
T. Ishii,
Y. Horio,
K. Morishige,
N. Takahashi,
M. Yamada,
T. Yamashita,
H. Kiyama,
K. Sohmiya,
S. Nakanishi,
and
Y. Kurachi.
A novel ATP-dependent inward rectifier potassium channel expressed predominantly in glial cells.
J. Biol. Chem.
270:
16339-16346,
1995[Abstract/Free Full Text].
34.
Taylor, W. R.,
and
N. M. Green.
The predicted secondary structure of the nucleotide-binding sites of six cation-transporting ATPases lead to a probable tertiary fold.
Eur. J. Biochem.
179:
241-248,
1989[Abstract].
35.
Wang, W.,
and
G. Giebisch.
Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct.
J. Gen. Physiol.
98:
35-61,
1991[Abstract].
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