From the Institute of Physiology II, Friedrich Schiller University Jena, Teichgraben 8, 07740 Jena, Germany
Received for publication, August 17, 2002, and in revised form, December 2, 2002
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ABSTRACT |
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Phosphatidylinositol polyphosphates (PIPs) are
potent modulators of Kir channels. Previous studies have implicated
basic residues in the C terminus of Kir6.2 channels as interaction
sites for the PIPs. Here we examined the role of the N terminus and
identified an arginine (Arg-54) as a major determinant for
PIP2 modulation of ATP sensitivity in
KATP channels. Mutation of Arg-54 to the neutral
glutamine (R54Q) and, in particular, to the negatively charged
glutamate (R54E) impaired PIP2 modulation of ATP
inhibition, while mutation to lysine (R54K) had no effect. These data
suggest that electrostatic interactions between PIP2 and
Arg-54 are an essential step for the modulation of ATP sensitivity.
This N-terminal PIP2 site is highly conserved in Kir
channels with the exception of the pH-gated channels Kir1.1, Kir4.1,
and Kir5.1 that contain a neutral residue at the corresponding
positions. Introduction of an arginine at this position in Kir1.1
channels rendered the N-terminal PIP2 site functional
largely increasing the PIP2 affinity. Moreover, Kir1.1
channels lose the ability to respond to physiological changes of the
intracellular pH. These results explain the need of a silent
N-terminal PIP2 site in pH-gated channels and highlight the
N terminus as an important region for PIP2 modulation of
Kir channel gating.
Kir channels are a superfamily of eukaryotic channel proteins that
are expressed in many tissues and responsible for important physiological processes such as cell excitability, insulin secretion, K+ homeostasis, vascular tone, and regulation of the heart
rate. Four subunits assemble to a channel. Each subunit contains two transmembrane segments with cytoplasmic N- and C-terminal domains and a
connecting loop forming the pore (1). Some members of the Kir channel
family are endowed with gating mechanisms such as ATP gating
(KATP channels) (2) and pH gating (Kir1.1 and Kir4.1
channels) (3). These gating mechanisms are central for the diverse
functions of Kir channels in physiology and the understanding of the
related pathophysiology. Kir1, Kir4, and Kir5 channels, that are
predominantly expressed in epithelia, are exquisitely sensitive to
changes in intracellular pH in the physiological range (3-5). This pH
sensitivity is mediated by the protonation of a lysine in the N
terminus (Lys-80 in Kir1.1) that induces closure of the channel's pore
by an allosteric mechanism (pH gating) (3, 6). Even small changes in
the pH sensitivity can cause severe kidney defects such as the Bartter
syndrome (3), highlighting the physiological importance of proper pH
gating in Kir1.1 channels. Kir6 channels display a very ubiquitous
expression pattern and, in coassembly with the sulfonylurea receptor
(SUR),1 represent the
ATP-sensitive K+ channels (KATP channels) (7).
Intracellular ATP closes KATP channels by binding to the
Kir6.2 subunits (ATP gating), whereas the SURs act as regulatory
subunits endowing the channel with sensitivity to MgADP and
pharmacological compounds. The ATP/ADP dependence of KATP
channels couples cell metabolism to membrane excitability, which plays
an important role in the physiology of many tissues (e.g.
pancreas, heart, brain) (1, 2, 8). Highly negatively charged membrane
phospholipids, in particular the phosphatidylinositol polyphosphates
(PIPs), such as phosphatidylinositol 4,5-bisphosphate
(PIP2), were found to interact with Kir channels, and in
general they stabilize the open state of the channel (9-14). In
addition, PIPs were shown to interfere with the different gating mechanisms of Kir channels. A recent report indicated modulation of pH
sensitivity of Kir1.1 channels by PIP2 since a mutation in
the C terminus (R188Q) that reduced PIP2 binding also
changed the pH sensitivity (15). Further, PIPs are effective modulators of KATP channels because they reduce the sensitivity to
inhibition by intracellular ATP (12, 13). The effect on ATP sensitivity is of particular physiological importance since the amount of ATP
inhibition determines the activity of KATP channels in
cells. Moreover, regulation of PIPs levels via signal transduction
pathways represents an effective means to regulate KATP
channels by various receptors (12, 13, 16-18). PIP2 was
shown to interact with basic residues in the C terminus of Kir6.2. For
Kir6.2/SUR channels the basic residues cluster in two regions of the C
terminus (176-222 and 303-314) (11-13, 19). Further, two regions in
the cytoplasmic C terminus (20, 21) and one region in the N terminus
(21) have been identified where mutations markedly reduce ATP
sensitivity. Intriguingly, the two C-terminal regions (near 182-185
and 333-338) are in proximity (at least in primary sequence) to the
regions that are implicated in PIP2 binding (176-222 and
303-314); however, the mechanistic basis of this coincidence is not
clear. The region near Arg-50 in the N terminus has been implicated in
ATP inhibition (21), and we mutated, therefore, basic residues in this
region (Lys-47-Lys-67) to screen for PIP2 binding sites.
This approach identified Arg-54 as an important determinant for
PIP2 binding in KATP channels that appeared to
be vital for mediating the antagonizing effect of PIP2 on
ATP inhibition. Further, we demonstrated that the N-terminal
PIP2 site is silent in pH-gated Kir channels
(e.g. Kir1.1) because they lack a positively charged residue
at the relevant position. Introduction of a positively charged residue (e.g. arginine) largely increased PIP2 binding
and, more importantly, impaired pH gating explaining the need of a
silent N-terminal PIP2 site for pH-gated Kir channels.
Mutagenesis, cRNA Synthesis, and Oocytes Injection--
Murine
Kir6.2, rat SUR2A, and Kir.1.1 (ROMK1) were used in this study.
Site-directed mutagenesis was performed as described (22) and verified
by sequencing. For oocyte expression, constructs were subcloned into
the pBF expression vector (23). Capped cRNAs were synthesized in
vitro using SP6 polymerase (Promega, Heidelberg, Germany) and
stored in stock solutions at Electrophysiology--
Giant patch recordings (22) in inside-out
configuration under voltage-clamp conditions were made at room
temperature (~23 °C) 3-7 days after cRNA injection. Neomycin and
ATP were purchased from Sigma.
L-a-Phosphatidyl-D-myo-inositol-4,5-phosphate
(PIP2 from bovine brain) was purchased from Roche Molecular
Biochemicals, stored as stocks (1 mM) at Arg-54 in the N Terminus of Kir6.2 Is a Major Determinant for
PIP2 Binding--
The region near Arg-50 (Lys-47-Lys-67)
was screened for residues that might contribute electrostatically to
the binding of PIP2 or ATP (Fig.
1A). Basic residues in this
region (Lys-47, Arg-50, Arg-54, Lys-67) were mutated to the negatively
charged amino acid glutamate. This charge-reversing substitution should reduce the binding of PIP2 or ATP via electrostatic
repulsion if the residue is located close to the respective binding
sites. We found that ATP sensitivity was reduced for R50E (as reported previously, (21)) and, surprisingly, increased for R54E, whereas K47E
and K67E channels displayed ATP sensitivities similar to WT channels
(see below, Fig. 3D). To assess the impact of the different
mutants on PIP2 binding we compared the current amplitude briefly after patch excision with that obtained after application of
PIP2. This type of assay has been used previously (19) and is based on the finding that PIP2 increases the channel's
open probability (Po). For WT channels
PIP2 increased the current amplitude (thus
Po) only marginally (Figs. 1C and
4B). This suggests that the affinity for PIPs is so high
that the endogenous PIP2 already maximally opens the
channels. Similar results were obtained for the mutants K47E, R50E, and
K67E (Fig. 1C). In distinction, R54E channels showed only
little initial activity, but application of PIP2 increased
the current amplitude by a factor of 29 ± 9 suggesting a marked
reduction in PIP2 affinity (Figs. 1, B and C and 4C). To investigate whether the charge at
position 54 is critical, Arg-54 was mutated to a neutral (R54Q) and to
a positively charged amino acid (R54K). R54K channels showed WT
behavior, whereas the current produced by R54Q channels was increased
by a factor of 4.5 ± 1.5 by PIP2 (Fig.
1C). The impact of mutations on PIP2 modulation
in the order R54E > R54Q > R54K = WT suggest
electrostatic interactions between the charge at position 54 and
PIP2. The mutation R176A in the C terminus of Kir6.2 has
been previously shown to reduce PIP2 binding (11, 13) and
is shown here for comparison. The current amplitude produced by R176A
channels was increased by PIP2 by a factor of 3.2 ± 0.5 (Fig. 1C).
Neomycin Inhibition as an Assay for PIP2 Affinity in
Kir Channels--
Neomycin is a polycation that binds specifically to
PIP2 and for this reason has been used to determine the
PIP2 content in biological membranes (24). In
electrophysiological experiments, neomycin was shown to reverse the
effects of PIP2 on KATP channels causing
inhibition of channel activity and reduction of ATP sensitivity (11,
25). Thus, the neomycin sensitivity of a Kir channel might be a measure
of its PIP2 affinity. Accordingly, channels with a high
PIP2 affinity are expected to be less sensitive to neomycin
than those with low PIP2 affinity. To test this assumption we measured neomycin inhibition of KATP and Kir1.1 channels
since Kir1.1 channel are thought to bind PIP2 more tightly
than KATP channels. In good agreement, KATP
channels (IC50 = 17.1 ± 2.2 µM) were
about 3-fold more sensitive to neomycin than Kir1.1 channels (IC50 = 43 ± 10 µM) (Fig.
2C). Using this assay, we
tested for the PIP2 affinities of the different N-terminal
(K47E, R50E, R54E, R54Q) mutants and the C-terminal mutant R176A of
Kir6.2. Some of the mutated channels produced only small currents
(R54E, R54Q, R176A) necessitating the measurement of neomycin
inhibition subsequent to application of PIP2 (10 µM for 45 s) (Fig. 2A). The mutations R54E, R54Q, and R176A altered neomycin inhibition considerably: the
concentration-response curves were much steeper and most of the
inhibition occurred between 1 µm and 10 µM
(IC50 of about 3 µM) (Fig. 2, A
and B). For K47E channels neomycin sensitivity was somewhat
increased (IC50 = 38 ± 10 µM) and for
R50E channels somewhat reduced (IC50 = 318 ± 183 µM) compared with WT channels (IC50 = 122 ± 98 µM after PIP2) (Fig.
2C), whereas the shape of the concentration-response curves
was not changed. These results are qualitatively in good agreement with
the findings on PIP2-amplitude modulation (Fig. 1). R54E,
R54Q, and R176A largely affected PIP2-amplitude modulation
and neomycin sensitivity, whereas K47E and R50E channels showed
basically WT behavior.
Arg-54 Determines PIP2 Modulation of ATP Sensitivity in
KATP Channels--
The effect of the N-terminal mutations
as well as the C-terminal mutant R176A on the PIP2
modulation of ATP inhibition was characterized by comparing the ATP
sensitivity before and subsequent to application of 10 µM
PIP2 for 45 s. This procedure shifted the
IC50 for ATP inhibition of WT channels by a factor of
71 ± 3 (Fig. 3, B,
C, and D). Similar values were obtained for K47E (shift factor: 55 ± 17), R50E (shift factor 98 ± 16), and
K67E (shift factor 73 ± 15) (Fig. 3C). For R176A the
shift factor was reduced to 28 ± 4 (Fig. 3C) in
agreement with previous findings on this mutant (13). R54E and R54Q had
by far the largest effects on the PIP2 modulation of ATP
inhibition with the corresponding shift factors of 2.5 ± 0.3 and
8.2 ± 1, respectively (Fig. 3, A, B, and
C). Thus, the mutations R54E and R54Q impaired the
modulation of ATP sensitivity by PIP2 with R54E being more
potent than R54Q. The impact of R54Q on the antagonistic effect of
PIP2 on ATP inhibition is shown directly in Fig.
4A. Application of 50 µM PIP2 readily removed inhibition of WT
channel produced by 1 mM ATP (n = 3, Fig.
4A). For R54Q channels even prolonged application (see time scale) of 50 µM PIP2 only marginally
antagonized ATP inhibition (n = 3, Fig. 4A).
Very similar results were obtained for R54E, whereas R54K showed WT
behavior (data not shown). For R176A channels PIP2
modulation of ATP inhibition was significantly reduced; however, clearly less pronounced compared with R54E/R54Q channels
(n = 3, Fig. 4A). Fig. 4, B and
C shows the effect of PIP2 and ATP on a single
WT and R54E channel. WT channels display high initial Po (>0.8) that was only marginally increased
upon application of PIP2. Given subsequent to
PIP2, 100 µM ATP produced virtually no
inhibition (Fig. 4B). In contrast, R54E channels have very low initial Po (<0.05), and PIP2
largely increased the Po to a level similar to
WT channels (>0.8); however, 100 µM ATP potently inhibited channel activity (Fig. 4C) in full agreement with
the macroscopic currents (Figs. 1 and 3). In summary, these results show that the mutations R54E/R54Q disrupt the ability of
PIP2 to antagonize ATP inhibition.
Role of the N-terminal PIP2 Site in Kir1.1
Channels--
The arginine at position 54 is highly conserved among
the members of the Kir channel superfamily; however, Kir1.1, Kir4.1, and Kir5.1 possess a neutral residue at the corresponding position (Fig. 5A). These channels have
in common that they are gated by intracellular protons (3-6, 26). We
chose Kir1.1 to study the role of the position Ile-63 that corresponds
to Arg-54 in Kir6.2. An Arginine was introduced at position 63 (I63R),
and the impact on PIP2 affinity was assayed by monitoring
the run down of channel activity upon exposure to a
Mg2+-containing solution (Fig. 5B).
Mg2+ is thought to induce a run down via a breakdown of
PIP2 though activation of phosphatases and lipases
associated with the patch (10). WT channels lost most of the channel
activity within 12 min. In I63R channels run down was markedly slower
indicative of an increased PIP2 affinity (Fig.
5B). Accordingly, the neomycin inhibition of I63R channels
was reduced dramatically from an IC50 of 43 ± 10 µM (WT channels) to 7.3 ± 1.7 mM (Fig.
5C).
To investigate the role of position 63 for pH gating, WT and I63R
channels were exposed to various pH values of the bathing solution.
Acidification from 7.5 to 6.0 resulted in complete but reversible
inhibition of Kir1.1 channels (Fig. 5E). Using a Hill equation the effective pKa and Hill coefficient for
pH inhibition were estimated to be 6.83 ± 0.04 and 4.4 ± 0.2, respectively, in good agreement with previous reports (Fig.
5D) (3, 6). The mutation I63R largely reduced the pH
sensitivity shifting the pKa to 5.77 ± 0.08 and the Hill coefficient to 2.2 ± 0.1 (Fig. 5, D and
F). If this shift in pH sensitivity was caused by an
increased binding of PIP2, then application of
PIP2 should cause a similar shift in pH sensitivity for WT
channels. However, application of PIP2 had no significant
effect on pH inhibition (Fig. 5D) suggesting that
PIP2 binding was already saturated before the addition of
exogenous PIP2. These results point to an effect of
I63R on pH gating in Kir1.1 channels that is distinct to the effect on
PIP2 affinity (see "Discussion").
We screened the proximal N terminus for residues that contribute
electrostatically to the binding of PIP2 or ATP by
substituting basic residues by the negatively charged glutamate (K47E,
R50E, R54E, K67E). Two of these mutations altered ATP inhibition
markedly. R50E reduced the ATP sensitivity about 12-fold, whereas R54E
increased the sensitivity about 3-fold. However, neither Arg-50 nor
Arg-54 are likely to contribute to ATP binding directly as detailed below.
Arg-54 Is an N-terminal Determinant for PIP2 Binding in
Kir6.2/SUR Channels--
The mutation R54E resulted in
KATP channels with very low Po upon
patch excision that was largely increased by addition of exogenous PIP2 (PIP2-amplitude
modulation) consistent with a reduced PIP2 affinity. In
contrast, WT channels as well as the mutants K47E, R50E, and K67E
showed nearly maximal Po upon patch excision, and exogenous PIP2 had little effect. The PIP2
affinity of the different mutant channels clearly correlated with the
charge at position 54 with WT and R54K channels having the highest
affinity, R54Q channels an intermediate, and R54E the lowest
PIP2 affinity. These results strongly suggest electrostatic
interaction between Arg-54 and PIP2. Previous work has
identified several residues in the C terminus of Kir6.2 as determinants
for PIP2 binding, e.g. R176A was shown to reduce
PIP2 binding (11, 13, 19). The effect of R176A on
PIP2-amplitude modulation was comparable to R54Q (Fig.
1C) suggesting similar importance for the binding of
PIP2 to KATP channels. PIP2 binding
of WT and mutant channels was also tested using neomycin inhibition as
a relative measure for the PIP2 affinity. R54E, R54Q, and
R176A channels showed a markedly increased sensitivity to neomycin
compared with WT, K47E, and R50E channels. These results further
substantiate the view that Arg-54 directly contributes to
PIP2 binding.
Arg-54 Plays a Key Role for the PIP2 Modulation of ATP
Sensitivity in KATP Channels--
The ATP sensitivity of
KATP channels depends on the concentration of PIPs in the
membrane. Increasing concentrations of PIP2 increase the
IC50 value for ATP inhibition by several orders of magnitude (12, 13), and thus, the shift in ATP sensitivity for a given
increase in membrane PIP2 should be related to the PIP2 affinity of the channel. We found that the mutations
K47E, R50E, and K67E had virtually no effect on the PIP2
modulation of ATP inhibition in contrast to R54E and R54Q.
R54E/R54Q dramatically reduced the effect of PIP2 on
ATP inhibition with R54E being more potent than R54Q. The results are
in excellent agreement with those on PIP2-amplitude
modulation and show that the charge at position Arg-54 determines the
effect of PIP2 on Po and on ATP sensitivity.
On the Mechanism of PIP2 Modulation of ATP
Inhibition--
As pointed out in the introduction, regions implicated
to be important for ATP inhibition and PIP2 binding appear
to coincide (at least in primary sequence) in the C terminus of Kir6.2
(19-21). This tendency seems to be even more striking with the
identification of Arg-54 as a determinant of PIP2 binding
in the N terminus since Arg-50 is an important determinant of ATP
inhibition (21). On the mechanistic basis of this finding two
explanations come to mind. First, ATP and PIP2 might bind
to overlapping sites, and secondly, PIP2 modulates ATP
inhibition allosterically by interaction with basic residues in regions
that are critical for the gating mechanism that links ATP binding to
channel closure. Both alternatives have been put forward recently
(27-29). As an argument against physically overlapping binding sites
it has been pointed out that mutations in the C terminus that affected
PIP2 binding in the most cases did not change ATP
inhibition (19). This holds valid also for the N terminus. R50E
markedly reduced ATP sensitivity but had no effect on PIP2
modulation of ATP inhibition. On the contrary, R54E largely reduced the
effect of PIP2 on ATP inhibition but had no direct effect
on ATP sensitivity. Assuming electrostatic repulsion between
R54E and PIP2 it is rather unlikely for ATP binding
to occur in close proximity to position 54 without sensing the charge
at this position. These results argue against overlapping binding sites
for PIP2 and ATP in the N terminus and favor an allosteric
mechanism. In other words the interaction of PIP2 with Arg-54 appears to disrupt the mechanism that allows ATP to induce channel inhibition. Accordingly, disabling the N-terminal
PIP2 site is expected to reduce the ability of
PIP2 to modulate the ATP sensitivity as seen with
R54E/R54Q channels. The identification of several
PIP2 sites in N and C terminus raises the question whether
the sites are functionally equivalent. Are there PIP2 sites
that preferentially affect ATP inhibition and others that are more
important for control of e.g. open-state stability?
Comparing R176A and R54Q suggests such functional heterogeneity. Both
mutations increased PIP2-amplidude modulation to a similar
extent (Fig. 1C) suggesting that Arg-54 and Arg-176
contribute about equally to the effect of PIP2 on
open-state stability. However, Arg-176 contributed obviously less to
the antagonizing effect of PIP2 on ATP inhibition compared
with Arg-54 (Figs. 3C and 4A). Moreover, none of
the other potential PIP2 binding sites in the C terminus identified so far appeared to be very critical for the PIP2
modulation of ATP sensitivity. Mutations at these positions produced
significant effects on PIP2-amplitude modulation, but no
marked effects on the ability of PIP2 to shift ATP
sensitivity have been observed (19). These findings suggest a pivotal
role of Arg-54 for mediating the antagonizing effect of
PIP2 on ATP inhibition.
Role of the N-terminal PIP2 Site for pH Gating in Kir
Channels--
The arginine at position 54 is highly conserved among
the Kir channel superfamily pointing to a general role for
PIP2 binding in Kir channels. Indeed, a recent paper
demonstrated that this arginine also contributes to PIP2
binding in Kir2.1 channels (30). Intriguingly, Kir1.1, Kir4.1, and
Kir5.1 present with a neutral residue at the corresponding position. A
distinctive property of these Kir channels is a strong effect of
intracellular protons on the open probability of the channel. This pH
gating is mediated by a lysine residue (Lys-80 in Kir1.1) in the N
terminus that serves as a pH sensor and is lacking in other Kir
channels (3, 6). Protonation of the pH sensor causes reversible
inhibition of channel activity in the physiological range. The unusual
acidic pKa (
It has been proposed that PIP2 binding to Kir1.1 alters the
pKa for pH gating (15). Thus, we tested whether the absence of the N-terminal PIP2 site might be a prerequisite
for Kir1.1 channels to operate in the physiological pH range
(6.8-7.5). We observed, indeed, that introduction of an arginine at
the N-terminal PIP2 site largely increased the
PIP2 affinity of Kir1.1 channels and shifted the
pKa for pH inhibition far out of the physiological
range (pKa for I63R
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
70 °C. Xenopus oocytes were surgically removed from adult females and manually dissected. About 50 nl of a solution containing cRNA specific for SUR2A and Kir6.2
subunits was injected into Dumont stage VI oocytes. Oocytes were
treated with collagenase type II (Sigma, 0.5 mg/ml) and incubated at
19 °C for 1-3 days and defolliculated prior to use.
20 °C,
diluted in Kint solution to final concentrations,
sonicated for 35 min, and used within 6 h. Pipettes used were made
from thick-walled borosilicate glass, had resistances of 0.2-0.4
M
(tip diameter of 20-30 µm), and were filled with
(in mM, pH adjusted to 7.2 with KOH) 120 KCl, 10 HEPES and
1.8 CaCl2. Currents were recorded with an EPC9 amplifier (HEKA electronics, Lamprecht, Germany) and sampled at 1 kHz with analog
filter set to 3 kHz. Solutions were applied to the cytoplasmic side of excised patches via a multibarrel pipette and had the following
composition in mM (Kint): 100 KCl, 10 HEPES, 2 K2EGTA (total K+ concentration was 120 mM, pH adjusted to 7.2 with KOH). Computational work was
done on Macintosh G4 using commercial software (IGOR, WaveMetrics) and
Excel 2001 (Microsoft).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Effect of PIP2 on the
amplitude of currents mediated by Kir6.2/SUR2A channels and mutants
expressed in Xenopus oocytes and studied in giant
inside out patches. The same protocol applies to all following
experiments (Fig. 1-Fig. 5). A, schematic model of a Kir6.2
subunit residues of interest are highlighted. B,
R54E currents measured at 80 mV (inward currents shown as upward
deflection), patch excision, and application of 10 µM
PIP2 are indicated. C, bars represent
the fold change of current amplitude ± S.E. (n > 3) upon
application of 10 µM PIP2 for 45 s.
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Fig. 2.
Neomycin inhibition of Kir1.1, Kir6.2, and
mutant channels in inside out patches. A, neomycin
inhibition subsequent to application of 10 µM
PIP2 for 45 s; neomycin concentration as indicated.
B, from data such as shown in A
concentration-response curves (CV curves) were obtained and fitted to a
standard Hill equation; error bars represent ± S.E.
C, from CV curves as in B the IC50
values were determined and plotted as bar with ± S.E.
Stars indicate that the IC50 values were
determined subsequent to application of 10 µM
PIP2 for 45 s.
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Fig. 3.
Effects of Kir6.2 mutations on
PIP2 modulation of ATP sensitivity.
A, R54E currents with applications of 10 µM
PIP2 and various ATP concentrations as indicated.
B, from data such as in A CV curves were obtained
and fitted to a standard Hill equation (13); error bars
indicate ± S.E. C, from CV curves in B
IC50 values were determined and plotted as bars with ± S.E. D, table with values of the IC50 values
before (contr.) and after application of 10 µM
PIP2 for 45 s.
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Fig. 4.
Antagonism of ATP inhibition by
PIP2 for WT and mutant channels.
A, for WT, R54Q, and R176A currents the effect of
PIP2 and ATP is shown with concentrations as indicated.
Similar results were obtained in at least two further experiments.
B, single channel recordings for WT and R54E channels
measured at 80 mV and filtered at 2 kHz. Application of
PIP2 and ATP as indicated. From the last 3 s before
ATP application the Po was estimated to be > 0.8 < 0.9 for WT and R54E. Po was
calculated from amplitude histograms (data not shown). The
Po of the R54E channels before PIP2
was below 0.05. Similar results were obtained in two further
experiments.
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Fig. 5.
Effects of I63R on PIP2
affinity and pH sensitivity of Kir1.1 channels.
A, sequence alignment of various Kir channels for the region
near Arg-54 in Kir6.2; pH-gated channels are indicated with
arrows (Alignment was done with the commercial software
DNA-Star). B, time course of channel run down for WT and
I63R channels induced by application of 2 mM
Mg2+; data points are mean ± S.E. C,
neomycin inhibition of WT and I63R channels. From experiments such as
in Fig. 2A CV curves were obtained and fitted to a standard
Hill equation; error bars indicate ± S.E. The
IC50 values for WT and I63R were 43 ± 10 µM and 7.3 ± 1.7 mM, respectively.
E, currents from WT and I63R channels (F) at the
pH values indicated. D, from recordings such as in
E and F CV-curves were obtained and fitted to a
standard Hill equation (open symbols); error bars
indicate ± S.E. pKa and Hill coefficient were 6.8 ± 0.02 and 4.2 ± 0.3 for WT and 5.77 ± 0.08 and 2.2 ± 0.1 for I63R channels. Filled symbols represent CV curves
subsequent to application of 20 µM PIP2 for
30 s; pKa and Hill coefficient were 6.79 ± 001 and 2.7 ± 0.3 for WT and 5.75 ± 0.04 and 1.8 ± 0.1 for I63R channels.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
6.8) of the lysine is thought to come
about from its proximity to two arginines in the N and C terminus of
the same subunit forming a "Arg-Lys-Arg triad" that shifts the
pKa of the lysine into the physiological range via
electrostatic interactions (3).
5.8). It has been shown
that reduction in PIP2 binding can shift the effective
pKa for pH gating in Kir1.1 channels to more
alkaline values. This was based on the finding that low
PIP2 concentrations in the membrane or reduction in
PIP2 binding, as seen with a mutant channel (R188Q), increased the pH sensitivity (15). We found that PIP2
binding is already saturated for WT Kir1.1 channels since addition of exogenous PIP2 caused no additional shift in the
pKa. This outcome does not conflict with previous
findings (15); however, it suggests that an increase in
PIP2 affinity (e.g. I63R) should not alter pH
gating and, thus, should not account for the effect of I63R on pH
gating. PIP2 interaction with the N-terminal site might
either reduce H+ binding to the pH sensor thus shifting the
pKa of lysine 80 (e.g. though a change of
the micro environment of Lys-80) or disturb the transduction mechanism
allowing protonation of the pH sensor to power channel closure. Indeed,
the reduction of the Hill coefficient for I63R (WT = 4.2 ± 0.3 and I63R = 2.2 ± 0.1, Fig. 5D) might indicate
reduced coupling between the pH sensor and the gate that controls
channel activity. In conclusion, ATP gating in KATP
channels and pH gating in Kir1.1 are controlled by a N-terminal
PIP2 site. While for KATP channels this site is necessary to allow potent modulation of ATP sensitivity by
PIP2, a silent N-terminal PIP2 site is a
prerequisite for intact pH gating in Kir1.1 channels. Even subtle
changes in the pH sensitivity of Kir1.1 channels cause severe kidney
defects as found in patients with the antenatal Bartter syndrome (3).
Thus, a high evolutionary pressure to preserve a neutral residue at the
N-terminal PIP2 site is expected and consistent with the
absence of a positively charged residue in all pH-gated Kir channels.
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ACKNOWLEDGEMENTS |
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We appreciate the excellent technical support by Dr. Yuan Ruan and thank Dr. Klaus Benndorf and Dr. Bernd Fakler for critically reading the manuscript.
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FOOTNOTES |
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* This work was supported by Grant Ba 1793 from the Deutsche Forschungsgemeinschaft (to T. B.).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.
Both authors contributed equally to this work.
§ Present address: flyion GmbH, Waldhäuserstrasse 34, 72076 Tübingen, Germany.
¶ To whom correspondence should be addressed. Tel.: 0049-3641-938860; Fax: 0049-3641-933202; E-mail: thbau@mti-n.uni-jena.de.
Published, JBC Papers in Press, January 4, 2003, DOI 10.1074/jbc.M208413200
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ABBREVIATIONS |
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The abbreviations used are: SUR, sulfonylurea; PIP, phosphatidylinositol polyphosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; WT, wild type.
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