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INTRODUCTION |
The modulation of membrane excitability is a significant cellular
property, which not only provides cells with an important strategy for
responding to their external stimuli but also enables them to monitor
their internal environment and intermediary metabolism (1). Inward
rectifier K+ channels play a part in this process in which
several intracellular molecules such as nucleotides and protons are
involved (2, 3). Whereas the concentration of protons is normally
controlled by several buffering and feedback-controlling systems, pH
can fall beyond its normal levels under certain pathophysiological conditions. For instance, CO2 retention, or hypercapnia,
can cause a reduction in intra- and extracellular pH leading to
respiratory acidosis (4).
It is known that CO2 sensing, which plays an important role
in the feedback regulation of CO2 and pH homeostasis in
mammalian systems, is carried out by chemoreceptors, especially the
central chemoreceptors (5-8). Through these chemoreceptors, a high
PCO2 level stimulates respiratory neuronal networks in the
brain stem and enhances respiratory motor output. During this process,
the information of PCO2 levels received by these
chemoreceptor cells may first be conveyed to the change in their
membrane excitability and then passed to the respiratory neuronal
networks through chemical or electrical synaptic transmissions (9, 10).
Hence, the change in membrane excitability constitutes an important
step in the CO2 sensing.
The alteration in membrane excitability with hypercapnia is known to be
mediated by specific ion channels. CO2 has been shown to
enhance the firing activity of locus ceruleus neurons by suppressing a
proton- and polyamine-sensitive inward rectifying K+
current (11). Our previous studies have shown that CO2
induces a depolarization in Lymnaea snail neurons by
inhibiting a K+ channel with modest inward rectification
(12). Recently, we have further demonstrated that CO2
inhibits specific inward rectifier K+ channels including
Kir2.3 (13). Because the Kir2.3 is expressed in the central nervous
system and contributes to the maintenance of membrane excitability
(14), detailed studies of the modulation of Kir2.3 during hypercapnia
may bring about an understanding of the molecular mechanisms for
CO2 sensing.
Hypercapnia can modulate channel activity directly by acting on channel
protein and indirectly by recruiting second messengers and other
intermediate molecules. Our previous studies showed that the Kir2.3
inhibition during hypercapnia is largely mediated by decreases in the
intra- and extracellular pH (pHi and pHo) and
demonstrated that this channel has two pH sensors on either side of the
plasma membranes (13). Whereas the pHo sensor has been well
studied (15), the pHi sensor, which contributes to more
than 80% of channel inhibition, works at near physiological pH levels,
and seemingly plays a more important role during hypercapnia, is still
not understood. Therefore, we designed these experiments using a
molecular genetic approach combined with patch clamp experiments to
have an intervention of the structural-functional relationship of the
Kir2.3 in CO2/pHi sensing.
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MATERIALS AND METHODS |
Frogs (Xenopus laevis) were anesthetized by bathing
in 0.3% 3-aminobenzoic acid ethyl ester. A few lobes of the ovaries
were removed after a small abdominal incision (~5 mm).
Xenopus oocytes were treated with 2 mg/ml collagenase (Type
I, Sigma) in OR2 solution (82 mM NaCl, 2 mM
KCl, 1 mM MgCl2, and 5 mM HEPES)
for 90 min at room temperature. After washing, the oocytes were then
incubated at 18 °C in ND-96 solution containing (in mM)
96 NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 HEPES,
and 2.5 sodium pyruvate with 100 mg/liter Geneticin added.
A vector for eukaryotic expression (pcDNA3.1, Invitrogen, Carlsbad,
CA) was used to express Kir2.1 and Kir2.3 channels. Chimerical constructs between Kir2.1 and Kir2.3 were prepared by an overlap extension at the junction of the interested domains using polymerase chain reaction (Pfu or Taq DNA polymerases,
Stratagene, La Jolla, CA). The resulting polymerase chain reaction
products were subcloned into pcDNA 3.1. Site-specific mutations
were made using a site-directed mutagenesis kit (Quickchange,
Stratagene). Correct mutations in both the overlapping polymerase chain
reaction and site-directed mutagenesis were confirmed with DNA sequencing.
Whole cell currents were studied on the oocytes 2-5 days after
injection. Two-electrode voltage clamp was performed using an amplifier
(Geneclamp 500, Axon Instruments, Foster City, CA) at room temperature
(22-24 °C). The extracellular solution contained 90 mM
KCl, 3 mM MgCl2, and 5 mM HEPES (pH
7.4). Cells were impaled using electrodes filled with 3 M
KCl. The current electrodes had a resistance of 0.3-0.6 megaohms.
Current records were low-pass filtered (Bessel, 4-pole filter, 3 dB at
5 kHz), digitized at 5 kHz (12-bit resolution), and acquired using
pClamp 6.0.3 (Axon Instruments) (16, 17). Oocytes were accepted for
further experiments only if they expressed large inward rectifying
currents (>3 µA), and their leak currents measured as the difference
before and after a leak subtraction were less than 10% of the peak currents.
Xenopus oocytes were placed in a semi-closed recording
chamber (BSC-HT, Medical System, Greenvale, NY) in which oocytes were placed on a supporting nylon mesh, so that the perfusion solution bathed both the top and bottom surface of the oocytes. CO2
(15% balanced with air) exposures were the same as described
previously (13). A high concentration of KHCO3 was used for
intracellular acidification (18, 19). In these experiments, bicarbonate at concentrations of 20, 50, and 90 mM was applied to the
bath solutions without changing the K+ concentration.
Intracellular pH levels were measured when these perfusates were
administrated to oocytes.
Patch clamp experiments were performed at room temperature (about
25 °C) as described previously (16, 17). In brief, fire-polished patch pipettes (0.5-2 megaohms) were made from 1.2-mm borosilicate capillary glass (Sutter P-94/PC puller). Macroscopic currents were
recorded from Giant inside-out patches. Current records were low-pass
filtered (2,000 Hz, Bessel, 4-pole filter,
3 dB), digitized (10 kHz,
12-bit resolution), and stored on computer disk for later analysis
(pClamp 6, Axon Instruments). Junction potentials between the bath and
pipette solutions were appropriately nulled before seal formation.
For macroscopic current recordings, the oocyte vitelline membranes were
mechanically removed after exposing hypertonic solution (400 mosmol)
for 15 min. The stripped oocytes were placed in a Petri dish containing
regular bath solution (see below). Recordings were performed using
solutions containing equal concentrations of K+ applied to
the bath and recording pipettes. The bath and recording pipette
solution contained (in mM): 40 KCl, 75 potassium gluconate, 5 potassium fluoride, 0.1 sodium vanadate, 10 potassium pyrophosphate, 1 EGTA, 0.2 ADP, 10 PIPES,1
10 glucose, and 0.1 spermine (FVPP solution, pH 7.4). This bath solution was chosen after several others had been tested regarding channel run-down in excised patches. In a control experiment, we found
that macroscopic currents recorded from giant inside-out patches were
very well maintained, showing less than 10% reduction over a 20-min
period of recordings made in such a bath solution.
A parallel perfusion system was used to administer agents to patches or
cells at a rate of ~1 ml/min with no dead space (16, 17). Low pH
exposures were carried out using the same bath solutions that had been
titrated to various pH levels as required by experimental protocols.
PIPES buffer was used because of its buffering range from pH 5.8 to
7.4.
Data are presented as means ± S.E. (n
4).
Differences in means were tested with the Student's t test
and were accepted as significant if p
0.05.
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RESULTS |
CO2 and Low pHi Inhibit Kir2.3
Currents--
Kir2.3 currents were studied by expressing these
K+ channels in Xenopus oocytes. In the voltage
clamp mode, whole cell Kir2.3 currents showed a strong inward
rectification and were highly sensitive to extracellular
Ba2+ (IC50 = 11 ± 2 µM,
n = 4). Exposure of the oocytes to 15% CO2 produced a marked inhibition of the whole cell Kir2.3 currents by
80 ± 2% (n = 7) (Fig.
1A). This effect was
reversible and dependent on concentrations of CO2 (5, 10, and 15%, data not shown). Selective intracellular acidification
without changing the extracellular pH also inhibited Kir2.3 to an
extent comparable with that of CO2 when changes in pH
levels were considered. The measured intracellular pH level during 15%
CO2 exposure was 6.58 ± 0.13 (n = 6)
using ion-selective microelectrodes (20). Selective intracellular acidification to this pH level inhibited Kir2.3 by 74 ± 4%
(n = 5).

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Fig. 1.
Inhibition of Kir2.3 currents by
hypercapnia. Whole cell currents were recorded from a
Xenopus oocyte 3 days after cDNA injection using a
two-electrode voltage clamp. Currents were recorded before, during, and
after 15% CO2 exposure. A, Kir2.3 currents were
reversibly inhibited by CO2. The currents decreased by 80%
of their base-line values during a 5-min CO2 exposure and
returned to the control level 5 min after the exposure ended.
B, a similar exposure however, had no effect on Kir2.1
currents.
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The inhibition of Kir2.3 by CO2 or intracellular
acidification can be a direct effect of protons on the channel protein
or an indirect effect through changes in concentrations of
intracellular second messengers, protein kinases, phosphatases, and
other cytosol-soluble factors. To delineate the mechanisms underlying
the channel modulation, we performed experiments using cell-free
excised patches. In the inside-out patch configuration, Kir2.3 currents
were strongly inhibited when the cytosolic surface of the plasma
membrane was exposed to low pH solutions. This inhibition was fast,
reversible, and concentration-dependent (Fig.
2). Another member of the Kir2 family,
Kir2.1, however did not respond to either high CO2 (Fig. 1B) or pHi 6.6 (Fig.
3), suggesting that this effect was
rather specific. Because cytosol-soluble factors were vastly diluted or
washed out under our experimental condition, the inhibition of
Kir2.3 channels is unlikely to be mediated by second messengers and
other cytosol-soluble factors. Moreover, because our intracellular solutions contained chemicals that were unfavorable to protein dephosphorylation (see "Materials and Methods"), the modulation of
Kir2.3 channel activity by pH levels may not be related to the fast
turn-over of protein phosphorylation and dephosphorylation.

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Fig. 2.
Inhibition of Kir2.3 by lowering
pHi. A, Kir2.3 currents were recorded from
an inside-out patch with symmetric K+ concentration on both
sides of the oocyte membrane. Pulse command potentials from 160 mV to
140 mV were applied to the patch with an increment of 20 mV at a
holding potential of 0 mV. Exposure of the internal membrane to
solutions with various pH levels produced a graded inhibition of inward
rectifying currents. B, the amplitude of these currents can
be expressed as a function of intracellular pH (pHi) using
the Hill equation: y = 1/(1 + (pK/x)h), where y is the
current amplitude, pK is the midpoint pH value for channel
inhibition, x is pHi, and h is the
Hill coefficient. The pK and h values here are pH
6.76 and 3.5, respectively.
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Fig. 3.
The crucial role of the N-terminal region in
pH sensing. A, because Kir2.3 (HIR) responds to
CO2 and low pHi but Kir2.1 (IRK) does not,
chimeras were constructed between these two channels to identify the
intracellular pH-sensing domain. In IN-HIR, the Kir2.3 N-terminal
region (from the N terminus to the beginning of M1) was substituted
with the counterpart of Kir2.1. This IN-HIR recombinant lost its
sensitivity to intracellular acidification. When the Kir2.1 N-terminal
domain was replaced with the corresponding sequence in Kir2.3, the
mutant channel became pHi-sensitive. Note that
pHi sensitivity of these chimeras was studied by
two-electrode clamp experiments in different oocytes. B,
concentration-dependent inhibition of K+
currents in inside-out patches. Whereas Kir2.3 currents showed strong
pHi sensitivity with pK 6.77, Kir2.1 and IN-HIR
had almost no response to a pHi change from 7.4 to 5.8. Although its pHi sensitivity decreased (pK
6.45), HN-IRK responded to pHi changes in a manner more
like Kir2.3 than Kir2.1. Data are presented as means ± S.E.
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The CO2/pH-sensitive Structure Is Located in the
N-terminal Region of Kir2.3--
Our results in excised patches
support an idea that the proton-sensitive mechanism for channel gating
is located on the Kir2.3 channel protein. To further test this idea, we
chose to use molecular genetic approaches to determine the molecular
basis of Kir2.3 channel modulation by intracellular protons. According
to the generally accepted membrane-spanning topology of inward
rectifier K+ channels, the Kir2.3 channel should have its N
and C termini in the cytosol. In addition, part of the pore (P or H5)
sequence should be accessible from the intracellular side. Based on
these characteristics of Kir channels, we constructed chimeras in which each of these three sequences was replaced by its counterpart in
Kir2.1. CO2 and pH sensitivities of these recombinant
HIR-IRK channels were then studied in whole cell voltage clamp and
excised patches. Substitution of the N terminus in Kir2.3 with that in Kir2.1 (IN-HIR) completely eliminated the channel response to low
pHi (Figs. 3A and 5). A chimerical Kir2.1
channel carrying an N-terminal sequence of Kir2.3 (HN-IRK) became
pH-sensitive, although the degree of the pH sensitivity was smaller
than that of wild-type Kir2.3 (Figs. 3A and 5). Switches of
the C terminus (IRK-HC, Fig. 5) or the H5 region from Kir2.3 to Kir2.1
(IN-HIR, Figs. 3A and 5), however, failed to produce any
CO2/pH sensitivity in these mutant channels. Thus, these
results indicate that the CO2/pHi-sensitive
mechanism is located on the N terminus rather than the H5 and C
terminus of Kir2.3.
Titratable Amino Acid Residues in the N Terminus Are Not
Involved--
It is known that a large number of protein molecules can
be modulated by pH levels. Binding of protons to certain titratable amino acids can cause changes in the electrical charges of these residues and thus alter the protein conformation and channel activity. If this is the case in Kir2.3, there should be amino acid residues on
the channel protein acting as receptive sites for protons. To test this
hypothesis, we examined titratable amino acids in the N terminus of
Kir2.3, especially histidines in which the side chain has a
pK value of 6.04. Using site-directed mutagenesis, these
histidines were changed to aspartates in which positive charges at
these positions were converted to negative charges during hypercapnia.
A simultaneous replacement of all three histidines (H2D, H4D, H11D) had
no effect on channel expression and sensitivity to CO2 and
pHi (Fig. 4, A and
B). Neither did replacements of individual ones, suggesting
that these histidines do not constitute the pH sensor in Kir2.3. To
further strengthen this idea, we deleted all of these histidines plus
the other nine residues at the beginning of the N terminus and found
that the CO2/pHi sensitivity was unaffected (see below). In addition to these histidines, several residues that
exist only in Kir2.3 and are potentially titratable at nonphysiological (extremely high or low) pH levels were studied. After these residues were switched to those in Kir2.1, including R6G, R14T, K16Q, and R17Q,
we found that the same pH sensitivity was retained in all of these
mutant Kir2.3 channels. Thus, none of these residues appears to be the
reception or initiation site for proton sensing.

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Fig. 4.
Titratable histidines in the N terminus in
CO2/pH sensing. A, simultaneous mutations
of all three histidines to negatively charged aspartic acids or
replacements of individual ones had no effect on the expression as well
as the sensitivity of these mutant channels to CO2,
indicating that these histidine residues in the Kir2.3 N-terminal
region are not the initiation site of pH sensing. B,
comparison map of amino acid sequences of the N termini between Kir2.3
and Kir2.1.
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CO2/pH Sensing Is Related to a Short Sequence Near the
M1 Domain--
To understand the pH-dependent mechanisms
for Kir2.3 gating, we divided the N-terminal region into three segments
and reconstructed several N-terminal chimeras using Kir2.3 and Kir2.1
sequences. Most of these chimeras were expressed in oocytes as
functional channels, although the current amplitude in some of them was
reduced. To eliminate the extracellular pH sensor, the sequence from M1 through M2 in Kir2.3 was replaced with a corresponding one in Kir2.1.
This mutant channel carrying N and C termini from Kir2.3 (HIH) showed
CO2 and pHi sensitivities almost identical to
the wild-type Kir2.3 (Fig. 6B). Thus, several chimeras were
constructed based on the HIH.
The first 12 amino acids at the N terminus of Kir2.3 (N1-12IH) were
not involved in the CO2 sensitivity. A truncation of these residues did not have any significant effect on the channel expression, base-line currents, and sensitivity to CO2 (70 ± 3%,
n = 4, p > 0.05) (Fig.
5). Although a deletion of the next 12 residues did not produce functional channels, chimera with the first 19 amino acids replaced by those in Kir2.1 (N1-19IH) showed a
CO2 sensitivity very close to that of the wild-type Kir2.3
(61 ± 4%, n = 6). The middle part of the
N-terminal region includes 20 residues with 9 of them different between
Kir2.3 and Kir2.1. Substitution of this sequence in Kir2.3 (N25-44IH)
moderately reduced the level of CO2 sensitivity (54 ± 6%, n = 4). Unlike the two N-terminal regions
mentioned above, an amino acid sequence near the M1 membrane-spanning domain containing about 10 residues was critical for CO2
sensitivity. Replacement of this short sequence alone (N51-60IH) was
sufficient to eliminate 80% of the CO2 sensitivity of the
mutant Kir2.3 (13 ± 2%, n = 4). When this short
sequence was constructed to Kir2.1 (N77-86IRK), we found that the
mutant channel gained a substantial sensitivity to CO2
(18 ± 5%, n = 4) and pHi (Figs. 5
and 7). Extension of this sequence to 41 residues prior to the M1
domain of Kir2.3 (N46-86IRK) gave the mutant Kir2.1 a CO2
sensitivity (36 ± 3%, n = 4) nearly the same as
the HN-IRK (40 ± 4%, n = 6, p > 0.05) (Fig. 5). Therefore, this 10-residue motif is critical for the sensing of CO2/pH in Kir2.3, although another 30 amino
acids (total 41 residues) around this motif are also needed to achieve
the full effect of the entire N terminus.

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Fig. 5.
Localization of a CO2-sensing
motif in the N terminus of Kir2.3. Even though the wild-type
Kir2.3 (HIR) was strongly inhibited, Kir2.1 (IRK) was not sensitive to
CO2. When its N terminus was replaced with that in IRK, the
mutant Kir2.3, IN-HIR, lost its pHi sensitivity (note that
the abbreviated names of chimeras are explained in the lower
panel). Transferring the N terminus from HIR to IRK made the
HN-IRK CO2 sensitive, whereas inducing the HIR C terminus
to IRK (IRK-HC) was ineffective. To further understand the
CO2-sensing motif, the N-terminal regions of Kir2.3 and
Kir2.1 were divided into three segments at two conserved areas. A
deletion of the first 12 residues in the N terminus did not
significantly affect the CO2 sensitivity of the N1-12IH.
Substitution of amino acids 1-19 in Kir2.3 with those in IRK
(N1-19IH) had only a modest effect on its CO2 sensitivity.
When the middle part of the N-terminal region (amino acids 25-44) was
replaced, chimera N25-44IH was still fairly CO2-sensitive,
although its sensitivity was slightly reduced. In contrast to these
three chimeras, a substitution of 10 amino acids near the M1
membrane-spanning sequence of Kir2.3 (N51-60IH) drastically reduced
its CO2 sensitivity. Construction of residues 20-60 of
Kir2.3 to Kir2.1 gave this mutant IRK (N46-86IRK) a CO2
sensitivity as great as that of the HN-IRK. This was seen even with the
introduction of 10 residues to IRK (N77-86 IRK). Data are presented as
means ± S.E.
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CO2/pH Sensing in Kir2.3 Depends on a Few Critical
Residues--
There is a high sequence homology in these 10 residues
between Kir2.3 and Kir2.1, in which Kir2.3 differs from Kir2.1 in only three residues (Thr53, Tyr57, and
Met60). Mutation of all of them into residues in Kir2.1
(T53I, Y57W, and M60V) greatly reduced the pH sensitivity by 0.6 pH
units and generally disabled this mutant channel in terms of
CO2 sensing (Fig. 6).
Replacements of two of these residues (T53I, Y57W) had the same effect
(Fig. 6B). Even with a single mutation (T53I), the
CO2 and pH sensitivities were severely diminished. Mutation of either tyrosine or methionine alone (Y57W or M60V) also caused a
reduction in the CO2 and pH sensitivities, though their
effects were much less than that of the T53I mutation. When these three residues were constructed to Kir2.1 (N77-86IRK), the effect was dramatic. The mutant Kir2.1 started to be inhibited at pHi
6.6 with a pK value of 6.0 and h (Hill
coefficient) of 2.3 (n = 5). Its pH sensitivity became
more like that of HN-IRK, a chimera carrying the full N terminus from
Kir2.3, than of the wild-type Kir2.1 (Fig.
7). Mutation of isoleucine 79 alone
(I79T-IRK) had an effect on converting the pH sensitivity of the Kir2.1
as effectively as the N77-86IRK mutation (pK 5.93, h 2.3, n = 5) (Fig. 7), demonstrating the
critical role of the threonine 53 of Kir2.3 in CO2 and
pHi sensing.

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Fig. 6.
Identification of critical amino acid
residues in Kir2.3 for CO2/pH sensing. A,
further studies of the amino acid sequence of the short motif near the
M1 domain show that there are only three residues that differ between
Kir2.3 and Kir2.1. Replacement of all three amino acids with those in
Kir2.1 almost totally disabled the mutant channel (M60V/Y57W/T53I) in
CO2 sensing. Individual mutation of three amino acids
showed that Thr53 appeared more critical. B,
concentration-dependent inhibition of K+ currents
in inside-out patches. The mutant M60V/Y57W/T53I reduced the
pK level by about 0.6 pH units from pK 6.77 to
6.18. Similar results were seen with the Y57W/T53I mutation. Even a
single mutation at Thr53 decreased proton sensitivity to pH
6.42. Data are presented as means ± S.E.
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Fig. 7.
Creation of pH sensitivity in Kir2.1 by
introducing three residues from Kir2.3. There are three amino acid
residues that differ between Kir2.3 and Kir2.1 in the 10-residue motif
near the M1 domain. After mutations of these residues in Kir2.1 to
those in Kir2.3 (N77-86IRK), the effect was dramatic. The mutant
Kir2.1 started to be inhibited at pHi 6.6 with
pK 6.0 and h 2.3 (n = 5). Its pH
sensitivity became more like that of HN-IRK, a chimera carrying the
entire N terminus of Kir2.3, than of the wild-type Kir2.1. A single
mutation of I79T (I79T-IRK) had almost the same effect on converting
the pH sensitivity of the Kir2.1 as HN-IRK (pK 5.93, h 2.3, n = 5). Data are presented as
means ± S.E.
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Because threonine 53 in Kir2.3 is a protein kinase C phosphorylation
site (21), channel sensitivity to CO2 was studied in the
presence of high concentration H7
(1-[5-isoquinolinesulfonyl]-2-methylpiperazine dihydrochloride, a
blocker of protein kinases C and A; Sigma). After bathing the oocytes
with 30 µM H7 for 20 min, exposure of the oocytes to 15%
CO2 produced the same degree of inhibition of Kir2.3
currents (74 ± 3% control versus 72 ± 1% with
H7, p > 0.05, paired Student's t test,
n = 4), a result consistent with our studies of
wild-type Kir2.3 using excised patches (see above).
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DISCUSSION |
In our current studies, we have demonstrated that a molecular
motif that is located on the N terminus consisting of about 10 amino
acids is responsible for gating the Kir2.3 channel by high
CO2 and low intracellular pH.
Direct Effect of Protons on Kir2.3 Currents--
Like
Ca2+, nucleotides, and other second messengers, proton is
an important regulator of cellular functions. A number of ion channels
have been known to be modulated by protons; these include large
conductance Ca2+-activated K+ channels, h
currents, gap junctions, and
-aminobutyric acid and
N-monomethyl arginine receptor channels (22-27). Changes in the activity of these ion channels can take place under several physiological and pathophysiological conditions when intra- and extracellular pH levels are low. One of these conditions is high CO2 or hypercapnia. CO2 sensing by the central
chemoreceptors is critical in feedback mechanisms controlling
CO2 levels in mammals (5, 7, 8, 28), which may involve the
modulation of K+ channel activity. Inhibition of inward
rectifier K+ channels can produce depolarization and an
increase in membrane excitability (2, 3). Although there are a number
of ways to inhibit these K+ channels during hypercapnia,
our current studies indicate that the mechanisms for the inhibition of
Kir2.3 are related to the specific structure of the channel protein. We
have found that fast and reversible inhibition of Kir2.3 activity by
high CO2 or low pH can be seen in cell-free excised
patches, indicating that cytosolic soluble factors such as second
messengers, Mg2+, and polyamines are not players. Also,
CO2/pH sensing is seen in Kir2.3 but not in Kir2.1, which
has a nearly 80% homology to Kir2.3 in amino acid sequence, suggesting
that a specific sequence in Kir2.3 protein underlies this sensitivity.
Moreover, our data do not support the idea that the inhibition of
Kir2.3 during hypercapnia or intracellular acidification is mediated by
phosphorylation of channel proteins. There are chemical compounds in
our intracellular solution to prevent channel rundown that are
inhibitors of phosphatases and phosphodiesterases. This observation
plus the lack of ATP and Mg2+ in our intracellular solution
strongly suggests that the turnover of protein phosphorylation and
dephosphorylation in gating Kir2.3 may not occur under such an
experimental condition. Also our results have shown that the inhibition
of Kir2.3 activity by CO2 is not affected by the blockade
of protein kinases C and A. Therefore, specific structures on Kir2.3
channel protein seem to be responsible for CO2 and pH sensing.
Mechanisms for Gating Kir2.3 Activity by Protons--
If protons
have a direct effect on channel functions, they may bind to the channel
protein through certain titratable amino acid residues and alter
conformation and activity of the channel. Also, protons can affect
channel activity if they are involved in the binding of the channel
protein with another protein. In any case, the channel protein should
have specific sequences for these interactions. To locate these sites,
we have studied the pH sensitivity of Kir2.3 channel by selectively
replacing amino acid residues and peptide sequences in this channel
with those in Kir2.1, which is not sensitive to a pH change in
physiological ranges. These molecular genetic interventions have led us
to look closely at the structure-functional relationship in the Kir2.3 channel in CO2/pH sensing.
Consistent with the idea that the sensing mechanism is related to the
channel protein, we have found that CO2/pH sensitivity requires the presence of the N-terminal region of Kir2.3. To find proton-binding sites in the N terminus, we have examined all of the
histidine residues by substituting them with aspartate, a negatively
charged amino acid, or by deleting all of them with another nine
residues at the very end of the N terminus. However, none of these
mutants has shown a change in its sensitivity to CO2 and
pH, indicating that these histidines are not the pH sensor. Interestingly, the extracellular pH sensor does not involve titratable histidine residues either. Although an extracellular domain including a
histidine is crucial in pHo sensing, it does not seem to
have a proton-binding site. Indeed, selective substitution of the
histidine residue in this cluster has no effect on the pHo
sensitivity of Kir2.3 (15). Thus, Coulter et al. (15)
believe that an unidentified cysteine residue may play a role.
Using these chimerical and site-directed mutant Kir channels, we have
identified a consensus sequence in the N-terminal region of Kir2.3 that
is crucial for Kir2.3 gating by CO2 and pHi.
This motif is located in a conserved region near the M1
membrane-spanning domain. Although there are only three residues that
are different between Kir2.3 and Kir2.1, this motif may involve some
neighboring residues. Mutation of any of these residues causes a marked
reduction in CO2 sensitivity. Of these three residues, the
Thr53 is the key. Mutation of this residue leads to an
almost complete loss of channel sensitivity to pHi and
CO2, whereas creation of this residue in Kir2.1 makes the
mutant channel pH-sensitive. Interestingly, threonine is not a
titratable amino acid, and thereby pH change should not affect either
its charges or its polarity. Thus, protons may not act directly on this
residue to change channel conformation and activity. Then how can this
residue be involved in CO2/pH sensing and the modulation of
channel activity? We believe that there are at least three potential
ways by which this threonine residue can participate in the modulation
of channel activity during hypercapnia. 1) This residue may be engaged
in the maintenance of the pK value of another amino acid
residue(s) titratable at high or low pH, as suggested previously in
Kir1.1 (20, 29). 2) This residue may be involved in a motif that is
required for protein-protein interactions in CO2/pH
sensing. 3) Phosphorylation at this residue may occur during
CO2/pHi exposure.
Is it possible that protons bind to ionizable amino acids other than
histidine residues in the N terminus? To answer this question, we have
changed nonconserved lysine and arginine residues in the Kir2.3 N
terminus into corresponding ones in the Kir2.1 (R6G, R14T, K16Q, and
R17Q). Our data reveal that mutations of these residues do not affect
CO2/pHi sensitivity. Also, we have systematically divided the N termini of Kir2.3 and Kir2.1 into several
segments and rejoined them alternatively to construct chimerical N
termini. These extensive interventions would allow us to interrupt the
pH sensitivity of Kir2.3 if there were such a proton-binding site,
whether on positively or negatively charged residues. Although some of
these chimeras, such as the N51-60IH, are shown to be more critical
than others in CO2/pH sensing, residues potentially
titratable in physiological pH levels do not exist in the sequence of
Kir2.3. Interestingly, there are three residues (Asp52,
Arg54, and Arg56) that are titratable at
extremely high or low pH in the short motif. We have tried to mutate
some of them into neutral and polar residues (asparagine) in the Kir1.1
channel. Because they are highly conserved in all Kir channels, these
mutations did not produce any functional channels (30). Therefore,
whether some of them are proton sensors with pK levels that
can be affected by the Thr53 is still unknown.
Because mutations of Thr53 and Tyr57 into
nonpolar isoleucine and tryptophan dramatically reduced the
CO2 sensitivity of Kir2.3, the polarity of positions 53 and
57 seems important in pH sensing. The periodicity of the three crucial
residues (Thr53, Tyr57, and Met60)
may suggest a helical structure in the region, but our search using the
Chou-Fasman and Robson-Granier analyses did not show any helical
structure in this short motif.
Our results indicate amino acid sequences other than this motif are
also needed to fulfill a complete CO2 and pHi
sensitivity. In addition to these 10 residues, we have observed that a
sequence composed of a total 41 amino acids can produce an effect of
CO2/pH as effectively as the entire N terminus. Also, the
presence of the C terminus enhances the CO2/pH sensitivity
of the mutant Kir channels, although it has no effect on
CO2/pH sensing by itself. A simple explanation of these
data would be that the C terminus interacts with the N terminus in
gating the channel, as suggested recently in Kir1.1 by Schulte et
al. (31). Our data, however, do not support the idea that
CO2/pH sensing in Kir2.3 is a result of protein
phosphorylation, as has been described above. Therefore, it is possible
that CO2/pH sensing in Kir2.3 is mediated by an interaction
of amino acid residues on the Kir2.3, which is affected by protons and
controls the channel activity. Clearly, further understanding of the
molecular mechanisms for Kir2.3 modulation by protons may provide
information of channel gating under a variety of pathophysiological
conditions, and the demonstration of these critical residues in
CO2/pH sensing in our current studies constitutes an
important step toward this goal.
Physiological Significance of Kir2.3 Sensitivity to Intracellular
Acidification--
We believe that the molecular basis of pH sensing
in Kir2.3 may underlie the CO2 chemoreception in the
central nervous system. Studies on central chemoreceptors have shown
that a large number of these cells depolarize and their membrane
excitability increases during hypercapnia. This change in membrane
excitability is not mediated by a change in synaptic transmission,
because the depolarization persists with the removal of extracellular
Ca2+ or exposure to tetrodotoxin (32). Although several
ionic mechanisms may be involved in the regulation of membrane
excitability during hypercapnia, K+ channels, especially
the inward rectifier K+ channels, seem to be the major
player (11, 12, 33). The inhibition of these inward rectifier
K+ channels during hypercapnia produces depolarization and
increases membrane excitability (13). With two pH sensors on Kir2.3,
cells can sense pH changes on either side of the plasma membranes, in their internal and external environment. Because the primary sensor is
a K+ channel, the changes in intra- and extracellular pH
can be readily coupled to membrane excitability in the nerve cells that
are endowed with this pH sensing mechanism and in the neuronal networks
to which these cells project. Because Kir2.3 is expressed in the central nervous system (14, 34), the determination of molecular mechanisms in proton sensing may be applicable to the search for unidentified homologues of the Kir2.3 channel in brain stem neurons, which may eventually result in the identification of the
CO2 central chemoreceptors.