From the Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010
Received for publication, October 23, 2000, and in revised form, January 22, 2001
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ABSTRACT |
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ATP-sensitive K+
(KATP) channels may be regulated by protons in addition to
ATP, phospholipids, and other nucleotides. Such regulation allows a
control of cellular excitability in conditions when pH is low but ATP
concentration is normal. However, whether the KATP changes
its activity with pH alterations remains uncertain. In this
study we showed that the reconstituted KATP was
strongly activated during hypercapnia and intracellular acidosis using whole-cell recordings. Further characterizations in excised patches indicated that channel activity increased with a moderate drop in
intracellular pH and decreased with strong acidification. The channel activation was produced by a direct action of protons on the
Kir6 subunit and relied on a histidine residue that is conserved in all
KATP. The inhibition appeared to be a result of channel
rundown and was not seen in whole-cell recordings. The biphasic
response may explain the contradictory pH sensitivity observed in
cell-endogenous KATP in excised patches. Site-specific mutations of two residues showed that pH and ATP sensitivities were independent of each other. Thus, these results demonstrate that
the proton is a potent activator of the KATP. The
pH-dependent activation may enable the KATP to
control vascular tones, insulin secretion, and neuronal excitability in
several pathophysiologic conditions.
Hypercapnia and acidosis affect vascular tone, skeletal muscle
contractility, insulin secretion, epithelial transport, and neuronal
excitability, which may be mediated by
KATP1 (1-5).
However, previous studies on the pH sensitivity of these K+
channels were controversial and even contradictory. In the absence of
ATP, acidic pH was shown to stimulate cell-endogenous KATP (6, 7), inhibit it (8, 9), and have little or no effect (10, 11). This
inconsistency is further complicated by the indirect effect of
ATP or Mg2+ and tissue-specific KATP species
(8-12). Consequently, it is unclear whether KATP is
modulated during hypercapnia and acidosis and what molecular mechanisms
are underlying the modulations. The cloned KATP channels
are ideal for addressing these questions because they allow for
fine dissection of the modulatory mechanisms and elaborate manipulation
of PCO2 and pH in an expression system (13, 14).
Therefore, we studied the modulation of the cloned KATP
(Kir6 with SUR, Ref. 15) by CO2 and acidic pH. To
locate the pH sensors, we also studied Kir6.2 with a truncation of 36 amino acids at the C terminus (Kir6.2 Oocytes from Xenopus laevis were used in
the present studies. Frogs were anesthetized by bathing them in 0.3%
3-aminobenzoic acid ethyl ester. A few lobes of ovaries were removed
after a small abdominal incision (~5 mm). Then, the surgical incision was closed and the frogs were allowed to recover from the anesthesia. Xenopus oocytes were treated with 2 mg/ml collagenase (Type
I, Sigma) in an OR2 solution consisting of (in mM)
NaCl 82, KCl 2, MgCl2 1, and HEPES 5 (pH 7.4) for 90 min at
room temperature. After three washes (10 min each) of the oocytes with
the OR2 solution, cDNAs (25-50 ng in 50 nl of water) were
injected into the oocytes. For coexpression, Kir6.x and SUR1 were
injected in a 1:2 ratio. The oocytes were then incubated at 18 °C in
the ND-96 solution containing (in mM) NaCl 96, KCl
2, MgCl2 1, CaCl2 1.8, HEPES 5, and sodium
pyruvate 2.5 with 100 mg/liter geneticin added (pH 7.4).
Rat Kir6.1 (uKATP, GenBankTM/EBI accession number D42145) and mouse
Kir6.2 (mBIR, GenBankTM/EBI accession number D50581) cDNAs were
generously provided by Dr. S. Seino. Hamster SUR1
(GenBankTM accession number L40623) was a gift from Dr. L. Bryan. The cDNAs were subcloned to a eukaryotic expression vector
(pcDNA3.1, Invitrogen Inc., Carlsbad, CA) and used for
Xenopus oocyte expression without cRNA synthesis.
Site-specific mutations were produced using a site-directed mutagenesis
kit (Stratagene, La Jolla, CA). The orientation of the constructs and
correct mutations were confirmed with DNA sequencing.
Whole-cell currents were studied on the oocytes 2-4 days after
injection using a two-electrode voltage clamp with an amplifier (Geneclamp 500, Axon Instruments Inc., Foster City, CA) at room temperature (~24 °C). The extracellular solution contained
(in mM) KCl 90, MgCl2 3, and HEPES 5 (pH 7.4).
Extracellular acidification was done by titrating the extracellular
solution to desired pH levels. The HEPES buffer was chosen because of
its buffering range and membrane impermeability, as shown in our
previous studies (17, 18). In intracellular acidification experiments,
90 mM KCl was replaced with the same concentration of
KHCO3 (pH titrated to 7.4), so that the K+
concentration remained the same in these experiments (19). When oocytes
were exposed to these perfusates, intracellular pH (pHi) and
extracellular pH (pHo) were measured using ion-selective
microelectrodes as described previously (14). Whole-cell currents were
also studied with nigericin (10 µM) at various bath pH
levels. This protonophore forms cation channels permeable primarily to
protons (20, 21). Using 90 mM K+ in the bath
solution as permeable cation, pHi becomes the same as
pHo in the presence of nigericin (20). Exposure to nigericin
increased oocyte-endogenous currents at each pH point. The current
changes in these pH points were measured in oocytes without any
injection, averaged (n = 4), and subtracted from
current records of the Kir6-expressing cells because the alterations
were due to nigericin rather than changes of pHi in these cells.
Experiments were performed in a semiclosed recording chamber (BSC-HT,
Medical Systems Corp., Greenvale, NY) in which oocytes were placed on a
supporting nylon mesh; the perfusion solution bathed both the top and
bottom surface of the oocytes. The perfusate and the superfusion gas
entered the chamber from two inlets at one end and flowed out at the
other end. There was a 3 × 15-mm gap on the top cover of the
chamber, which served as the gas outlet and an access to the oocytes
for recording microelectrodes. At baseline the chamber was ventilated
with atmospheric air. Exposure of the oocytes to CO2 was
carried out by switching a perfusate that had been bubbled for at least
30 min with a gas mixture containing CO2 at various
concentrations balanced with 21% O2 and N2 and superfused with the same gas (14, 17, 18). The high dissolvability of
CO2 resulted in a detectable change in intra- or
extracellular acidification as fast as 10 s in these oocytes.
Macroscopic and single-channel currents were recorded in excised
patches at room temperature (~24 °C) as described previously (22,
23). In brief, the oocyte vitelline membranes were mechanically removed
after being exposed to hypertonic solution (400 mosmol) for 5 min. Recordings were performed on the stripped oocytes using the same
solution applied to bath and recording pipettes. The solution contained
(in mM) KCl 10, potassium gluconate 130, potassium fluoride
5, EGTA 1, and HEPES 10 (pH 7.4). A parallel perfusion system was used
to deliver low pH perfusates at a rate of ~1 ml/min with no dead
space (22, 23). Macroscopic and single-channel currents were analyzed
using the pClamp 6 software as detailed previously (22, 23).
Data are presented as means ± S.E. analysis of variance or
Student's t test was used. Differences of CO2
and pH effects before versus during exposures were
considered to be statistically significant if p Expressions of Kir6.2 together with SUR1 (Kir6.2+SUR1) in
Xenopus oocytes produced K+ currents with clear
inward rectification in whole-cell recordings and
ATP-dependent inhibition in inside-out patches. When
oocytes expressing Kir6.2+SUR1 were exposed to 15% CO2
(14, 18), massive activation of the whole-cell inward rectifying
currents occurred (129 ± 31%, n = 5). Similar
channel activation was observed in Kir6.2
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C36) because it expresses functional channel without the SUR subunit and retains fair ATP sensitivity (16).
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
0.05.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
C36 (143 ± 15%,
n = 11), whose CO2 sensitivity did not show
any significant difference from the Kir6.2+SUR1 and Kir6.2
C36+SUR1 (p > 0.05), indicating that the
CO2-sensing mechanism is located on the Kir6.2 subunit
(Fig. 1, A and B).
The effect was reversible and dependent on CO2
concentrations. An apparent increase in the current amplitude was seen
with PCO2 as low as 7.6 torr (1%), and higher
PCO2 resulted in stronger activation (Fig.
1C). Interestingly, the Kir6.1+SUR1 showed a similar
CO2 sensitivity (141 ± 28%, n = 6;
Fig. 1B), suggesting that various KATP channels
consisting of Kir6.1 or Kir6.2 are likely to be activated by
CO2. In contrast, Kir2.1 had no response to 15%
CO2, whereas Kir1.1, Kir2.3, and Kir4.1 were inhibited
(Fig. 1B).
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Fig. 1.
Augmentation of cloned KATP
currents during hypercapnia and intracellular acidification.
A, whole-cell K+ currents were studied with a
series of voltage commands (from 160 mV to 140 mV with 20-mV
increments at a holding potential of 0 mV) using a bath solution
containing 90 mM K+. The Kir6.2
C36 currents
show a clear inward rectification of ~4 µA at
160 mV. When the
oocyte was exposed to 15% CO2, the inward rectifying
currents were markedly enhanced. Washout led to a complete recovery.
B, similar channel activation was observed in Kir6.2+SUR1,
Kir6.2
C36+SUR1, and Kir6.1+SUR1, all of which showed CO2
(15%) sensitivity indistinguishable from Kir6.2
C36
(p > 0.05). In contrast, the same CO2
exposure had no effect on Kir2.1 and caused inhibition of Kir1.1,
Kir2.3, and Kir4.1. Data are presented as means ± S.E.
C, activation of Kir6.2
C36 currents with various
concentrations of CO2. Significant increase in the current
amplitude was seen with 1% CO2 (7.6 torr). Higher
PCO2 resulted in much stronger activation.
Asterisk, exposure to the atmospheric air showing that all
changes are statistically significant (p < 0.05).
pHi was measured as described previously (14). The pHi
values were obtained from four different oocytes at each
CO2 level, averaged and shown in parentheses
following the CO2 concentrations. D,
In another cell, intracellular acidification was produced
using 90 mM KHCO3 (pHo 7.4). Such a
manipulation reduced pHi to 6.6, and reversibly enhanced the
Kir6.2
C36 currents. E, Intracellular acidification to
pHa 6.6 using high concentration bicarbonate or nigericin (10 µM in the KD90 solution) caused an activation of the
Kir6.2
C36 that was almost identical to that produced by 15%
CO2. Extracellular acidification to the pH level seen
during 15% CO2 exposure (pHo 6.2) using
membrane-impermeable HEPES buffer had little effect, however, which is
significantly different from channel activation by CO2 and
acidic pHi (asterisk, p < 0.001).
I, current; WS, washout.
To understand whether the channel activation was produced by pH
changes, currents were studied with a selective decrease in intracellular pH (pHi) to 6.6 or extracellular pH (pHo) to 6.2 as we have previously measured during CO2 (15%)
exposure (14). Selective intracellular acidification using bicarbonate (14, 18, 19) without changing pHo activated the Kir6.2C36
currents by 162 ± 31% (n = 4), which showed no
significant difference from the hypercapnic effect (p > 0.05, Fig. 1, D and E). Lowering pHo
to 6.2 without changing pHi, however, increased the currents
only modestly (8 ± 2%, n = 4; Fig.
1E), suggesting that the channels are stimulated
predominantly by intracellular protons. The pH sensitivity of
whole-cell Kir6.2
C36 currents was examined by permeabilization of
the plasma membranes using protonophore nigericin (10 µM)
at various pHo levels (20, 21). Graded activation of
Kir6.2
C36 currents was seen with graded acidification (Fig.
2B). When the maximal
activation was reached at pH ~6.6, the current amplitude increased by
150 ± 11% (n = 6), which should mainly be
produced by a drop in pH because acidic pHo had little effect
on the currents. Thus, a consistent enhancement of the channel activity
was demonstrated at pHi 6.6 in the presence or absence of
CO2.
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To determine whether the channel activation is carried out by the
inherent mechanism of Kir6.2 or mediated by cytosolic factors, Kir6.2C36 and Kir6.2+SUR1 currents were studied in excised
inside-out patches in the absence of ATP, ADP, and other cytosolic
soluble factors (22, 23). Exposure of the internal surface of patch membranes to acidic pH augmented the macroscopic inward rectifying currents (Fig. 2A). The peak activation occurred at
pHi 6.6-6.8 (Fig. 2B). A further decrease in
pHi caused rapid inhibition of the Kir currents (Fig. 2,
A and B). The inhibition appeared to be channel
rundown because it was not seen in whole-cell recordings and channel
activity showed little or no recovery during washout at pH 7.4, particularly after a long period of exposure in patches (>30 s). Thus,
extremely acidic pH may accelerate KATP rundown as
described previously (7, 11-13). A similar bell-shaped current-pHi relationship was observed in Kir6.2+SUR1 (Fig.
2B), further supporting the observation that
pHi sensitivity is independent of the SUR subunit.
Single-channel recordings showed that the current stimulation was
mainly caused by augmentation of the open state probability
(NPo) with concurrent moderate suppression (by
10.7 ± 0.2% measured at pH 6.2, n = 4) of the
single-channel conductance. When the NPo was
plotted as a function of pHi a bell-shaped
NPo-pH relationship was also obtained, which was
almost identical to the response of macroscopic currents to acidic
pHi (Figs. 2B and 3).
The relationship of channel activity (i.e. macroscopic
currents and NPo) versus pHi
can be described using a sum of two Hill equations with one for channel
activation (pKa 7.15, h 2.0) and the
other for channel rundown (pKa 6.48, h
6.0) (Fig. 2B). The pHi-dependent
channel activation was virtually superimposed with that of whole-cell
currents, consistent with the idea that the pH sensing mechanism exists
inherently in the channel protein.
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If the pHi sensing is indeed an intrinsic property of the
channel protein, there should be specific protein domains or amino acid
residues responsible for the proton detection. To test this hypothesis,
we performed site-directed mutagenesis on potentially titratable
residues of histidine, an amino acid with its side chain
pK (6.04) closest to the channel activation
pKa (7.15). Among nine histidine residues studied in
the intracellular N- and C-terminal domains, we found that His-175
was critical for the pHi sensitivity of Kir6.2C36. Mutation
of this residue to lysine (residue found in other Kir channels, H175K) or alanine (H175A) completely eliminated the proton-induced channel activation (Fig. 4). These mutants were
even inhibited during hypercapnia in whole-cell recordings (Fig. 4,
A and C) and by acidic pHi in excised
patches (Fig. 4B), which were also observed in mutants
expressed with SUR1. Single-channel conductance of the H175K was
significantly smaller than its wild-type counterpart (64.4 ± 0.9 picosiemens, n = 4; p < 0.05).
Interestingly, a relief of channel rundown was seen in some of the
His-175 mutations. In the H175K, NPo remained
above 0.1 after 200 s of perfusion with a solution of pH 6.2 (n = 3). In contrast, NPo
dropped to below 0.01 within 40 s in the wild-type channel
(n = 12) and H175A mutant under the same condition
(n = 3). Mutations of other histidine residues had no
significant effect on the CO2 sensitivity
(p > 0.05, Fig. 4C).
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Finally, we examined if ATP and pH sensitivities depended on each
other. It is known that the K185E mutation greatly reduces the ATP
sensitivity of Kir6.2C36 (24). We found that the K185E had identical
pH sensitivity to the wild-type Kir6.2
C36, whereas the H175K
mutation did not affect the ATP sensitivity (Fig.
5).
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DISCUSSION |
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KATP channels are regulated by ATP, ADP, and
phospholipids (11, 2428). Such modulations allow them to
control cellular activity during energy insufficiency. In the present
studies, we have demonstrated the proton as another KATP
regulator. We have found that hypercapnia and acidic pH at near
physiological levels augment KATP activity in striking
contrast to other members of the Kir family that are either inhibited
by acidic pH or lack response (Fig. 1B) (29).
The effect of hypercapnia is likely to be mediated by a decrease in intracellular pH, inasmuch as similar channel activation occurs with a drop in pHi but not pHo. In cell-free excised patches we have found a moderate decrease in pHi augments KATP activity consistent with our whole-cell recordings in which pHi drops to 6.6 with 15% CO2 (14). A further decrease in pHi causes channel rundown in excised patches consistent with previous observations (7, 11-13). Such channel stimulation followed by inhibition may explain by and large the contradictory results of the pH sensitivity obtained previously in the cell-endogenous KATP using excised patches, i.e. when the KATP is studied with moderate acidification in a short period of exposure, channel activity increases. On the other hand, lower pH levels with a longer period of exposure produces channel inhibition with poor reversibility (8, 9). In the presence of ATP, however, channel rundown is largely diminished so that only activation remains (6-12). Because the biphasic response to acidic pH is not seen in our whole-cell recordings with severe hypercapnia (15% CO2) and acidification (pH 5.8), the channel inhibition or rundown may not occur in intact cells.
Our current studies have also begun to shed insight into the molecular mechanisms underlying KATP activation during acidosis. We have shown that the pH-sensing mechanisms are located on the Kir6 subunit rather than in the SUR. Indeed, we have demonstrated that mutation of a single histidine residue (H175A or H175K) is sufficient to eliminate completely the acid-induced channel activation. Instead of stimulation, the His-175 mutants show a significant inhibition by hypercapnic acidosis. The inhibition appears to suggest that the pH-dependent channel rundown may include a proportion of channel inhibition that is not seen in the wild-type channels under whole-cell recordings when the channel activation is dominant. The channel inhibition manifests itself when the channel stimulation is eliminated, as for His-175 mutations. When this inhibition is considered, the activation phase of the pH-current relationship curve shown in Figs. 2B and 5A can be even steeper. The inhibition, however, cannot totally account for the pH-dependent rundown, because rundown, though relieved in the H175K, is still seen in the H175A mutation. The histidine-dependent pH sensitivity thus is consistent with the proton-mediated regulation of a large number of proteins. Because the histidine is conserved in both Kir6.1 and Kir6.2 in all known species (Fig. 4D), it is very likely that the pH-sensing mechanism exists in KATP channels in various tissues. This unique site may offer an approach to control the KATP selectively without interference of other Kir channels.
Our data indicate that pH sensitivity is independent of ATP for the following reasons: 1) acid-induced channel activation is seen in the absence of ATP; 2) channel pH sensitivity in excised patches resembles that in whole-cell recordings; 3) the K185E mutation greatly reduces ATP sensitivity without affecting the pH sensitivity; and 4) the H175K mutation does not compromise the ATP sensitivity. Therefore, proton sensing in the KATP is very unlikely to be mediated by ATP, although it may be modulated by ATP.
Because a drop in pH often accompanies metabolic stresses and is more
frequently seen than sole energy depletion, pH sensitivity enables the
KATP channels to play a role in a wide variety of pathophysiological conditions. Pharmacological manipulation of the
KATP in coronary arteries and cerebral vasculature has been shown to affect vascular tones during hypercapnia and acidosis (5, 30).
KATP may be activated by lactoacidosis during skeletal muscle fatigue (31), contributing to the decrease in tetanic force and
the protection against injury (3). Excessive neuronal activity also
reduces pHi (32), which may activate the KATP,
leading to a suppression of hyperexcitability and a cessation of
seizure activity (2). Therefore, the demonstration of KATP
modulation by pHi has a profound impact on understanding cellular functions during metabolic stress and offers a potential intervention to control the cellular activity by manipulating the
inherent pH sensing mechanism of the KATP channels in the treatment and prevention of stroke, epilepsy, diabetes mellitus, and
coronary heart diseases.
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ACKNOWLEDGEMENTS |
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We thank Dr. S. Seino for providing Kir6.1 and Kir6.2 cNDAs and Dr. L. Bryan for the SUR1 cDNA.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant HL58410, American Diabetes Association Grant 01039, and American Heart Association Grant 9950528N.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.
These two authors contributed equally to this work.
§ To whom correspondence should be addressed: Dept. of Biology, Georgia State University, 24 Peachtree Central Ave., Atlanta, GA 30303-4010. Tel.: 404-651-0913; Fax: 404-651-2509; E-mail: cjiang@gsu.edu.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M009631200
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ABBREVIATIONS |
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The abbreviations used are: KATP, ATP-sensitive K+; SUR, sulfonylurea receptor; pHi, intracellular pH; pHo, extracellular pH; NPo, open state probability.
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