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INTRODUCTION |
The physiological roles of potassium (K+) channels in
the regulation of water and electrolyte transport in kidney are well known (1). In the thick ascending limb
(TAL)1 of Henle's loop, the
recycling of K+ ions across the low-conductance apical
K+ channels is essential for NaCl reabsorption through the
apical Na-K-2Cl cotransporter (1). The importance of the apical
K+ channels for NaCl reabsorption in TAL is underscored by
the genetic disease, Bartter's syndrome, in which loss-of-function
mutations of the channels result in impairment of NaCl transport in
this nephron segment (2). In the cortical collecting ducts (CCDs), secretion of K+ into urinary space is mediated by active
transport of K+ into the cell through basolateral
Na+-K+-ATPase, followed by passive movement of
K+ into the tubular fluid through apical K+
channels (1). cDNAs for ROMK1 and its isoforms ROMK2 and -3 have
been isolated (3, 4). Based on the distribution of mRNA and
proteins, and biophysical characterization, it is known that ROMK1 and
ROMK2 encode the low-conductance secretory K+ channels in
CCDs and TALs, respectively (4).
The apical K+ channels in CCDs and TALs are regulated by
multiple signaling pathways, including protein kinase A (PKA), protein kinase C (PKC), cGMP, calcium-calmodulin activated kinase II, intracellular pH, arachidonic acid, etc. (5). These signaling pathways
play important roles in hormonal regulation of the K+
channels. The PKA pathway is important for the regulation of the
K+ channels in TALs and CCDs by vasopressin (6, 7). The
importance of direct phosphorylation by PKA for channel function is
further supported by the finding that one of the mutations in
Bartter's syndrome is at a PKA phosphorylation site (2). The PKC
pathway is likely important for regulation of K+ transport
in CCDs by many PLC-activating hormones and growth factors, including
prostaglandin E2, bradykinin, and epidermal growth
factor (8-11). Activation of PKC by phorbol 12-myristate 13-acetate
(PMA) inhibits apical K+ channels in CCDs (11, 12).
Recently, we and others reported a novel mechanism for regulation of
ROMK and other inward rectifying K+ channels via direct
interaction with membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2) (13-15). This direct interaction
occurs between PIP2 and several positively charged residues
in the proximal C termini of the inward rectifying K+
channels (15) and likely regulates channel opening by stabilizing the
structure of the cytoplasmic entrance of the pore. The importance of
PIP2 for inward rectifier K+ channels is
further supported by many studies showing that PIP2 influences the regulation of the channels by other signaling or gating
molecules. PIP2 modulates the regulation of GIRK channels by G
and intracellular Na+ and Mg2+ ions
(15, 16), the regulation of KATP by
intracellular ATP (17, 18), and the regulation of ROMK by PKA (19) and
pHi (20). We found that phosphorylation of ROMK by PKA does not directly activate ROMK1 channels in membranes that are depleted of
PIP2 (19). Rather, it lowers the concentration of
PIP2 necessary for activation of the channels, suggesting
that PKA activates ROMK1 by enhancing PIP2-channel interaction.
The molecular mechanism by which PKC inhibits the activity of ROMK
channels remains poorly understood. It was reported previously that PKA
antagonizes PKC inhibition of K+ channels in CCD (12). In
the present study, we examine the hypothesis that PKC inhibits ROMK1
channels via a PIP2-dependent mechanism.
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EXPERIMENTAL PROCEDURES |
Molecular Biology--
Wild type ROMK1 cDNA was in the
pSPORT plasmid (3). Site-directed mutagenesis of ROMK1 was performed
using a commercial mutagenesis kit (QuikChange from Stratagene, La
Jolla, CA), and confirmed by nucleotide sequencing as previously
described (15, 19, 20). mCAP cRNAs of the wild type and mutant ROMK1
channels were transcribed in vitro using T7 RNA polymerase
(15, 19, 20).
Two-electrode Voltage-Clamp Recording--
Xenopus
laevis oocytes were prepared as previously described (15,
19, 20). Oocytes were injected with cRNA for wild type or mutant ROMK1.
Current-voltage (I-V) relationships (
100 to +100 mV, in 25 mV steps)
were measured in oocytes at ~23 °C by two-electrode voltage-clamp
using an OC-725C oocyte clamp amplifier (Warner Instrument), pCLAMP7
software, and Digidata 1200A digitizer (Axon Instrument). The
resistance of current and voltage microelectrodes (filled with 3 M KCl solution) was 1-2 M
. The bath solution contained (in mM) 96 KCl, 1 MgCl2, 1 CaCl2, 5 Hepes (pH 7.5 by KOH).
Cell-attached and Excised Inside-out Patch Clamp
Recording--
Patch clamp pipettes (pulled from borosilicate glass,
Warner Instrument Co., Hamden, CT) were filled with solutions
containing (in mM): 100 KCl, 1 MgCl2, 2 CaCl2, 5 Hepes (pH 7.4 with KOH). Pipette tip resistance
ranged from 3 to 5 megaohms. For cell-attached recordings, bath
solution contained 100 KCl, 1 MgCl2, 1 CaCl2, 10 Hepes (pH 7.4 with KOH). For inside-out recordings, we used the
Mg2+-free bath solution containing 100 KCl, 5 EDTA, and 5 Hepes (to prevent fast run-down) (15, 19, 20). In the experiments applying PKC in inside-out membranes (Fig. 7), purified rat brain PKC
(from Calbiochem; 1 unit/ml) was applied in a Mg2+-free
bath solution containing 500 nM Ca2+ (4.86 mM CaCl2 and 5 mM EDTA) and 50 µM 1-oleoyl-2-acetyl-sn-glycerol (OAG).
Single-channel currents were recorded with an Axopatch 200B patch clamp
amplifier (Axon Instruments, Foster City, CA), low-pass filtered at 1 kHz using an 8-pole Bessel filter, sampled every 0.1 ms (10 kHz) with
Digidata-1200A interface and stored directly onto the computer hard
disk (100 GB) using pCLAMP8 software. Data were transferred to CD for
long-term storage. For analysis, event list files were generated using
the Fetchan program and analyzed for open probability, amplitude, and
dwell-time histograms using pCLAMP6 pSTAT (version 6.0.5, Axon
Instruments). Open probability (NPo) was analyzed on
segments of continuous recording (duration as indicated, respectively)
as previously described (20, 21).
Measurement of 32P-labeled PIP2 by
TLC--
32P-Labeled PIP2 in oocyte membrane
was measured by TLC as described previously for cultured cells (22, 23)
with minor modifications. Briefly, PIP2 in oocytes (~10
each group) were labeled by incubating in ND96 (1 ml) containing
[32P]PO4 (40-50 µCi) for 4 h.
Oocytes were further incubated in the isotope-free ND96 for 2 h
before treatments by Me2SO (vehicle), PMA and/or
calphostin-C as indicated. Oocytes were homogenized in a buffer
containing 1 ml of 5% trichloroacetic acid and 1 mM EDTA
and spun in a microcentrifuge. Pellets were extracted in a
buffer containing CHCl3, MeOH, 10 N HCl
(20:20:0.2). The organic (lower) phase was collected, dried, and
dissolved in 20 µl of CHCl3. Samples (5 µl) and lipid
standards were resolved by TLC using 1-propanol,
H2O, NH4OH (65:15:20). 32P-Labeled
lipids were detected by autoradiography. Lipid standards were detected
with iodine vapor.
Measurement of PIP2 Mass by High Pressure Liquid
Chromatography (HPLC)--
PIP2 mass was measured by
separation of deacylated phospholipids using anion-exchange HPLC as
previously described (24, 25). Briefly, oocytes with or without PMA
treatment were homogenized and phospholipids were extracted in 1 ml of
cold CHCl3, MeOH, 10 N HCl (20:40:1).
Phospholipids were deacylated with monomethylamine and the head groups
were separated by anion-exchange HPLC (Ionpac AS-11-HC2 mm column and
AG11-HC2 mm guard column) using a three-stage NaOH gradient from 10 to
80 mM. Elution of the deacylated phospholipids (in
comparison with the standards) was monitored by detection of the
glycerol head group by suppressed conductivity.
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RESULTS |
Reduction of PIP2-channel Interaction Increased the
Sensitivity of ROMK1 to Inhibition by PKC in Oocytes--
To test the
hypothesis that PIP2 plays an important role in modulating
PKC inhibition of ROMK1 channels, we compare the effects of PKC
activators, PMA and OAG, on wild type and mutant ROMK1 channels with
reduced PIP2 affinity. We have previously shown that
mutation of a PKA consensus site, serine 219, to alanine (S219A)
reduces the affinity of ROMK1 for PIP2 (19). As shown in
Fig. 1B, the activity of S219A
mutant was partially inhibited by addition of PMA (300 nM)
to the extracellular bath solution. The decrease in channel activity
was apparent within 10 min after administration of PMA and leveled at
~60% inhibition by 30 min. Pretreatment with PKC inhibitor
calphostin-C (0.5 µM) prevented the inhibition of
channels by PMA (Fig. 1C). Another PKC activator OAG
inhibited S219A channel activity similarly (Fig. 1D). As
reported previously by others (26), activation of PKC by PMA did not cause a significant inhibition of wild type ROMK channels expressed in
oocytes (Fig. 1A). These results suggest that reduction of PIP2-ROMK1 interaction increases the sensitivity of
channels to inhibition by PKC. Lack of inhibition by PKC on the wild
type channel in oocytes is likely because of that ROMK1 has a higher baseline affinity for PIP2 (see "Discussion").

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Fig. 1.
Effect of PMA on wild type ROMK1 and S219A
mutant with reduced PIP2 affinity. A,
effect of PMA (300 nM) on wild type ROMK1. K+
currents through ROMK were measured by two-electrode voltage-clamp. PMA
was added immediately after recording of K+ currents at
t = 0 min. +Ba2+ indicates
addition of 1 mM BaCl2. B, effect of
PMA on S219A mutant channel. C, effect of calphostin-C (0.5 µM) on PMA-mediated inhibition of S219A currents. Inward
K+ currents (Ik) at 100 mV were
normalized to currents before PMA. * indicates p < 0.05 by unpaired t test. D, effect of OAG (50 µM) on S219A currents.
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The role of PIP2-channel interaction in modulating PKC
inhibition of ROMK1 is further supported by the following studies using additional mutants with reduced PIP2 affinity. Arginine 188 of ROMK1 is critical for electrostatic interaction with
PIP2 (15, 19). PIP2-channel interaction is
reduced by neutralization of arginine 188 to glutamine (R188Q), but not
by a conserved charge substitution by lysine (R188K) (15, 19). Mutation
of another PKA phosphorylation site, serine 313, to alanine (S313A)
also reduces PIP2 affinity (19). As shown in Fig.
2, addition of PMA caused inhibition on
S313A and R188Q mutants but not on R188K mutant. Pretreatment with
calphostin-C prevented the inhibition (Fig. 2, A-C). OAG
caused a similar inhibition on S313A and R188Q (not shown).

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Fig. 2.
Effect of PMA on ROMK1 mutants with reduced
or normal PIP2 binding affinity. A, S313A
mutant. B, R188Q mutant. C, R188K mutant.
Experimental paradigm is the same as described in the legend to Fig. 1.
S313A and R188Q have reduced PIP2 binding affinity
(19). R188K has normal PIP2 binding affinity (15,
19). n = 5-8 for each experiment. * indicates
p < 0.05. NS, not significant.
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Activation of PKC Reduced Open Probability for ROMK1--
We
performed cell-attached patch clamp recording to examine whether PKC
affects single channel conductance and/or open probability (NPo) of the channel. In oocytes expressing S219A
mutants, PMA caused a reduction in NPo (Fig.
3A). The
NPo of S219A was reduced to 25 ± 11% in 30 min (Fig. 4A). PMA did not
alter the current-voltage relationship (not shown) or unitary conductance of single channels (36 ± 2.7 pS versus
35 ± 1.9 pS; between
50 and
100 mV). In time control
experiments, Me2SO (vehicle) had no effect on the channel
(not shown). Pretreatment with calphostin-C prevented the reduction of
NPo caused by PMA (Fig. 3B). The effect
of PMA to reduce NPo was further confirmed on S313A
and R188Q mutants. As shown in Fig. 4, B and C,
PMA reduced NPo of S313A and R188Q by 65 and 68%,
respectively.

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Fig. 3.
Effect of PMA on S219A channels in
cell-attached recording. A, representative tracing for
the effect of PMA added as indicated. Single channel recording holding
at 100 mV. C indicates current level at the closed state.
1, 2, and 3 indicate current levels
with one, two, or three channel openings. B, representative
tracing after pretreatment by calphostin-C.
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Fig. 4.
Effect of PMA on the activity of S219A
(panel A), S313A (panel B), and R188Q
(panel C) mutant in cell-attached recording.
n = 5-7 for each experimental condition. The activity
of channel (measured as NPo) after 10 min addition
of PMA was normalized to the pre-PMA level (100%).
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PIP2 Reversed PMA-induced Inhibition of Channel
Activity--
The activity of S219A mutant channels was first recorded
in on-cell patch clamp recording. As shown earlier, addition of PMA caused a reduction of NPo of channel in ~10 min
(Fig. 5A). After channel
inhibition by PMA, inside-out membrane patches were then excised and
PIP2 (50 µM) was applied to the cytoplasmic face. Application of PIP2 to the cytoplasmic face of the
excised inside-out membrane increased NPo of the
channel. These results suggest that PMA inhibits the activity of the
S219A mutant ROMK by reducing PIP2-channel interaction.
After recovery of channel activity by PIP2, further
application of PMA to the excised patch did not cause inhibition of the
channel (Fig. 5A). The lack of effect for the second
application of PMA in the inside-out membrane is not because of the
fact that PKC was not present in the excised membranes (see Fig. 7,
below). Fig. 5B summaries the results of five similar
experiments. As shown, PMA decreased NPo to 15 ± 4% of the control level (Fig. 5B, time point
2). Application of PIP2 to the inside-out
membranes increased NPo to 68 ± 18% of the
control level (time point 3), which was not affected by
re-application of PMA to the excised membranes (63 ± 11%; time point 4). ROMK channels run down slowly in the excised
membranes in Mg2+-free bath solutions (15, 19, 20). The
incomplete recovery by PIP2 in the excised membranes is
likely because of run-down of channels over time.

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Fig. 5.
Reversal of PMA-mediated inhibition of S219A
channel by PIP2 in inside-out patch. A,
single channel recording was initially performed in the cell-attached
mode (labeled On-cell) and followed by excised inside-out
mode. The presence of PMA (300 nM) or PIP2 (50 µM) was indicated by the respective horizontal
bar. Upper panel, each vertical column
represents averaged NPo over 20 s. Lower
panel, tracing in expanded time base as indicated by time points
labeled by numbers 1-4. B, averaged
NPo (mean ± S.E., n = 5) at
the indicated time points 1-4.
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PMA Caused Reduction of Membrane PIP2 Content--
The
reduction of PIP2-ROMK interaction by PKC may be because of
reduction of membrane PIP2 content. We examined the effect of PKC activation on PIP2 content in oocyte membrane.
Phospholipids in oocyte membranes were labeled by incubating with
[32P]PO4, extracted, and resolved by thin
layer chromatography. As shown in Fig.
6A, PMA caused a reduction of
32P-labeled PIP2 and PIP. Pretreatment by
calphostin-C prevented the reduction. The average reductions of
32P-labeled PIP2 and PIP by PMA were 57 (p < 0.05; Fig. 6B) and 62% (not shown),
respectively. To assure that the effect of PMA is not because of
alteration of the kinetics of labeling by
[32P]PO4, we also measured PIP2
and PIP mass using anion exchange high pressure liquid chromatography
(24, 25). In agreement with the results from measurement of
32P-labeled PIP2 and PIP, we found that PMA
reduced PIP2 and PIP mass in oocyte membranes by 43 and
64%, respectively.

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Fig. 6.
Effect of PMA on membrane PIP2
content in oocytes. A, representative autoradiograph of
measurement of 32P-labeled PIP2 and PIP by TLC.
B, averaged (mean ± S.E., n = 4)
32P-labeled PIP2 before and after PMA (300 nM, 10 min) with or without pretreatment with calphostin-C
(0.5 µM). PIP2 content after PMA was
normalized to the level before PMA (called baseline = 100%). *
indicates p < 0.05 versus baseline.
NS, not significant.
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Application of Purified PKC Had No Effect on S219A Channels in
Excised Inside-out Patches--
The effect of PKC on S219A channels
was further studied using purified PKC in the excised inside-out
patches. As shown in Fig. 7, application
of purified PKC (1 units/ml) to the cytoplasmic face of the membrane
did not cause inhibition of the channel over 10 min. The activity of
channel in the membrane patches after application of purified PKC (Fig.
7B, open square) was not different from that in
the time-control experiments (closed circle). These results
suggest that the pathway(s) involved in inhibition of channels by PKC
is disrupted in the excised membranes.

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Fig. 7.
Effect of purified PKC on S219A channel in
inside-out patch. A, representative tracing.
B, averaged normalized NPo (mean ± S.E., n = 5 for each) with (open square) or
without (closed circle) application of PKC (see
"Experimental Procedures").
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Activation of PKC via PLC-coupled Receptor Inhibited S219A Channel
and Caused Reduction of Membrane PIP2
Content--
Finally, we examined the role of PKC in the regulation of
ROMK channel by phospholipase C (PLC)-coupled receptors. Type 1 muscarinic receptor (M1R) activates PLC via Gq (27). In
many cell types including Xenopus oocytes, elevation of
intracellular Ca2+ following receptor activation of PLC
increases outwardly rectifying Cl
currents by activating
the endogenous Ca2+-dependent Cl
channels (27). Addition of carbachol (indicated by the upward arrow in the left panel of Fig.
8A) caused an immediate
increase in outwardly rectifying Cl
currents, confirming
the expression of M1 receptors in oocytes. In oocytes co-expressing M1R
and S219A, carbachol inhibited the activity of the channel over 7-10
min (Fig. 8B). Compared with the time course of hydrolysis
of PIP2 by PLC (based on the rise in the intracellular
Ca2+ and activation of outwardly rectifying
Cl
currents in Fig. 8A), the time course of
inhibition of S219A channel was much slower. This delayed inhibition of
channel by carbachol correlated with the effect observed for activation
of PKC by PMA. In support of the interpretation that carbachol inhibits the channel via activation of PKC, pretreatment with calphostin-C prevented the inhibition (not shown). As in the experiments using PMA,
carbachol did not affect wild type ROMK1 channel co-expressed with M1
muscarinic receptor in oocytes (not shown).

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Fig. 8.
Effect of receptor activation of PLC and PKC
on S219A channel and PIP2 content in oocytes.
A, carbachol (CCh) causes an immediate increase
in outward rectifying Cl currents in oocytes expressing
M1 receptor. Voltage ramp from 100 to 100 mV (in 300 ms) was applied
every 30 s (see right panel). Outward currents at +100
mV are shown in the left panel. CCh (50 µM)
was added as indicated by the upward arrow. Five experiments
with similar results were performed. B, effect of CCh on
K+ currents (shown as normalized Ik
holding at 100 mV) in oocytes co-expressing S219A and M1R. Currents
were recorded by two-electrode voltage-clamp. *, p < 0.05 versus time control (i.e. no addition of
CCh). C, effect of CCh on 32P-labeled
PIP2 in oocytes expressing M1R. Results shown are mean ± S.E. (n = 4) of % normalized to the level without
CCh (i.e. control).
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The effect of carbachol on PIP2 content was examined (Fig.
8C). Expression of M1R by itself (without addition of
carbachol) did not alter [32P]PIP2 content in
oocytes (labeled M1R; 95 ± 7% of control).
Application of carbachol reduced [32P]PIP2
content to 65 ± 13% of the control level at 10 min (labeled M1R+CCh; p < 0.05 versus
Control). The reduction of
[32P]PIP2 content by carbachol at 10 min was
prevented by pretreatment with calphostin-C (labeled M1R+Cal-C + CCh; 109 ± 15% of the control level). The complete reversal
of PIP2 to the control level by calphostin-C is consistent
with the notion that reduction of PIP2 by PLC-mediated
hydrolysis is transient (lasting <5 min) (28). Together, these results
suggest that hydrolysis of PIP2 by phospholipase C by
itself is not sufficient to cause inhibition of the channel and that
activation of PKC is necessary.
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DISCUSSION |
PKC inhibits the activity of K+ channels in rat CCDs
(12), but the molecular mechanism for this regulation is not known. We
have shown previously that PIP2 is critical for the
activity of ROMK channel (15, 21) and the regulation of ROMK by other pathways such as PKA (19) and intracellular pH (20) interacts with
PIP2 regulation of the channel. In the present study, we found that mutations of ROMK1 with reduction of affinity for
PIP2 increase the sensitivity of the channel to inhibition
by PKC. We also found that application of PIP2 to the
cytoplasmic surface reverses PKC-mediated inhibition and that
activation of PKC reduces membrane PIP2 content. Together,
these results suggest that PKC inhibits ROMK channels by reducing
membrane PIP2 content.
Others have also reported that for other inward rectifier
K+ channels mutation of the PIP2-binding site
increases the sensitivity of channels to reduction in membrane
PIP2 content. Kobrinsky et al. (27) reported
that receptor-regulated PIP2 hydrolysis (via M1 muscarinic
receptor) causes desensitization of K+ currents through G
protein-coupled inward rectifying K+ channels (GIRK).
Another inward rectifying K+ channel, IRK1, has a higher
PIP2 affinity relative to the GIRK channel (15). Kobrinsky
et al. (27) reported that hydrolysis of PIP2
under the same experimental conditions does not affect the activity of
IRK1. They also found that mutations of PIP2-binding residues of IRK1 render the channel sensitive to receptor-regulated reduction of PIP2 in the membrane (27). The affinity of
ROMK1 for PIP2 is the same as that of IRK1 (15). Thus, it
is perhaps no surprise that a partial reduction of PIP2 by
PMA does not affect the activity of the wild type ROMK1 channel in oocytes.
Then, why does activation of PKC cause inhibition on K+
channels in CCD (12) but not on the wild type ROMK1 channel expressed in Xenopus oocytes (see Ref. 26 and Fig. 1A
also)? Our study suggests that this difference may be explained by a
lower baseline PIP2-K+ channel interaction in
CCD than in Xenopus oocytes, which renders channels in CCDs
more sensitive to PKC regulation. The lower baseline PIP2-K+ channel interaction in CCD may be
because of lower PIP2 content in the apical membrane of CCD
and/or that native K+ channels are partially phosphorylated
by PKA and thus have a lower affinity for PIP2. It is
difficult to directly compare PIP2 content in the apical
membrane of CCD with that in oocyte membrane. Our finding that PKA site
mutants S219A and S313A are sensitive to inhibition by PKC at least
provides support for the latter possibility.
Many hormones, including bradykinin, prostaglandin E2, and
epidermal growth factor inhibit K+ transport in CCDs
(8-11, 29-31). Bradykinin and prostaglandin E2 activate
PLC-
via G proteins and epidermal growth factor activates PLC-
via the intrinsic receptor tyrosine kinase (29-31). These PLC-activating hormones may inhibit K+ transport through
either hydrolyzing PIP2 and/or through activating PKC.
However, some hormone receptors, such as epidermal growth factor
receptors, are present only in the basolateral membrane (30) in
contrast to the localization of the K+ channel in the
apical membrane of CCD. Furthermore, the reduction of PIP2
in the plasma membranes caused by hydrolysis via PLC alone is generally
limited in magnitude and duration (28) and is probably not sufficient
to cause a decrease in the activity of the channels with a high
affinity for PIP2, such as ROMK. In this study, we found
that hydrolysis of PIP2 following M1 receptor activation is
not sufficient to cause inhibition of the S219A ROMK mutant (which has
a lower affinity for PIP2 probably equivalent to the native
channels) and activation of PKC is necessary for inhibition of the
channel via stimulation of M1 receptors (Fig. 8). Activation of PKC
likely does so by amplifying the reduction of PIP2
initiated by hydrolysis by PLC. Thus, activation of PKC is likely the
mechanism by which PLC-activating hormones inhibit K+
channels in the apical membrane of CCD. Regulation of K+
channels by these hormones is important in certain physiological and
pathophysiological states. For example, inhibition of K+
channels may help to prevent excess kaliuresis during natriuresis (occurs as a result of inhibition of Na+ reabsorption at
the proximal sites). A recent review suggests that loss of this direct
inhibitory action in K+ secretion by prostaglandin
E2 may be helpful in mitigating hyperkalemia associated
with administration of nonsteroidal anti-inflammatory drugs (29).
One common mechanism for PKC to regulate protein function is by direct
phosphorylation of target proteins. Indeed, direct phosphorylation is
important for PKC regulation of some inward-rectifier K+
channels (32, 33). Our present study does not contradict with the
possibility that phosphorylation of ROMK by PKC may be important for
channel inhibition. Rather, it suggests that reduction of
PIP2 content by PKC is important for inhibition of ROMK1 in oocytes. Hill and Peralta (34) recently reported that activation of PKC
by PMA and M1 receptor inhibits GIRK channels expressed in oocytes.
Interestingly, the authors found that mutation of potential PKC
phosphorylation sites does not prevent the inhibition of GIRK channels
by PKC. Our work opens new possibilities for regulation of
PIP2-regulated ion channels and other proteins by PLC-activating hormones.
Nasuhoglu et al. (25) recently reported that PMA and
diacylglycerol decrease membrane PIP2 content in guinea pig
ventricles and in a mouse CCD cell line, but not in all cell types. The
mechanism by which activation of PKC alters membrane PIP2
content remain unknown. Potential mechanisms include regulation of PLC
(35, 36), regulation of phosphatidylinositol transfer protein (37), and
regulation of activity and location of lipid phosphatases and/or lipid
kinases (25, 38). The molecular identity of lipid kinases and
phosphatases regulating PIP2 metabolism in the cell surface
remains controversial. Identification of the lipid kinases and
phosphatases involved may help future studies to investigate the
mechanism by which PKC alters PIP2 content in the cell surface.