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J Biol Chem, Vol. 275, Issue 14, 10182-10189, April 7, 2000
From the Department of Medicine, University of Texas, Southwestern
Medical Center, Dallas, Texas 75235-8856
ROMK channels are responsible for
K+ secretion in kidney. The activity of ROMK is
regulated by intracellular pH (pHi) with acidification causing
channel closure (effective pKa ~6.9). Recently,
we and others reported that a direct interaction of the channels with
phosphatidyl-4,5-bisphosphate (PIP2) is critical for
opening of the inwardly rectifying K+ channels. Here, we
investigate the relationship between the mechanisms for regulation of
ROMK by PIP2 and by pHi. We find that disruption of
PIP2-ROMK1 interaction not only decreases single-channel open probability (Po) but gives rise to a ROMK1
subconductance state. This state has an increased sensitivity to
intracellular protons (effective pKa shifted to pH
~7.8), such that the subconductance channels are relatively quiescent
at physiological pHi. Open probability for the subconductance
channels can then be increased by intracellular alkalinization to
supra-physiological pH. This increase in Po for
the subconductance channels by alkalinization is not associated with an
increase in PIP2-channel interaction. Thus, direct
interaction with PIP2 is critical for ROMK1 to open at full
conductance. Disruption of this interaction increases pHi
sensitivity for the channels via emergence of the subconductance state.
The control of open probability of ROMK1 by pHi occurs via a
mechanism distinct from the regulation by PIP2.
Potassium channels play important roles in the regulation of
potassium transport in kidney (1). Recently, cDNAs for the renal
K+ channels and splice isoforms, ROMK1, -2, and -3, have
been isolated (2-4). ROMKs belong to a large family of inward
rectifier K+ channels, which also includes the strongly
rectifying IRK1, the G protein-gated GIRK1, and the pancreatic Opening of the G protein-gated GIRK channels requires G protein ROMK1 channels are also regulated by cAMP-dependent protein
kinase (PKA) (14, 15) via direct phosphorylation of the channels (16).
Activation of ROMK channels by PKA underlies the regulation of renal
potassium transport by arginine vasopressin (17, 18). Recently, we
found (19) that regulation of ROMK by PKA requires PIP2.
Without PIP2 in the membrane, PKA phosphorylation is not sufficient to activate the channels. PKA phosphorylation of ROMK, however, enhances channel interaction with PIP2. This study
further supports a critical role of direct PIP2 binding in
channel function. However, it is not known how direct lipid-channel
interaction contributes to channel activation. Recently it was reported
that subconductance ROMK channels result from mutation of PKA sites or
partial, Mg2+-induced run-down (20). Given that
PIP2 can reactivate run-down channels and that PKA
modulates PIP2-channel interaction, it is likely that
interaction between PIP2 and ROMK1 is important for the
channels to open at the full conductance.
Another important physiological regulator for ROMK is intracellular pH
(pHi). ROMK channels are unique among members of inward
rectifier K+ channels in having a very high sensitivity to
intracellular protons. Intracellular acidification reversibly reduces
open probability (Po) of native K+
channels in cortical collecting ducts as well as that of ROMK1 channels
expressed in oocytes, with an effective pKa value of
~6.9 (21-27). This reduction in K+ conductance by
acidification plays a key role in K+ homeostasis during
metabolic acidosis (1). The amino acid responsible for the steep pH
dependence for ROMK has been identified as lysine 80 (24, 25).
Substitution of lysine 80 by methionine abolishes the sensitivity of
ROMK1 to intracellular protons. It has been proposed that the ability
of lysine (with an effective pKa value of 10.5 as a
free amino acid) to function as a pH sensor in the physiological pH
range requires an acid shift in effective pKa value
induced by local chemical environment (24). Consistent with this idea,
mutation of threonine 51 in ROMK2 (equivalent to threonine 70 of ROMK1)
to either glutamate or lysine results in a shift in effective
pKa toward the alkaline or the acid direction,
respectively (25). Given the critical role for PIP2 in
maintaining a stable open-channel conformation, it is conceivable that
changes in PIP2-channel interaction may affect the local
channel structure around lysine 80 and thereby alter pH sensitivity.
In this study, we first tested the hypothesis that the disruption of
PIP2-channel interaction contributes to the molecular basis
for the subconductance state. Having found that PIP2 is critical for the stable open-channel conformation, we then examined whether and how PIP2-channel interaction affects the
sensitivity of the channels to pHi.
Molecular Biology--
Wild type ROMK1 cDNA was in the
pSPORT plasmid (2). Site-directed mutagenesis of ROMK1 was performed
using a commercial mutagenesis kit (Quickchange from Stratagene, La
Jolla, CA) and confirmed by nucleotide sequencing as described
previously (8, 19). mCAP cRNAs of the wild type and mutant ROMK1
channels were transcribed in vitro using T7 RNA polymerase
(8).
Giant Patch Clamp Recording--
Xenopus oocytes were
injected with ~5 ng of cRNA for the wild type or mutant ROMK1, and
giant patch clamp recording was performed as described previously (8,
19). The pipette (extracellular) solution contained (in mM)
100 KCl, 2 CaCl2, 5 Hepes (pH 7.4). Bath (cytoplasmic)
solution contained either 100 KCl, 5 Hepes (pH 7.4), 5 EGTA, and 1 MgCl2 (Mg2+ solution) or 100 KCl, 5 Hepes, 5 EDTA, 4 NaF, 3 Na3VO4, and 10 Na4P2O7 (FVPP solution, mixture of
fluoride, vanadate, and pyrophosphate) as indicated for each
experiment. Inward K+ currents (at Single Channel Patch Clamp Recording--
Patch clamp pipettes
(pulled from borosilicate glass, Warner Institute 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 M Disruption of PIP2-ROMK1 Interaction Decreases
Po for the Full Conductance Opening and Produces a
Subconductance State--
Regulation of the ROMK1 inward rectifier
K+ channels by PIP2 involves direct binding of
PIP2 to a region of the C terminus which includes amino
acid arginine 188 (Arg-188) (8). Depletion of membrane PIP2
via activation of the Mg2+-dependent lipid
phosphatases causes a progressive reduction in macroscopic ROMK
currents (run-down) over ~3-5 min in giant patch clamp recording
(8). Fig. 1A shows that wild
type ROMK1 channels recorded on-cell from oocytes have a near-maximal
single-channel open probability (Po = 0.91 ± 0.08, n = 5). The single-channel current-voltage
relationship for channels recorded on-cell is weakly inward-rectifying
with an inward slope conductance of 36 ± 2.3 pS
(n = 5, 0 to
After complete run-down in Mg2+ solution, application of
exogenous PIP2 over ~1-3 min to the cytoplasmic face of
the excised patch restored the open probability for the full
conductance state to the level of that recorded in the on-cell
configuration (Fig. 1C). As shown in Fig. 1C, the
subconductance state was frequently observed during this period of
PIP2-mediated recovery from run-down. Similar to run-down
in Mg2+ solution, application of anti-PIP2
antibody (at a submaximal concentration) produced the subconductance
state and decreased Po for the full conductance
opening (not shown). These results suggest that both the subconductance
state and the closed state are favored by disruption of
PIP2-channel interaction. Similar to the earlier report by
MacGregor et al. (20), we also observed a second
subconductance state with unitary conductance ~17% of the full
conductance channels during Mg2+-induced run-down. Because
of the low amplitude and the low opening frequency, the second
subconductance state was not analyzed in this study.
Mutation of arginine 188 of ROMK1 to glutamine (R188Q) reduces
channel's affinity for PIP2 (8). In contrast to the wild type ROMK1 channel which has a high Po for full
conductance state and only rarely shows subconductance state on-cell,
R188Q mutant exhibited frequent subconductance openings as well as a
low Po for the full conductance state in on-cell
recording (Fig. 2A, see below
for discussion of the flickery opening). These results suggest that,
due to the reduction in the affinity for PIP2, R188Q mutants cannot be fully activated (i.e. high
Po for full conductance opening) by the
endogenous PIP2 that prevails in the oocyte membrane on-cell. Consistent with this idea, raising PIP2
concentration to a supra-physiological level by application of
exogenous PIP2 to inside-out patches (in which the
endogenous PIP2 was not depleted by run-down) overcame the
effect of R188Q mutation (Fig. 2B). We have previously shown
that mutant channels with lysine substituted for arginine 188 (R188K)
have normal PIP2-channel interaction, suggesting that the
nature of this interaction is electrostatic (19). Here, we also found
that R188K, like wild type ROMK1, has a high Po
and exists nearly exclusively in the full conductance state (Fig.
2C). The mean open and closed times for R188K were not
different from those of wild type channels (
The apparent open-channel noise (flickery opening) for R188Q mutants
(Fig. 2A) is higher as compared with that for the run-down channels and remained unchanged even though the low channel activity was reversed by application of exogenous PIP2 (Fig.
2B). Open-channel noise for R188K, however, was not
qualitatively different from that for the wild type channels (Fig.
2C). Thus, charge neutralization by the R188Q mutation
probably alters channel gating and/or the interaction of K+
ion with the pore during permeation, besides disrupting
ROMK-PIP2 interaction. The apparent reduction in the mean
amplitude for the full conductance opening of R188Q (indicated by a
broken line in the expanded-time trace in Fig.
2A) is likely due to incomplete resolution of these flickery
opening events. Fig. 2D is an amplitude histogram for R188Q,
showing two distinct peaks corresponding to full- (labeled
O) and subconductance (labeled S) states. As above, the current amplitude for the full conductance state appears reduced as compared with wild type ROMK1. The current amplitude for the
R188Q substate, however, is similar to the amplitude for the wild type
ROMK1 substate after partial run-down. This finding, taken together
with the finding of elimination of this substate (but not of the
flickery opening) by application of exogenous PIP2,
suggests that the R188Q subconductance state is truly resulting from
disruption of PIP2-channel interaction rather than an
artifact of incomplete resolution of full opening events. Fig.
2E shows that mean Po (for full
conductance opening) for R188Q is significantly reduced compared with
that for wild type (WT), R188K, or R188Q after application of exogenous
PIP2. Thus, the electrostatic interaction between
PIP2 and the C-terminal region of ROMK1, which includes arginine 188, is critical for maintaining channel opening at high Po and full conductance.
Disruption of PIP2 Channel Interaction Increases the
Sensitivity of the Channels to Intracellular
Protons--
Intracellular acidification leads to channel closure. The
critical role of PIP2 in maintaining ROMK activity raises
the possibility that pHi regulation may also be modulated by
PIP2-channel interaction. In the following experiments
(Fig. 3), wild type or mutant channels
were recorded in on-cell, giant patches and then excised into a
Mg2+-free bath solution that also contained a mixture of
the phosphatase inhibitors, fluoride, vanadate, and pyrophosphate
(FVPP). This solution prevents run-down of ROMK current probably by
inhibiting both Mg2+-dependent protein
phosphatases as well as lipid phosphatases thus slowing channel
dephosphorylation and membrane PIP2 depletion (6, 8, 19,
28). As shown in Fig. 3A, activity of wild type channels
(measured as inward K+ currents at
Phosphorylation of ROMK by PKA is known to enhance
PIP2-channel interaction (19). Disruption of the PKA
phosphorylation sites serine 219 or serine 313 by point mutation to
alanine (S219A or S313A) decreases PIP2-channel interaction
compared with wild type, although to a lesser extent than does the
R188Q mutation (19). In an analogous fashion, pHi sensitivity
for S219A and S313A, like that for R188Q, is shifted in the alkaline direction although to a lesser extent (pKa, 7.3 ± 0.05, n = 3, and 7.28 ± 0.04, n = 3, for S219A and S313A, respectively). Similar
shift in pKa by mutation of PKA sites has also been
observed for ROMK2 (29). Thus, disruption of PIP2-channel interaction through PKA site mutation also leads to an increased pHi sensitivity.
Sensitivity to pHi is also increased after PIP2
depletion by exposure to cytoplasmic Mg2+. As shown in Fig.
4A, wild type channels in
inside-out giant membranes were allowed to run down in Mg2+
solution and were then exposed to FVPP solutions at different pH.
Activity of the run-down current was partially recovered by alkalinization to pH 9.4. The apparent incomplete recovery of macroscopic current with alkalinization is due to recovery of channels
to the subconductance state (see Fig.
5C, below). Subsequent acidification below pH 9.4 inhibited the channels. The pHi sensitivity of ROMK1 current recovered (by alkalinization) after Mg2+-induced PIP2 depletion was increased
compared with ROMK1 before run-down (pKa 7.73 ± 0.02, n = 5, after run-down, versus pKa 6.84 ± 0.04 before run-down, Fig.
4B). Fig. 4C shows the steady state I-V
relationships for currents (taken from the experiment shown in Fig.
4A) recorded on-cell (
The magnitude of the shift in pKa by R188Q mutation
is more than that by Mg2+ run-down (pKa
7.9 versus 7.73, respectively). We cannot exclude the
possibility that effects other than the reduction in
PIP2-channel interaction may also contribute to the shift
in pHi sensitivity induced by R188Q mutation. Our results, nevertheless, provide evidence that reduction of ROMK1-PIP2
interaction can render the channels more sensitive to intracellular
protons. Because of the shift in effective pKa to
~7.8, channels with disrupted PIP2-interaction are nearly
quiescent at the physiological pHi 7.4 but can be activated by
alkalinization. As shown in Fig. 7 below, this increase in sensitivity
is mediated by the titratable amino acid lysine 80.
Cytoplasmic Alkalinization Increases the Open Probability for the
ROMK1 Subconductance State After Disruption of PIP2 Channel
Interaction--
Both full and partial conductance states were present
at low frequency for R188Q recorded both on-cell (Fig. 2A)
and after excision into FVPP at pHi 7.4 (Fig. 5A, top
panel and Fig. 5B). Cytoplasmic alkalinization of R188Q
to pH 9.4 increased Po of predominantly the
subconductance state (Fig. 5A, bottom panel and Fig.
5B). Similarly, alkalinization of ROMK1 channels with
disrupted PIP2 interaction brought about by run-down also revealed the subconductance state (Fig. 5C). A membrane
patch containing a single wild type channel was first excised into FVPP solution at pH 9.4 (Fig. 5C, top panel), allowed to run down
in Mg2+ solution at pH 7.4 to the level of no detectable
channel activity (~5 min, not shown), and then re-exposed to FVPP at
pH 9.4 (bottom panel). Whereas the full conductance opening
was observed before run-down (top panel), alkalinization
after Mg2+-induced run-down led to opening of the
subconductance state (bottom panel). These results confirm
that the alkalinization-induced increase in activity for channels with
reduced PIP2 interaction is mostly from an increase in
Po for the subconductance state. They also
suggest that the increase in pH sensitivity resulting from reduced
PIP2-channel interaction is primarily due to emergence of
the subconductance state.
Fig. 5D shows single channel I-V relationships for the full
conductance state recorded at FVPP (pH 9.4) before run-down (labeled Full) and for the subconductance state reactivated by FVPP
(pH 9.4) after Mg2+ run-down (labeled Sub). The
weak inward rectification observed for both full and subconductance
channels is due to Na+ ions in FVPP solution (30) and
disappeared when inside-out membranes were bathed in Na+-
and Mg2+-free KCl solutions (not shown). Thus, whether
PIP2-ROMK1 interaction is disrupted by
Mg2+-induced run-down or by point mutation of Arg-188,
pH-dependent regulation is still intact. Nevertheless, a
critical level of PIP2 in the membrane and/or a critical
level of phosphorylation on the channels may be required for channel
activity, i.e. when channels were allowed to run down in
Mg2+ solution for >30 min, alkalinization failed to cause
activation (not shown).
Increase of ROMK Activity by Alkalinization Is Not through
Increased PIP2-ROMK Interaction--
Anti-PIP2
antibodies bind PIP2 in the membrane (8, 19, 31, 32) and
uncouple PIP2-channel interaction (8-10, 19). The
sensitivity of the channels to inhibition by anti-PIP2
antibody (assessed based on the time for antibody to cause 50%
inhibition of current, t1/2, see Fig.
6) correlates well with the affinity of
the channels for PIP2 (8, 19), i.e. R188Q
displays an increased sensitivity to inhibition by anti-PIP2 antibody at pH 7.4 as compared with the wild type
ROMK1, indicating a reduced interaction with PIP2 (8).
In the following experiments (Fig. 6A), R188Q channels in
giant patches were excised and stabilized in FVPP at pH 9.4 or 7.4 as
indicated. Anti-PIP2 antibodies were then applied in the
bath solution to inhibit the channels. Despite causing a marked
increase in activity (as in Fig. 3B), alkalinization to pH
9.4 did not alter the R188Q sensitivity to anti-PIP2
antibody (Fig. 6A, t1/2 22 ± 3 s for pH 9.4, n = 5, versus 14 ± 3 s for pH 7.4, n = 5). Thus, activation of R188Q
by alkalinization does not occur through enhanced PIP2
interaction. As the weak PIP2-channel interaction (shown
here as fast inhibition by anti-PIP2 antibody) correlates with the subconductance state, these results agree with single-channel recordings showing that alkalinization of R188Q is associated with an
increase in Po for predominantly the
subconductance state (Fig. 5B). Similarly, for wild type
ROMK1 channels activated by alkalinization to pH 9.4 after run-down in
Mg2+, inhibition by anti-PIP2 antibody was
rapid (Fig. 6B, t1/2, 26 ± 4 s, n = 3) compared with inhibition of ROMK1 at pH 9.4 before run-down (Fig. 6C, t1/2, 112 ± 17 s, n = 3). These results confirm that
alkalinization-induced increase in channel activity for the run-down
channels occurs despite reduced PIP2-ROMK1 interaction and
results from an increase in Po of the subconductance state.
Mutation of the Lysine 80 pH Sensor Abolishes the Alkaline Shift in
pH Sensitivity Caused by Disruption of PIP2 Channel
Interaction--
Intracellular protons inhibit wild type ROMK1
channels through titration of lysine 80 in the N-terminal cytoplasmic
domain (24). We next investigated whether lysine 80 is important for pH
sensing when PIP2-channel interaction is reduced. As shown previously (24), mutation of this "pH sensor" to methionine greatly
decreases the ability of protons to inhibit the wild type ROMK1
channels, where PIP2-ROMK1 interaction is intact (Fig.
7A, compare K80M,
Abolition of the alkaline shift in pH sensitivity by methionine
substitution of Lys-80 predicts that R188Q/K80M double mutants will
have higher activity than R188Q single mutants at the physiological pHi (~7.32, see Ref. 29). Indeed, we found that the single-channel activity recorded on-cell for R188Q/K80M mutants was
much higher than that for R188Q mutants (R188Q/K80M in the bottom panel of Fig. 7B versus
R188Q in Fig. 2A). The K80M mutation, by itself,
did not affect single-channel Po (Fig. 7B,
top panel) or kinetics ( Oxidation of Cytoplasmic Domains Prevents the
Alkalinization-induced Increase in Subconductance Activity--
When
ROMK1 channels are closed by intracellular acidification, movements in
the N and C terminus cause these domains to be accessible for
water-soluble oxidants and sulfhydryl reagents (27). To see whether
disruption of PIP2-channel interaction also renders the
channels susceptible to oxidation, wild type ROMK1 channels recorded in
giant inside-out membranes were allowed to run down in Mg2+
solution (pH 7.4) in the presence of the oxidant Cu-Phen. Oxidation by
Cu-Phen prevented the reactivation of the channels by alkalinization (Fig. 8A). Subsequent
application of the reducing agent DTT reversed the effect of oxidation
and allowed the channels to be partially reactivated by alkalinization
(mean currents before DTT were 19 ± 3% of the on-cell level
versus 56 ± 10% of the on-cell level after DTT,
n = 8, p < 0.02). Single-channel
recordings confirm these findings. As shown in Fig. 8B, the
subconductance Po after run-down in
Mg2+ solution and oxidation by Cu-Phen was increased by
alkalinization in the presence of DTT (bottom panel) but not
in the absence of DTT (top panel). The mean
Po for the alkalinization-induced subconductance state with or without DTT was 0.48 ± 0.18 (n = 5)
and 0.06 ± 0.02 (n = 5) (p < 0.05), respectively. Thus, similar to acidification-induced channel
closure, disruption of PIP2-channel interaction causes conformational changes in the cytoplasmic domains and renders them
susceptible to oxidation.
Without alkalinization, DTT alone did not increase activity of channels
run down in Mg2+ solution and oxidized by Cu-Phen (not
shown). Furthermore, Cu-Phen had no effect on channels recorded on-cell
or in excised inside-out membranes stabilized in FVPP solution (not
shown). In the absence of Cu-Phen, oxidation of the cytoplasmic domains
by environmental O2 did not occur over 10 min for channels
run down in Mg2+ solution and pH 7.4 (compare Fig.
5C with Fig. 8, A and B). This differs
from previous reports that channels closed by intracellular acidification in excised inside-out patches were readily oxidized (<5
min) by ambient O2 (25-27). Cysteine 49 and cysteine 308 are susceptible to modification by sulfhydryl reagents after channel closure by acidification; however, the cysteine residues involved in
the oxidation of ROMK remain unknown (27). We have attempted to
identify the residues involved in oxidation of the channels closed by
disruption of PIP2-channel interaction. Mutation of cysteine 49 and cysteine 208 singly or in combination, however, did not
prevent the effects of Cu-Phen on these channels (data not shown).
Opening of ROMK channels requires a direct interaction with
PIP2. We have previously shown that this interaction
involves the C terminus of ROMK which includes arginine 188 (8). We now
report that this interaction is critical for control of not only open
probability but also of single channel conductance. Wild type ROMK
channels display high open probability for full conductance opening at
the physiological membrane PIP2 concentration. Disruption
of PIP2-channel interaction favors transitions to a subconductance or closed state. This transition can be reversed by
application of exogenous PIP2 to the excised membrane
patches, further supporting that the ROMK subconductance state
represents an intermediate conducting conformation brought about by
disruption of PIP2-channel interaction. Although block by
protons or other ions can cause partial conducting states (33), the
following findings suggest that ion block is not giving rise to the
subconductance state observed in this study. First, the subconductance
state is observed even when both pipette and bath solutions contain only KCl, EDTA, and Hepes. Second, the unitary I-V relationships for
subconductance and full conductance states overlap when scaled to the
same amplitude, suggesting that voltage-dependent block by
some agent is not contributing. Third, the Po
for the subconductance state of run-down channels or R188Q is increased
by intracellular alkalinization, inconsistent with what one would
expect if proton block were occurring. Whether disruption of
PIP2-channel interaction gives rise to partial conductance
channels for other inward rectifier K+ channels, such as
IRK1 and GIRK1, remains to be examined.
Besides PIP2, intracellular pH also plays an important role
in regulation of ROMK. Although the regulation by PIP2 and
by pH both cause conformational changes in the cytoplasmic domains of
the channels and render them accessible for oxidation, these two
mechanisms are mostly separate (see Fig.
9). Binding of PIP2 to the
C-terminal region in the vicinity of arginine 188 stabilizes an
open-channel conformation that is featured by full conductance (Fig.
9A). This interaction also contributes to the regulation of
Po for the full conductance state by
PIP2. Intracellular pH, through titration of lysine 80, regulates opening of the channels through movements in both N and C
termini (27). Partial disruption of PIP2-channel
interaction causes the channels to enter a subconductance state and
reduces open probability for the full conductance state (Fig.
9B). Through changes in local channel structure around
lysine 80, the subconductance channels are more sensitive to
intracellular protons (effective pKa pH ~ 7.8) and thus only rarely open at the physiological pHi ~7.4.
Open probability for the subconductance state, however, can be
increased by intracellular alkalinization to supra-physiological pH
(via titration of lysine 80). This regulation of
Po by pHi is not due to a primary effect
on PIP2-channel interaction (see below). Complete depletion of PIP2 in the membrane causes the channels to enter the
closed state (Fig. 9C), which may be reactivated by
PIP2 but not by alkalinization. Determining the
stoichiometry of PIP2 binding to partial and full conductance states and the relationship between the two states in the
gating schemes for pHi and PIP2 control of the channels will require further experimentation.
Although pHi control of ROMK can be separated from the control
by PIP2, the movements in the C terminus in response to
changes in pHi could conceivably alter the structure of the
PIP2 binding region and affect its interaction with
PIP2. Indeed, it has been shown that intracellular
acidification leads to both emergence of subconductance channels and a
reduction in Po for the full conductance
channels (25, 26). The appearance of the subconductance state during
acidification, however, is variable and relatively infrequent as
compared with the magnitudes of reduction in Po
for the full conductance state and the reduction in the total current.
Thus, it is likely that alteration in PIP2-channel interaction is not the primary effect of pH on channels. Our results that alkalinization of channels with reduced PIP2
interaction increases Po primarily for the
subconductance state and fails to alter the sensitivity of the channels
to anti-PIP2 antibody also support this idea. Collectively,
these results also suggest that the increase in pHi sensitivity
resulting from disruption of PIP2-channel interaction is
due to emergence of the subconductance state.
Recently, several studies reported that PIP2 regulates ATP
sensitivity for KATP channels and that this control by
PIP2 may be important for physiological regulation of the
KATP channels by intracellular ATP (12, 13). Our present
findings that pH sensitivity is increased by disruption of
PIP2-channel interaction may also be of potential
physiological importance. Channels with normal PIP2
interaction have an effective pKa value of ~6.9
and are therefore near maximally active and less sensitive to small
changes in pH around the resting physiological pHi ~7.4
(especially in the alkaline direction). Reduction of PIP2 concentration in the membrane may shift effective
pKa for the channels toward pH 7.4 and render them
more sensitive to changes in pHi within a physiologically
important range. The importance of this shift in pHi
sensitivity in regulation of K+ transport in the cortical
collecting duct by the phospholipase C-coupled hormone bradykinin (14)
will require further experimentation.
We thank Drs. Gerhard Giebisch, Donald W. Hilgemann, and Gordon G. MacGregor for reading of the manuscript; Dr.
Bernd Fakler for discussion of the shift in pH sensitivity for R188Q
mutation in the early stages of the work; and Wei Ding and the late Em Phan for technical assistance.
*
This work was supported in part by National Institutes of
Health Grant RO1-DK-54368 (to C.-L. H.) and by a grant-in-aid from the
American Heart Association, National Center.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.
§
Supported in part by National Taiwan University and the Ministry of
Education of Taiwan.
¶
Supported in part by National Institutes of Health
Institutional Training Grant T32 DK-07257.
The abbreviations used are:
PIP2, phosphatidylinositol 4,5-bisphosphate;
pHi, intracellular pH;
DTT, dithiothreitol;
Cu-Phen, Dinitrato-1,10-phenanthroline copper(II);
PKA, cAMP-dependent protein kinase.
Phosphatidylinositol 4,5-Bisphosphate and Intracellular pH
Regulate the ROMK1 Potassium Channel via Separate but Interrelated
Mechanisms*
,,
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-cell
inward rectifier (5). These cDNAs encode polypeptides of
~300-500 amino acids, which share ~40% or more amino acid
identity and have the common structure of a cytoplasmic N terminus, two
hydrophobic segments that span the membrane as 
-helices, one
pore-forming partial membrane-spanning region, and a long cytoplasmic C terminus.
subunits. Other inward rectifier K+ channels, such as ROMK1
and IRK1, are constitutively open (5). Inward rectifier K+
channels run down (close) when inside-out membrane patches are excised
into ATP-free, Mg2+-containing solution. We and others
(6-9) recently found that direct interaction of inward rectifier
channels with the membrane lipid phosphatidylinositol 4,5-bisphosphate
(PIP2)1 is
critical for channel opening. Reduction of membrane PIP2
via activation of the Mg2+-dependent lipid
phosphatases causes channel run-down. Direct application of
PIP2-containing liposomes to membrane patches reactivates run-down channels (6-9). Furthermore, PIP2 is important
for regulation of the G protein-gated channels by G and by
intracellular Na+ (8-11) and modulates ATP sensitivity of
KATP channels (12, 13).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
30 mV holding
potential, at 23-25 °C) were recorded onto a chart recorder using
an Axopatch 200B amplifier, pCLAMP software, and Digidata 1200A
digitizer (Axon Instrument, Foster City, CA). Chart recording strips
were digitally scanned and analyzed in a computer for presentation.
Each vial of anti-PIP2 monoclonal antibody stock
(Perspective Biosystems, Framingham, MA) was reconstituted in 0.5 ml of
distilled water and diluted 40-fold into experimental solutions to
yield a final concentration of 40 nM. PIP2
(Roche Molecular Biochemicals) was diluted in water (1 mg/ml) and
sonicated to form liposomes (8). Dinitrato-1,10-phenanthroline
copper(II) (cupric phenanthroline) was from Aldrich. Dithiothreitol
(DTT) was from Sigma.
. In some experiments, the pipette solution contained 100 KCl, 5 EDTA, and 5 Hepes (pH 7.4 with KOH). The Mg2+-free, Mg2+-containing, and
FVPP bath solutions were the same as for the giant patch recording.
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 computer hard disc
(20 gigabytes) using pCLAMP7 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 pCLAMP7 pSTAT (version 6.0.5, Axon
Instruments, Foster City, CA). Open probability was analyzed on
segments of continuous recording (at least 5 min) from patches that
contained only one active channel during the lifetime (>20 min) of the
recording. Po for full and subconductance
opening in the same patch was determined using the criteria of
threshold crossing of the current levels assigned for closed, sub-, and
full states (i.e. base line for closed, current level 1 and
current level 2 for sub- and full states, respectively). The current
level for each state was assigned based on visual inspection of a long
segment of recording for best fit and comparison with the current peaks
in the amplitude histogram. For amplitude histogram analysis, only
periods of recordings containing bursts of channel activity were used.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
100 mV; not shown, but for similar I-V for channel in inside-out patch see Fig. 5D). Fig.
1B shows a representative recording (from the same channel
in Fig. 1A) 5 min after excision of the on-cell membrane
patch into Mg2+ solution to allow run-down. As described
previously by others (20), we found that Mg2+-induced
run-down caused channels to enter a subconductance state of 16 ± 1.1 pS (approximately 44% of the full, open state). In addition,
Mg2+-induced run-down decreased Po
for the full conductance opening. After partial run-down, the
subconductance state remained even when recordings were performed using
Mg2+-free solution (in mM, 100 KCl, 5 EDTA, and
5 Hepes) in both the pipette and the bath (data not shown), indicating
that Mg2+ or other non-permeant ions are not directly
responsible for maintaining the subconductance state. Fig.
1D shows an amplitude histogram for a single channel
recorded during partial Mg2+ run-down, demonstrating full
(labeled O for full opening) and subconductance (labeled
S for subconductance) channels with ~36 and ~16 pS
unitary conductance, respectively. The subconductance state is only
rarely observed in on-cell recordings of wild type channels
(e.g. <10 ms total open time in a 3-min recording, not shown).
View larger version (57K):
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Fig. 1.
Mg2+-induced run-down produces
subconductance ROMK1 channels. A, on-cell
single-channel recording of a single ROMK1 channel at 100 mV.
B, recording the same channel in A after excision
of inside-out membrane and run-down in Mg2+ solution for 5 min. C, after run-down to no channel activity,
PIP2 liposomes (50 µM, in
Mg2+-free solution) were perfused onto the cytoplasmic face
of the excised membrane patch shown in B (representative
tracings, n = 4 separate experiments). Lower
traces in A-C show recordings with expanded time base.
Dotted lines show closed (labeled C),
subconductance (labeled S), and open (labeled O)
channel levels. D, representative amplitude histogram
constructed from 3 min of continuous single channel in Mg2+
solution as in B. Bin width = 0.1 pA. Dashed
line shows fit to sum of 3 Gaussian distributions.
o = 19.3 ± 1.9 and c = 1.32 ± 0.11 ms for R188K,
n = 12, versus o = 20.8 ± 1.9 ms and c = 1.40 ± 0.16 ms for wild type,
n = 8).
View larger version (48K):
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Fig. 2.
Electrostatic interaction between arginine
188 of ROMK1 and PIP2 is important for full conductance
opening. A, on-cell recording of a single R188Q mutant
channel, 100 mV. This mutation reduces ROMK1 affinity for
PIP2 and thus increases the probability of entering closed
or subconductance state for the channel. The broken line in
the expanded time trace indicates the level of the current for the full
conductance opening (~2.9 pA) that matches the mean amplitude of the
full conductance opening shown by the histogram in D. See
text for discussion of the apparent reduction in the current amplitude
for the full conductance state. B, PIP2 (50 µM, in Mg2+-free solution) was applied to the
cytoplasmic face of the R188Q channel (same channel in A
after excision from on-cell membrane without run-down in
Mg2+ solution). The effect of PIP2 shown here
supports that the recording in A contains only one channel
fluctuating between closed, subconductance, and full conductance
states. C, on-cell recording from oocytes expressing a
single arginine 188 to lysine (R188K) mutant channel. D,
amplitude histogram of a single R188Q channel recorded on-cell.
Multiple periods of recording containing bursts of channel activity
were used. Bin width = 0.1 pA. Dotted line indicates
fit by the sum of three Gaussian distributions. O, S, and
C indicate full conductance, subconductance, and closing
levels, respectively. The area indicated by * may represent
incompletely resolved closing events or the second substate of smaller
conductance (20). E, mean open probability of full
conductance state for wild type ROMK1 (on-cell), R188Q
(on-cell), R188Q+PIP2 (PIP2 applied
to the inside-out membranes without run-down), and R188K
(on-cell). Mean ± S.E. shown, n = 3-5 for each
column.
30 mV holding
potential) was near maximal on-cell (where the resting pHi is
~7.32, see Ref. 29) and was not significantly increased when the
cytoplasmic face of the excised inside-out membrane patches was
alkalinized to pH 9.4. Subsequent acidification inhibited current in a
steep, pH-dependent manner. The effective
pKa for inhibition of ROMK1 by intracellular protons
was 6.84 ± 0.04 (mean ± S.E., n = 5; Fig.
3C). The PIP2-binding site mutant, R188Q, has
low activity on-cell (Fig. 2, A and E) and in the
excised inside-out membranes at pH 7.4 (Fig. 3B). Activity of R188Q was, however, markedly increased when the excised membrane patch was alkalinized to pH 9.4 (Fig. 3B). Subsequent
acidification also caused a steep pH-dependent inhibition
of the R188Q mutant channels. The effective pKa for
inhibition of R188Q mutant by intracellular protons, however, was
shifted to 7.9 ± 0.03 (n = 6; Fig. 3,
B and C). As mentioned above, the
PIP2 affinity for R188K is equivalent to that for wild type
channels, and we have found that R188K displayed pH sensitivity
identical to that of wild type channels (effective
pKa for R188K: 6.94 ± 0.04, n = 4; Fig. 3C). These results show that
PIP2-channel interaction regulates pHi sensitivity
of ROMK such that when PIP2 binding is reduced channels
become more sensitive to cytoplasmic protons. The alkaline shift in
effective pKa revealed by R188Q is opposite in
direction to that expected if Arg-188 itself was acting as a
pHi sensor (see Fig. 7A, below).
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Fig. 3.
Mutation of PIP2 binding residue
or PKA consensus site shifts pH sensitivity of ROMK1 toward alkaline
pH. A, pH-dependent inhibition of wild type
ROMK1 current recorded in giant patches. ROMK1 current was first
recorded on-cell and then excised into FVPP solution titrated to the
pHi value indicated. The resting pHi on-cell is ~7.32
(29). ROMK1 current was inhibited by exposure to progressively more
acidic pHi. After inhibition, membrane patches were alkalinized
to pH 9.4 to assess the level of remaining current. Experiments with
less than 85% of the total inhibitable currents at the end of an
experiment were not used for analysis. The small, transient rise in
current visible at the time of solution change is a perfusion artifact
(probably as a result of activation of the stretch channels). Others
have reported that ROMK channels are oxidized after 1 to 2 min of
intracellular acidification to pH ~6.5 and are unable to be
reactivated by alkalinization (25-27). We found that ROMK1 current can
recover from inhibition by low pHi even after periods as long
as 15 min, suggesting a much slower rate of oxidation under our
experimental conditions (see also Fig. 8). B,
pH-dependent inhibition of R188Q. Experimental paradigm as
in A. C, dependence of the wild type and mutant
ROMK1 channels on pHi. Relative currents (normalized to the
maximal currents at pHi 9.4, I/Imax) at different pHi
values were fitted with the Hill equation (26) using the Sigma-Plot
program. Experimental points for R188K, S219A, and S313A were obtained
from experiments similar to those in A and B and
represent mean ± S.E. n = 3-6 separate
experiments for each channel.
), after run-down in
Mg2+ solution (
), partially recovered by pH 9.4 (
),
and after subsequent inhibition by pH 7.4 (
).
View larger version (14K):
[in a new window]
Fig. 4.
Reduction of membrane PIP2 by
Mg2+-induced run-down shifts pH sensitivity.
A, pH-dependent inhibition of the wild type
ROMK1 current after run-down in Mg2+ solution. Experimental
paradigm as in Fig. 3A. B, dependence of the
ROMK1 current on intracellular pH after run-down (labeled as Mg
run-down). Currents were normalized as described for Fig.
3C. The curve labeled No run-down is identical to
that labeled WT in Fig. 3C. Points represent
mean ± S.E., n = 5. C, representative
steady state I-V relationships for currents recorded in
A.
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Fig. 5.
Intracellular alkalinization increases
opening for the subconductance channels. A, single
R188Q channel recorded in an inside-out patch (without run-down) with
FVPP solution at pH 7.4 (top panel) or FVPP at pH 9.4 (bottom panel). B, mean Po
for the full conductance state (open column) and the
subconductance state (shaded column) for single R188Q mutant
channel at pH 7.4 or pH 9.4. Po for the sub- and
full conductance states was analyzed using pSTAT program as described
under "Materials and Methods" and under "Results" for Fig. 2.
As shown in Fig. 7C, Po determined as
such correlates very well with the relative distribution of the two
states revealed in the amplitude histogram. C, a single wild
type ROMK1 recorded in an inside-out patch with FVPP pH 9.4 before
run-down (top panel) or reactivated by FVPP pH 9.4 after
run-down in Mg2+ solution (bottom panel).
D, single channel I-V curves for full and subconductance
states determined from records like those in C.
View larger version (11K):
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Fig. 6.
The effect of alkalinization on the
sensitivity of the channels to anti-PIP2 antibody.
A, anti-PIP2 antibody sensitivity for R188Q
mutants excised and stabilized in FVPP with pH 9.4 (continuous
line) or pH 7.4 (broken line). Current traces from
separate experiments in either pH 9.4 or pH 7.4 were normalized to the
maximal currents before application of antibody
(I/Imax) and superimposed for
presentation. The maximal current before antibody at pH 7.4 is
typically only ~5-10% of the maximal current at pH 9.4 (see Fig.
3B). B and C, anti-PIP2
antibody sensitivity for wild type ROMK1 excised and stabilized in FVPP
pH 9.4 (C) or reactivated by FVPP pH 9.4 after run-down in
Mg2+ solution (B). See text for
t1/2 values.
, with WT, ). Fig. 7A shows that mutation
of this residue also abolished the alkaline shift in pH sensitivity
induced by disruption of ROMK1-PIP2 interaction via the
R188Q mutation (compare R188Q/K80M, , with
R188Q, ). Thus, pH-dependent regulation of
ROMK under conditions of reduced PIP2 interaction is still mediated through the titration of lysine 80. Disruption of
PIP2-channel interaction likely causes alteration of the
local chemical environment around lysine 80 which results in an
alkaline shift of its effective pKa.
View larger version (20K):
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Fig. 7.
Lysine 80 is the pH sensor for channels with
reduced PIP2 interaction. A, mutation of
lysine 80 to methionine (K80M) abolishes the shift in pH sensitivity
induced by R188Q mutation. Experimental paradigm as in Fig.
3A. Points in A are mean ± S.E., with
n = 3-6 separate experiments for each channel.
B, on-cell single-channel recording of K80M (top)
and R188Q/K80M mutants (bottom). Membrane patch contains
only one channel. Vm = 100 mV. C, mean
Po for the full (open column, labeled
F) and subconductance (shaded column, labeled
S) states for R188Q (recorded as in Fig. 2A) and
the R188Q/K80M mutants (recorded as in B). n = 3-5, error bars = S.E. Representative amplitude
histograms for each mutant are shown above the corresponding
bar graphs. Dotted lines indicate fits to the sum of three
Gaussian distributions. F, S, and C
indicate amplitude peaks corresponding to full conductance,
subconductance, and closed states, respectively.
o = 19.8 ± 1.7 ms and
c = 1.35 ± 0.13 ms, n = 8). Similar to
the alkalinization-induced increase in the activity for R188Q (see Fig.
5, A and B) and run-down ROMK1 channels (see Fig.
5C), the increase in channel activity for R188Q by K80M
mutation is mostly due to an increase in Po of
the subconductance state (Fig. 7C).
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Fig. 8.
Oxidation prevents reactivation of run-down
ROMK1 current by alkalinization. A, oxidation by
Cu-Phen (100 µM) applied to the cytoplasmic face of giant
inside-out patches during run-down prevented reactivation of ROMK1
macroscopic current at pHi 9.4. Subsequent application of 2 mM DTT, however, allowed for recovery to take place.
B, single ROMK1 channel was first recorded on-cell
(top left and bottom left) and then excised into
Mg2+ solution at pHi 7.4 in the presence of Cu-Phen
to allow run-down (top right and bottom right).
Application of FVPP (pH 9.4) revealed only minimal channel opening
(top right). Significant recovery to the subconductance
state, however, was seen if recovery from oxidation was measured with
FVPP (pH 9.4) in the presence of 2 mM DTT (bottom
right). Vm = 100 mV.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (18K):
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Fig. 9.
Working model for dual regulation of ROMK by
pH and PIP2. Like all inward rectifier K+
channels, ROMK contains a short N-terminal cytoplasmic domain, two
transmembrane domains (M1 and M2), a pore-forming region and a long
C-terminal cytoplasmic domain. Based on the crystal structure of the
KcsA channels (34), M1 and M2 should be positioned as outer and inner
helices instead of illustrated as flanking the pore region in this
simplified drawing. K80 indicates the pH sensor lysine-80 in the N
terminus. R188 indicates the PIP2 binding residue
arginine-188 in the C terminus of ROMK1. The two arrows
indicate separate regulation by "PIP2" and "pH"
respectively. "C", "O", and "S" indicate closed,
full-conductance and the subconductance states, respectively. Oxidation
of the cytoplasmic domains of channels with reduced
PIP2-channel interaction prevents movements of these
domains in response to pH (not shown in the model).
ACKNOWLEDGEMENTS
FOOTNOTES
Both authors contributed equally to this work.
To whom correspondence and reprint requests should be
addressed: Dept. of Medicine, H5-112, MC-8856, UTSW Medical Center, Dallas, TX 75235-8856. Tel.: 214-648-8627; Fax: 214-648-2071; E-mail:chuan1@mednet.swmed.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Wang, W.,
Sackin, H.,
and Giebisch, G.
(1992)
Annu. Rev. Physiol.
54,
81-96[CrossRef][Medline]
[Order article via Infotrieve]
2.
Ho, K.,
Nichols, C. G.,
Lederer, W. J.,
Lytton, J.,
Vassilev, P. M.,
Kanazirska, M. V.,
and Hebert, S. C.
(1993)
Nature
362,
31-38[CrossRef][Medline]
[Order article via Infotrieve]
3.
Zhou, H.,
Tate, S. S.,
and Palmer, L. G.
(1994)
Am. J. Physiol.
266,
C809-C824 4.
Boim, M. A.,
Ho, K.,
Shuck, M. E.,
Bienkowski, M. J.,
Block, J. H.,
Slightom, J. L.,
Yang, Y.,
Brenner, B. M.,
and Hebert, S. C.
(1995)
Am. J. Physiol.
268,
F1132-F1140 5.
Nichols, C. G.,
and Lopatin, A. N.
(1997)
Annu. Rev. Physiol.
59,
171-191[CrossRef][Medline]
[Order article via Infotrieve]
6.
Hilgemann, D. W.,
and Ball, R.
(1996)
Science
273,
956-959[Abstract]
7.
Fan, Z.,
and Makielski, J. C.
(1997)
J. Biol. Chem.
272,
5388-5395 8.
Huang, C. L.,
Feng, S.,
and Hilgemann, D. W.
(1998)
Nature
391,
803-806[CrossRef][Medline]
[Order article via Infotrieve]
9.
Sui, J. L.,
Petit-Jacques, J.,
and Logothetis, D. E.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
1307-1312 10.
Zhang, H.,
He, C.,
Yan, X.,
Mirshahi, T.,
and Logothetis, D. E.
(1999)
Nat. Cell Biol.
1,
183-188[CrossRef][Medline]
[Order article via Infotrieve]
11.
Ho, I. H.,
and Murrell-Lagnado, R. D.
(1999)
J. Physiol. (Lond.)
520,
645-651 12.
Baukrowitz, T.,
Schulte, U.,
Oliver, D.,
Herlitze, S.,
Krauter, T.,
Tucker, S. J.,
Ruppersberg, J. P.,
and Fakler, B.
(1998)
Science
282,
1141-1144 13.
Shyng, S.-L.,
and Nichols, C. G.
(1998)
Science
282,
1138-1142 14.
Wang, W.,
and Giebisch, G.
(1991)
Proc. Natl. Acad. Sci. U. S. A.
88,
9722-9725 15.
McNicholas, C. M.,
Wang, W.,
Ho, K.,
Hebert, S. C.,
and Giebisch, G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
8077-8081 16.
Xu, Z.-C.,
Yang, Y.,
and Hebert, S. C.
(1996)
J. Biol. Chem.
271,
9313-9319 17.
Cassola, A. C.,
Giebisch, G.,
and Wang, W.
(1993)
Am. J. Physiol.
264,
F502-F509 18.
Wang, W.
(1994)
Am. J. Physiol.
267,
F599-F605 19.
Liou, H.-H.,
Zhou, S.-S.,
and Huang, C.-L.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
5820-5825 20.
MacGregor, G. G.,
Xu, J. Z.,
McNicholas, C. M.,
Giebisch, G.,
and Hebert, S. C.
(1998)
Am. J. Physiol.
275,
F415-F422 21.
Wang, W.,
Schwab, A.,
and Giebisch, G.
(1990)
Am. J. Physiol.
259,
F494-F502 22.
Schlatter, E.,
Haxelmans, S.,
Hirsch, J.,
and Leipziger, J.
(1994)
Pfluegers Arch.
428,
631-640[CrossRef][Medline]
[Order article via Infotrieve]
23.
Tsai, T. D.,
Shuck, M. E.,
Thompson, D. P.,
Bienkowski, M. J.,
and Lee, K. S.
(1995)
Am. J. Physiol.
268,
C1173-C1178 24.
Fakler, B.,
Schultz, J. H.,
Yang, J.,
Schulte, U.,
Brändle, U.,
Zenner, H. P.,
Jan, L. Y.,
and Ruppersberg, J. P.
(1996)
EMBO J.
15,
4093-4099[Medline]
[Order article via Infotrieve]
25.
Choe, H.,
Zhou, H.,
Palmer, L. G.,
and Sackin, H.
(1997)
Am. J. Physiol.
273,
F516-F529
26.
McNicholas, C. M.,
MacGregor, G. G.,
Islas, L. D.,
Yang, Y.,
Hebert, S. C.,
and Giebisch, G.
(1998)
Am. J. Physiol.
275,
F972-F981
27.
Schulte, U.,
Hahn, H.,
Wiesinger, H.,
Ruppersberg, J. P.,
and Fakler, B.
(1998)
J. Biol. Chem.
273,
34575-34579 28.
Friedman, Z. Y.
(1993)
J. Pharmacol. Exp. Ther.
267,
617-623 29.
Leipziger, J., Cooper, G. J., Xu, J., Hebert, S. C., and
Giebisch, G. (2000) Am. J. Physiol., in press
30.
Matsuda, H.
(1993)
J. Physiol. (Lond.)
460,
311-326 31.
Fukami, K.,
Matsuoka, K.,
Nakanishi, O.,
Yamakawa, A.,
Kawai, S.,
and Takenawa, T.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
9057-9061 32.
Gilmore, A. P.,
and Burridge, K.
(1996)
Nature
381,
531-535[CrossRef][Medline]
[Order article via Infotrieve]
33.
Hille, B.
(ed)
(1992)
Ionic Channels in Excitable Membranes
, 2nd Ed.
, pp. 390-422, Sinauer Associates, Inc., Sunderland, MA
34.
Doyle, D. A.,
Cabral, J. M.,
Pfuetzner, R. A.,
Kuo, A.,
Gulbis, J. M.,
Cohen, S. L.,
Chait, B. T.,
and MacKinnon, R.
(1998)
Science
280,
69-77
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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B.-I. Yeh, T.-J. Sun, J. Z. Lee, H.-H. Chen, and C.-L. Huang Mechanism and Molecular Determinant for Regulation of Rabbit Transient Receptor Potential Type 5 (TRPV5) Channel by Extracellular pH J. Biol. Chem., December 19, 2003; 278(51): 51044 - 51052. [Abstract] [Full Text] [PDF]
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 C. P. Ford, P. L. Stemkowski, P. E. Light, and P. A. Smith Experiments to Test the Role of Phosphatidylinositol 4,5-Bisphosphate in Neurotransmitter-Induced M-Channel Closure in Bullfrog Sympathetic Neurons J. Neurosci., June 15, 2003; 23(12): 4931 - 4941. [Abstract] [Full Text] [PDF]
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W.-Z. Zeng, X.-J. Li, D. W. Hilgemann, and C.-L. Huang Protein Kinase C Inhibits ROMK1 Channel Activity via a Phosphatidylinositol 4,5-Bisphosphate-dependent Mechanism J. Biol. Chem., May 2, 2003; 278(19): 16852 - 16856. [Abstract] [Full Text] [PDF]
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D. Schulze, T. Krauter, H. Fritzenschaft, M. Soom, and T. Baukrowitz Phosphatidylinositol 4,5-Bisphosphate (PIP2) Modulation of ATP and pH Sensitivity in Kir Channels. A TALE OF AN ACTIVE AND A SILENT PIP2 SITE IN THE N TERMINUS J. Biol. Chem., March 14, 2003; 278(12): 10500 - 10505. [Abstract] [Full Text] [PDF]
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J. Mao, J. Wu, F. Chen, X. Wang, and C. Jiang Inhibition of G-protein-coupled Inward Rectifying K+ Channels by Intracellular Acidosis J. Biol. Chem., February 21, 2003; 278(9): 7091 - 7098. [Abstract] [Full Text] [PDF]
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M. Lu, S. C. Hebert, and G. Giebisch Hydrolyzable ATP and PIP2 Modulate the Small-conductance K+ Channel in Apical Membranes of Rat Cortical-Collecting Duct (CCD) J. Gen. Physiol., October 29, 2002; 120(5): 603 - 615. [Abstract] [Full Text] [PDF]
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 W.-Z. Zeng, V. Babich, B. Ortega, R. Quigley, S. J. White, P. A. Welling, and C.-L. Huang Evidence for endocytosis of ROMK potassium channel via clathrin-coated vesicles Am J Physiol Renal Physiol, October 1, 2002; 283(4): F630 - F639. [Abstract] [Full Text] [PDF]
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L. Chen, T. Kawano, S. Bajic, Y. Kaziro, H. Itoh, J. J. Art, Y. Nakajima, and S. Nakajima A glutamate residue at the C terminus regulates activity of inward rectifier K+ channels: Implication for Andersen's syndrome PNAS, June 11, 2002; 99(12): 8430 - 8435. [Abstract] [Full Text] [PDF]
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A.-A. Konstas, M. Dabrowski, C. Korbmacher, and S. J. Tucker Intrinsic Sensitivity of Kir1.1 (ROMK) to Glibenclamide in the Absence of SUR2B. IMPLICATIONS FOR THE IDENTITY OF THE RENAL ATP-REGULATED SECRETORY K+ CHANNEL J. Biol. Chem., June 7, 2002; 277(24): 21346 - 21351. [Abstract] [Full Text] [PDF]
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T. Meyer, M.-C. Wellner-Kienitz, A. Biewald, K. Bender, A. Eickel, and L. Pott Depletion of Phosphatidylinositol 4,5-Bisphosphate by Activation of Phospholipase C-coupled Receptors Causes Slow Inhibition but Not Desensitization of G Protein-gated Inward Rectifier K+ Current in Atrial Myocytes J. Biol. Chem., February 16, 2001; 276(8): 5650 - 5658. [Abstract] [Full Text] [PDF]
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W.-Z. Zeng, H.-H. Liou, U. M. Krishna, J. R. Falck, and C.-L. Huang Structural determinants and specificities for ROMK1-phosphoinositide interaction Am J Physiol Renal Physiol, May 1, 2002; 282(5): F826 - F834. [Abstract] [Full Text] [PDF]
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