1 Department of Cellular and Molecular Physiology, Yale University, New Haven, Connecticut 06520; and 2 Division of Nephrology, Vanderbilt University, Nashville, Tennessee 37322 - 2372
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ABSTRACT |
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Close similarity between the rat native
low-conductance K+ channel in the apical membrane of renal
cortical collecting duct principal cells and the cloned rat ROMK
channel strongly suggest that the two are identical. Prominent features
of ROMK regulation are a steep pH dependence and activation by protein
kinase A (PKA)-dependent phosphorylation. In this study, we
investigated the pH dependence of cloned renal K+ channel
(ROMK2), wild-type (R2-WT), and PKA site mutant channels (R2-S25A,
R2-S200A, and R2-S294A). Ba2+-sensitive outward whole cell
currents (holding voltage 50 mV) were measured in two-electrode
voltage-clamp experiments in Xenopus laevis
oocytes expressing either R2-WT or mutant channels. Intracellular pH
(pHi) was measured with pH-sensitive microelectrodes in a
different group of oocytes from the same batch on the same day. Resting pHi of R2-WT and PKA site mutants was the same: 7.32 ± 0.02 (n = 22). The oocytes were acidified by adding
3 mM Na butyrate with external pH (pHo) adjusted to 7.4, 6.9, 6.4, or 5.4. At pHo 7.4, butyrate led to a rapid (
:
163 ± 14 s, where
means time constant, n = 4) and stable acidification of the oocytes (
pHi
0.13 ± 0.02 pH units, where
means change, n = 12). Intracellular acidification reversibly inhibited ROMK2-dependent
whole cell current. The effective acidic dissociation constant
(pKa) value of R2-WT was 6.92 ± 0.03 (n = 8). Similarly, the effective
pKa value of the N-terminal PKA site mutant
R2-S25A was 6.99 ± 0.02 (n = 6). The effective pKa values of the two COOH-terminal PKA site
mutant channels, however, were significantly shifted to alkaline
values; i.e., 7.15 ± 0.06 (n = 5) for R2-S200A
and 7.16 ± 0.03 (n = 8) for R2-S294A. The
apparent
pH shift between the R2-WT and the R2-S294A mutant was 0.24 pH units. In excised inside-out patches, alkaline pH 8.5 activated
R2-S294A channel current by 32 ± 6.7%, whereas in R2-WT channel
patches alkalinzation only marginally increased current by 6.5 ± 1% (n = 5). These results suggest that channel phosphorylation may substantially influence the pH sensitivity of ROMK2
channel. Our data are consistent with the hypothesis that in the native
channel PKA activation involves a shift of the pKa value of
ROMK channels to more acidic values, thus relieving a
H+-mediated inhibition of ROMK channels.
phosphorylation; potassium ion channel; inward-rectifier; potassium ion secretion; intracellular pH
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INTRODUCTION |
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ROMK CHANNELS expressed in Xenopus laevis oocytes exhibit biophysical and regulatory properties closely resembling those of the low-conductance K+ channel identified in the apical membrane of principal cells of the cortical collecting duct (CCD) (11). These include a single-channel conductance of 30-40 pS in symmetrical KCl solutions (6, 11), a high K+ selectivity (19), a high open probability at resting membrane voltages (6, 17), weak inward rectification, and a marked sensitivity to external Ba2+, but not to external tetraethylammonium (TEA) (6, 19, 22), inhibition by low cytosolic pH (2, 3, 19), and activation by protein kinase A (PKA) and inhibition by phosphatases (7, 10, 21). Furthermore, immunolocalization demonstrated expression of ROMK in the apical membrane of rat CCD (20). Together, this evidence most strongly supports the idea that the low-conductance K+ channel in the apical membrane of CCD principal cells and ROMK channels are identical. Functionally, ROMK channels are critically involved in the secretion of K+ across the apical membrane of CCD cells in the kidney (18). After cloning of ROMK1 (Kir 1.1a) (6) further splice variants at the 5' end were identified. The cloned renal K+ channel (ROMK2) (Kir 1.1b) lacks the first 19 amino acids of ROMK1, and ROMK3 (Kir 1.1c) contains a seven-amino acid extension (1, 22). In CCD principal cells, both ROMK1 and ROMK2 mRNA were identified (1).
In the present study, we have investigated the pH dependence of the cloned ROMK2 channel and three previously established PKA site mutants. These mutants have the serine amino acid at either position 25, 200, and 294 replaced with an alanine. They therefore mimic stably dephosphorylated ROMK2 channels (21). Our data indicate that the two COOH-terminal PKA site mutations (R2-S200A, R2-S294A) have their effective acidic dissociation constant (pKa) value significantly shifted to more alkaline values. These results indicate that the state of phosphorylation of ROMK channels alters their pH dependence.
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MATERIALS AND METHODS |
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Preparation and injection of oocytes. Oocytes were isolated from X. laevis frogs by partial ovarectomy under tricaine anesthesia (6.5 mM for 10-15 min). Subsequently, oocytes were manually dissected from ovarian lobes and defolliculated by treatment with 2 mg/ml collagenase (Worthington type 1) in a zero Ca2+ hypotonic solution [(in mM) 85 NaCl, 2 KCl, 1 MgCl2, 5 HEPES, pH 7.4] with gentle agitation for 1 h. After being washed in ND-96 solution [(in mM) 96 NaCl, 2 KCl, 1.8 CaCl2, 1 MgCl2 , 5 mM HEPES, pH 7.4], oocytes were placed in the same ND-96 solution to which 50 ng/ml gentamycin and 2.5 mM Na pyruvate were added. Twelve to twenty-four hours after isolation, healthy-looking stage V-VI oocytes were injected with 50 nl of water containing 4 ng cRNA of rat wild-type (R2WT) or mutant (R2-S25A, R2-S200A, or R2-S294A) ROMK2 as described previously (21). Intracellular pH (pHi) and voltage-clamp experiments were performed 2-6 days after injection.
Two-electrode voltage-clamp experiments.
Whole cell currents of ROMK2 (wild-type or PKA site mutants)-injected
oocytes were recorded using the Warner oocyte clamp (model OC-725C,
Warner Instruments, West Haven, CT). Microelectrodes were pulled on a
vertical puller (Narishige PP-88, Tokyo, Japan) from Kimax-51 glass
(Kimble Products) and had resistances of 0.5-2 M when filled
with 3 M KCl solution. Experiments were performed at 20-22°C.
Water- or noninjected oocytes had slope conductances (±20 mV) below 4 µS at their membrane voltage (Vm) (close to
30 mV). For measurements of ROMK2 and mutant channel currents, only those oocytes with slope conductances >33 µS [>1 µA at holding voltage (Vhold)
50 mV] around their measured
Vm of close to
80 mV were chosen.
Patch-clamp experiment.
For patch-clamp electrophysiology experiments (4, 5),
oocytes were injected with 12.5 ng of R2-WT or R2-S294A mutant channel
cRNA the same day of isolation, and were studied 1-4 days after
injection. Electrodes were pulled on a Narishige PP-83 from Dagan LG-16
glass capillaries and highly polished on a Narishige microforge
(MF-83). Electrodes had a resistance of 4-5 M when filled with
150 mM KCl pipette solution.
Measurement of pHi in oocytes.
We used ion-sensitive microelectrodes to measure oocyte
pHi. Detailed methods of this technique have been described
previously (12), hence only a brief outline is provided.
Measurement of pHi requires simultaneous measurement of
membrane potential with a Vm and pH electrode.
The microelectrodes were prepared from borosilicate capillary glass
(Warner Instruments) using a horizontal micropipette puller (Sutter
Instrument, Novato, CA). The Vm electrodes were
filled with 3 M KCl and had a resistances <5 M. The glass used to
make the pH electrodes was first cleaned in chromic acid before being
washed in distilled H2O and than rinsed in ethanol. The
electrodes were silanized at 200°C for 5 min, using
bis(dimethylamino)dimethylsilane (Fluka, Ronkonkoma, NY) and then
baked overnight. The tips of the electrodes were filled with
H+-selective ionophore (hydrogen ionophore I cocktail B,
Fluka). The pH electrodes were backfilled with a buffer (pH 7)
containing (in mM) 40 KH2PO4, 23 NaOH, and 150 NaCl. These electrodes were calibrated using solutions of pH 6.00 and
8.00 (Fisher Scientific, Fair Lawn, NJ) and had slopes of 55-60
mV/pH unit. A single-point adjustment using a pH 7.50 solution was made
to the offset of the pH electrode once it was placed in the perfusion
chamber. The Vm and pH electrodes were connected
to high-impedance electrometers. The Vm was the
difference between the Vm electrode and a
calomel reference electrode. The proton-dependent component of the
membrane potential was obtained from the subtraction of the
Vm and pH electrode signals. The signals were
digitized and recorded on an IBM-compatible PC using software written
in house.
Solutions and chemicals. Collagenase was obtained from Worthington Biochemical, Lakewood, NJ. All other chemicals were of the highest grade of purity available and were obtained from Sigma (St. Louis, MO). All whole cell voltage-clamp experiments were conducted in ND-96 solution [(in mM) 96 NaCl, 4 KCl, 1.8 CaCl2, 1 MgCl2, 5 HEPES, pH 7.4]. The patch pipette solution contained (in mM) 150 KCl, 1 CaCl2, 1 MgCl2 and 5 HEPES, pH 7.4. The patch experiment bath solutions contained (in mM) 150 KCl, 5 HEPES, and 5 EGTA, pH 7.4, 8.5 or 6.0 by KOH.
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RESULTS |
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To define the pH dependence of R2-WT vs. the three PKA site
mutants (R2-S25A, R2-S200A, and R2-S294A), we measured pHi
and Ba2+-sensitive whole cell currents. We used the sodium
salt of the permeant weak acid, butyrate (pKa
4.81 at 20°C) to acidify oocytes. Butyric acid was found
to be substantially more permeable through the oocyte membranes than
was acetic acid, which has been previously used for the same purpose
(W. F. Boron, unpublished observations; 2, 15). All
pHi acidification experiments were performed with 3 mmol/l
Na butyrate added to the ND-96 solution. To increase intracellular
acidification, external pH (pHo) was set to 7.4, 6.9, 6.4, and 5.4, giving free permeable butyric acid concentrations of 7.7, 24, 77, and 613 µmol/l. Without butyrate, these pHo changes
influenced neither pHi nor whole cell currents. Most whole
cell currents and pHi were measured in the same batch of
oocytes on the same day in either the pH or two-electrode voltage-clamp setup. Figure 1 shows an example of a
pHi measurement in an oocyte expressing the R2-S200A
mutant. The top trace depicts pHi, and the
bottom trace shows the simultaneously measured
Vm. Addition of 3 mmol/l butyrate in external pH
of 6.4 leads to a decrease in pHi from 7.32 to 7.03 after
4-5 min. Vm decreases simultaneously from
88 to
42 mV. This depolarization reflects the pH-dependent inhibition of R2-S200A current. Figure 2
shows the average initial pHi values and the effects of
butyrate at the varying pHo values. It is apparent that the
reduction in pHo of the butyrate solution produced a
parallel intracellular acidification. We observed no differences in
resting or butyrate-induced pHi changes between the R2-WT
and mutant channel-expressing oocytes; the data were therefore pooled.
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At 4 mM external KCl, the oocytes had a mean Vm
of 78.8 ± 0.4 mV (n = 62) with no differences
between the R2-WT and the mutant-expressing oocytes. However, with the
same amount of ROMK channel RNA injected, the level of functional
current expression differed significantly between R2-WT and the
different PKA site mutants. These data are similar to those originally
describing the difference of R2-WT and PKA site mutants and confirm the
reduction in whole cell current when PKA site mutants are expressed
(21).
The responses of R2-WT and R2-S294A to 3 mM butyrate at pHo
6.9 are compared in Fig. 3. The
top panel depicts the time course of pHi changes
in an oocyte expressing R2-WT when it was acidified with butyrate. The
middle and bottom panels show
Ba2+-sensitive whole cell currents
(Vhold-50 mV) for oocytes expressing R2-WT and
R2-S294A, respectively. Addition of a short pulse of 5 mM
Ba2+ nearly completely inhibited outward current, verifying
that the whole cell currents were mediated by the expressed ROMK
channels. Figure 3 shows that cell acidification reduced K+
current by 25% in R2-WT-expressing oocytes but nearly completely inhibited ROMK current with the R2-S294A mutant.
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The time course of the reduction in butyrate-induced pHi and whole cell current reduction was very similar. With 3 mmol/l butyrate and pHo 7.4, the time constant of the intracellular acidification was 163 ± 14 s. In oocytes selected from the same batch, the time constant for the butyrate-dependent decrease in whole cell current was 149 ± 5 s (n = 4).
Figure 4 shows I-V curves in
the absence and presence (after 5 min) of butyrate in oocytes
expressing either R2-WT (top) or R2-S294A
(bottom). I-V curves were obtained by varying
Vhold from 100 to +100 mV in 10-mV steps, with
each voltage applied for 500 ms. The acidifying step (mean
pH
decrease: 0.26 ± 0.05, n = 12) had little effect
on current in the R2-WT-expressing oocytes current, whereas it nearly
completely inhibited the whole cell current in the R2-S294A-expressing
oocyte.
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The results in Fig. 4 suggest that the lack of inhibitory effect of 3 mM butyrate in R2-WT- vs. R2-S294A-expressing oocytes might be due to
the high level of channel expression compared with R2-S294A expression.
To address this issue, we examined the effect of 3 mM butyrate in
R2-WT-expressing oocytes 1 day after injection when mean slope
conductance [102 ± 15 slope conductances (µS)] and outward
K+ current (3.07 ± 0.45 µA) at
Vhold were low. Butyrate (3 mM, pHo 6.9) inhibited 11% of ROMK-dependent outward current
(n = 4). In the same batch of oocytes
(investigated > 48 h after injection), when mean slope
conductance was 371 ± 36 µS, outward current was 11.1 ± 1 µA (at Vhold 50 mV), 3 mM butyrate
(pHo 6.9) inhibited 21% of ROMK-dependent outward current
(n = 11). Thus butyrate inhibited K+
current to a similar extent in low vs. high R2-WT-expressing oocytes,
and this level of inhibition was significantly less than that in
R2-S294A-expressing oocytes.
Subsequently we investigated the pH dependence of the PKA site mutants
and the R2-WT over a range of different pH values as described above
(see Fig. 2). The temporal responses to 3 mM butyrate (pHo
6.9) on R2-WT and all three PKA site mutant channel-expressing oocytes
(R2-S25A, R2-S200A, and R2-S294A) are shown in Fig.
5. Oocyte acidification produced greater
reductions in whole cell currents in PKA-site mutant ROMK channels
compared with R2-WT. These data indicate an increased sensitivity of
the PKA-site mutants to reductions in pHi.
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The comparison of the pH sensitivity of R2-WT and the different
PKA-site mutant channel-expressing oocytes were performed as follows.
Because pHi and current were measured in different oocytes,
we used steady-state pHi values and current values at 4-5 min after exposure to butyrate. Each single experimental data set was individually fitted with the normalized K+ current
(I/Imax, where I is
steady-state butyrate-inhibited K+ current and
Imax is K+ current before addition
of butyrate) as a function of intracellular H+
concentration using the modified Hill equation: I =
1/[1+([H]/K0.5)n],
where I is the normalized inhibited current, [H] is the
internal H+ concentration, K0.5 is
the H+ concentration of half-maximal inhibition, and
n is the Hill coefficient. Thus for each experiment the
H+ concentration of half-maximal inhibition was determined.
These values were compared using the unpaired t-test. Before
addition of butyrate, the R2-WT and the different constructs had the
following Imax values, expressed as microsiemens
measured between 0 and 50 mV holding voltage: R2-WT, 458 ± 23 (n = 23); R2-S25A, 191 ± 70 (n = 9); R2-S200A, 336 ± 42 (n = 13); and R2-S294A,
154 ± 26 (n = 17). Figure
6 depicts the summary of all data showing the normalized K+ current
(I/Imax) as a function of the
intracellular H+ concentration. At the lowest pH of 6.53, all R2-WT and PKA site mutant channels were completely and reversibly
inhibited. It is obvious that the R2-WT channel had an effective
pKa value of 6.92 ± 0.03 (n = 8). Similarly, the NH2-terminal PKA
site mutant R2-S25A showed an effective pKa
value of 6.99 ± 0.02 (n = 6). Compared with the
R2-WT channel, the two COOH-terminal PKA-site mutant channels were
found to have an effective pKa value
significantly (P < 0.005) shifted to more alkaline
values; i.e., R2-S200A, 7.15 ± 0.06 (n = 5) and
R2-S294A, 7.17 ± 0.03 (n = 8). The calculated Hill coefficients were 7.26 ± 0.9 for R2-WT, 7.75 ± 1.1 for
R2-S25A, 6.1 ± 0.8 for R2-S200A, and 6.7 ± 0.8 for
R2-S294A. These results are consistent with previous investigations of
ROMK2 pH dependence and indicate the high cooperativity of
H+-mediated channel inhibition (2, 3, 9).
These results suggest that the PKA-mediated phosphorylation state of
the COOH terminus of ROMK2 channels significantly alters their pH
dependence.
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In studies using the single-channel patch-clamp, the ROMK2-S294A has an
open probability (Po) 40% less than the fully
phosphorylated R2-WT channel (8). To determine the
sensitivity of the incompletely phosphorylated channel to pH, we
measured giant currents from excised inside-out patches from oocytes
expressing either R2-WT or R2-S294A channels (Fig.
7, A-D). Patches
from oocytes expressing R2-WT showed a mean conductance of 4.8 ± 0.9 nS (n = 5) in cytoplasmic solution, pH 7.4. When
this was replaced with a solution pH 8.5, there was a small increase in
the conductance (mean 5.1 ± 1.0 nS, n = 5) of
6.5 ± 1.0%. Excised patches containing R2-S294A channels
had a mean conductance of 3.0 ± 0.7 nS (n = 5) in
pH 7.4, but at more alkaline solution pH 8.5, the mean conductance of
the patches changed to 3.9 ± 0.9 nS (n = 5), an
increase of 32.9 ± 6.7%. This increase in current from the
mutant R2-S294A channel is significantly different from oocytes
expressing R2-WT (P < 0.05). This suggests that the
mutant channel is more sensitive to pHi and is partially
inhibited at the normal oocyte resting pH of 7.3.
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DISCUSSION |
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The regulation of ROMK channels is complex and involves a number of different factors. Prominent features of this regulation are the state of channel phosphorylation (21) and a very steep pH dependence (2, 3, 10, 19). In this study, we have investigated the pH dependence of R2-WT and the PKA site mutant channels in oocytes. Previous data provided strong evidence that phoshorylation of the channel polypeptide by PKA is involved in channel regulation and that PKA-dependent phosphorylation is essential for ROMK channel activity. PKA-dependent phosphorylation of ROMK2 at S25, S200, and S294 has been demonstrated (21). In this previous study, three PKA site mutant constructs were produced, which replaced serine by alanine at each of the positions (R2-S25A, R2-S200A, and R2-S294A). Each PKA site mutant reflects a stable single-site dephosphorylated state of the channel. Functional expression of single PKA site mutants has previously been shown and is confirmed in this study (8, 21). The present study also confirms that channel activity is reduced in all three single PKA site mutants expressed in oocytes. Channels containing either two or three PKA site mutants could not be shown to function in whole cell experiments (21) and thus were not used.
The effective pKa value of R2-WT channels of pH 6.92 is in close agreement with those reported by others (2, 3). In contrast, the two COOH-terminal PKA site mutants have effective pKa values substantially shifted to more alkaline pHi values compared with R2-WT. Thus the state of PKA-dependent phosporylation of ROMK2 appears to substantially influence the pH-mediated regulation of this channel. This conclusion depends on the assumptions that substitution of alanine for serine per se does not influence pHi sensitivity and that the ROMK2 channels expressed in oocytes are completely (or nearly completely) PKA phosphorylated. At present we have no means of excluding the former because substitution with an amino acid other than alanine would present a similar inability to distinguish the effects of amino acid substitution per se from that of phosphorylation. The latter is likely because we have previously shown that elimination of two or more PKA phosphorylation sites renders the ROMK2 channel inactive. In other words, significant further dephosphorylation of R2-S25A, R2-S200A, or R2-S294A would make the channel inactive and therefore would not influence baseline current measurements.
The secretory K+ channel in principal cells of the CCD can be stimulated by PKA activation (16, 17). One may therefore hypothesize that the native secretory K+ channel is only partially phosphorylated. Our present results may provide further insight into how channel phosphorylation leads to channel activation. PKA-dependent phosphorylation of serine residues shifts the pHi dependence to more acidic values, and this would activate these K+ channels. The resting physiological pHi of intact CCD principal cells is expected to be close to 7.2 as is the case for many other mammalian cells. Measured values have been reported to be 7.23 (13) and 7.31 (14). Therefore, the fully phosphorylated ROMK channel with an effective pKa value close to 7.0 would be completely active at physiological pH values. In contrast, the COOH-terminal PKA site mutant R2-S294A with an effective pKa value close to 7.2 could be partially inhibited at an assumed resting pH around 7.2-7.3. A recent study demonstrated that the R2-S25A mutant had a Po close to 1, whereas the R2-S200A and R2-S294A mutants had reduced Po values close to 0.5 (8). The present study showing different pHi dependence of the PKA site mutants suggests that the reduced Po values of the two COOH-terminal PKA site mutant channels were likely due to partial pH inhibition. We suppose that PKA site mutations on the NH2 terminus affect channel density (8), whereas mutations on the COOH terminus affect Po and confer changes in pH sensitivity. In excised inside-out patches, we show that the R2-WT channel current remains largely unaffected by pH 8.5 alkaline solutions, whereas the R2-S294A construct channels can be significantly activated by alkaline solutions. This supports the idea that these PKA-site mutant channels are partially pH inhibited at oocyte resting pH.
It has recently been demonstrated that a lysine residue (K61 for ROMK2 and K80 for ROMK1) is of primary importance to confer the steep pH dependence of the ROMK channel (3). Replacement of lysine at position 80 to methionine in ROMK1 completely abolished pH sensitivity. The authors assume that a titratable lysine side chain in the neutral pH range accounts for their findings. To explain the apparent discrepancy between the pKa of lysine as pure amino acid (10.5) and that observed in ROMK1 channel inhibition, it was suggested that the chemical environment in the protein structure could account for such a shift in pKa. Further studies have confirmed the critical importance of the same lysine at position 61 also in ROMK2 channels (2). In this latter work, the authors could furthermore identify a threonine at position 51 in ROMK2 channels, which significantly shifted the apparent pKa to more acidic values when exchanged with the positively charged lysine. Furthermore, the exchange of T51 for the negatively charged glutamate shifted the apparent pKa value to a more alkaline value. The authors propose that because of the close proximity to position K61 the T51 mutants may influence the chemical environment of the critical proton binding site and thereby lead to the observed shifts in pKa (2). The previous work thus demonstrated the critical role for the pH sensor on the NH2 terminus (2, 3).
One might speculate that any of the phosphorylation sites could have spatial proximity to the critical lysine 61 residue within one subunit or to one of the neighboring subunits and thereby modulate the channel's pKa value as a function of phosphorylation. In the tetrameric channel, the NH2 and COOH termini could directly interact, thereby allowing them to influence the important lysine 61 residue.
Thus the pH regulation of ROMK channels is complex. Here we suggest that the critical pH sensor on the NH2 terminus is modulated as a function of channel phosphorylation. In addition, recent data also indicate that cytosolic acidification increases the ability of ATP to inhibit the channel (9).
In summary, our data demonstrate that the state of phosphorylation of the two COOH-terminal PKA sites may substantially influence the pH value for half maximal inhibition of the steeply pH-sensitive ROMK2 channel.
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ACKNOWLEDGEMENTS |
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We are very thankful to Carmel McNicholas for support during the initial phase of the project. The pHi measurements were conducted in the Laboratory of W. F. Boron, whose kind help and collaboration we gratefully acknowledge. The authors thank C. L. Huang and D. W. Hilgemann (University of Texas Southwestern Medical Center at Dallas) for advice on the giant patch-clamp technique. G. G. MacGregor is a National Kidney Foundation/ American Society of Nephrology/Baxter Healthcare-Renal Division Fellow.
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FOOTNOTES |
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This project was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-17433 (to G. G. MacGregor) and DK-37605 (to S. C. Hebert).
Address for reprint requests and other correspondence: J. Leipziger, Physiologisches Institut,
Albert-Ludwigs-Universität, Hermann-Herder-Strae 7, 79104 Freiburg, Germany (E-mail: leipzige{at}ruf.uni-freiburg.de).
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.
Received 8 February 1999; accepted in final form 17 July 2000.
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