K+ Binding Sites and Interactions between
Permeating K+ Ions at the External Pore Mouth of an
Inward Rectifier K+ Channel (Kir2.1)*
Ru-Chi
Shieh
§,
Jui-Chu
Chang
, and
Chung-Chin
Kuo¶
From the
Institute of Biomedical Sciences, Academia
Sinica, Taipei 11529 and the ¶ Department of Physiology,
National Taiwan University College of Medicine and Department of
Neurology, National Taiwan University Hospital,
Taipei 100, Taiwan, Republic of China
 |
ABSTRACT |
The arginine at position 148 is highly conserved
in the inward rectifier K+ channel family. Increases
of external pH decrease the single-channel conductance in mutant R148H
of the Kir2.1 channel (arginine is mutated into histidine) but not in
the wild type channel. Moreover, in 100 mM external
K+, varying external pH induced biphasic changes of open
channel noise, which peaks at around pH 7.4 in the R148H mutant but not in the wild type channel. The maximum single-channel conductances are
higher in the wild type channel and R148H mutant at pH 6.0 than those
in the R148H mutant at pH 7.4. However, the maximal conductance is
achieved with much lower external [K+] for the latter.
Interestingly, the single-channel conductances and open channel noise
of the wild type channel at pH 6.0 and the R148H mutant at pH 6.0 and
7.4 become the same in [K+] = 10 mM. These
results indicate that the residue at position 148 is accessible to the
external H+ and probably is involved in the formation of
two K+ binding sites in the external pore mouth. Effective
repulsion between permeating K+ ions in this area requires
a positive charge at position 148, and such
K+-K+ interaction is the essential mechanism
underlying high K+ conduction rate through the Kir2.1
channel pore.
 |
INTRODUCTION |
Inward rectifier K+ channels are important in
maintaining stable resting membrane potentials and controlling
excitability of many cells. The physiological function of these
channels is closely related to their unique inward rectification
mechanisms, which allow much larger inward current than outward current
through the channels. Another important "asymmetrical" feature of
the inward rectifier K+ channels is that external but not
internal K+ seems to interact with and thereby activate the
channel (1). The mechanisms underlying inward rectification have been
ascribed to voltage (Vm)-dependent
blockade by internal cations such as Mg2+ (2, 3) and
polyamines (4, 5) and to a pHi-dependent intrinsic
gate (6). On the other hand, the ion permeating process itself, namely
interactions between K+ ions and between the permeating
K+ ion and the inward rectifier K+ channel, is
less characterized.
The arginine at position 148 is highly conserved in the inward
rectifier K+ channel family and seems to play an important
role in the K+ ion permeation process. Kubo (7) showed that
mutation of the arginine at position 148 into tyrosine (R148Y) in the
cloned inward rectifier K+ channel (Kir2.1) (8) resulted in
a reduction of the [K+]o-dependent
activation of inward currents and a negative shift of the
conductance-voltage relationship. Sabirov et al. (9)
reported that although mutation of the arginine at position 148 into
histidine (R148H) resulted in no channel activity, coexpression of the
mutant with the wild type
(WT)1 cRNA demonstrated
populations of channels with reduced single-channel conductances (
).
Moreover, the single-channel conductance is dramatically increased in a
recently cloned inward rectifier K+ channel (Kir7.1) when
the neutral amino acid methionine at position 125 (the equivalent
position of the arginine at site 148 in Kir2.1) was mutated into a
positively charged amino acid (10). All these studies suggest that the
positive charge at position 148 (148
) in the inward rectifier
K+ channel family is important in determining
K+ ion conduction and channel activation, yet detailed
mechanisms underlying the effects of arginine at position 148 with a
positive charge on K+ ion permeation through the Kir2.1
channel pore remain unexplored.
With successful expression of the Kir2.1 mutant R148H in
Xenopus oocytes, we studied the effect of pHo and
[K+]o on the single-channel conductance and the
open channel "noise" in WT channel and R148H mutant. We found that
the side chain of the amino acid at position 148 is accessible to the
external H+. Also, the amino acid at position 148 probably
is involved in the formation of a set of two K+ binding
sites in the external pore mouth. Effective repulsion between
permeating K+ ions in this area requires a positive charge
at position 148, and such K+-K+ interaction may
be an important mechanism underlying high K+ conduction
rate through the Kir2.1 channel pore.
 |
EXPERIMENTAL PROCEDURES |
Molecular Biology and Preparation of Xenopus Oocytes--
Mouse
macrophage Kir2.1 in pCDNAI/Amp (generously provided by Dr. Lily
Jan (University of California at San Diego)) digested with
HindIII and StuI was sublconed into the
HindIII-SmaI sites in pALTER/Tet (Promega,
Madison, WI). Site-directed mutation was then generated using the
Altered Sites II in vitro mutagenesis systems (Promega,
Madison, WI). The mutant DNA was sequenced with the ABI
PrismTM dRhodamine Terminator Cycle Sequencing Ready
Reaction Kit (PE Applied Biosystems, Foster City, CA) to confirm the
presence of histidine at position 148. The WT Kir2.1 subcloned in
pBSIISK(+) (a generous gift from Drs. Scott A. John and James N. Weiss)
and R148H DNAs were linearized with NotI and
ScaI, respectively, and approximately 1 µg of purified
linear DNA was used for in vitro T7 (for WT DNA) and Sp6
(for R148H DNA) transcription reactions (mMessage mMachine, Ambion,
Dallas, TX).
Xenopus oocytes were prepared as described previously (11).
In brief, Xenopus oocytes were isolated by partial
ovariectomy from frogs anesthetized with 0.1% aminobenzoic acid ethyl
ester. The day after isolation, Xenopus oocytes were
pressure-injected with either 10-100 pg of WT cRNA or 100-1000 pg of
R148H cRNA. Oocytes were maintained at 18 °C in Barth's solution
containing 88 mM NaCl, 1 mM KCl, 2.4 mM NaHCO3, 0.3 mM
Ca(N2O6), 0.41 mM CaCl2, 0.82 mM MgSO4, 15 mM HEPES, 20 µg/ml gentamicin, pH 7.6, and used 1-3 days
after RNA injection.
Single-channel Recordings--
Single-channel currents were
recorded at room temperature (22-24 °C) on cell-attached or
inside-out patches (12) using an Axopatch 200A amplifier (Axon
Instruments, Burlingame, CA). Glass electrodes were pulled using a
horizontal electrode puller (Sutter, Novato, CA) from borosilicate
glass tubing (WPI Inc., Saratoga, FL). The diameters of the pipettes
ranged from 1 to 5 µm. The command voltage pulses and data
acquisition functions were processed using a Pentium 100 computer, a
DigiData board, and pClamp6 software (Axon Instruments, Burlingame,
CA). Data were sampled at 5-10 kHz and filtered at 1-5 kHz.
For experiments performed in cell-attached patches, the pipette
solution contained 100 mM KCl + KOH, 2 mM EDTA.
For experiments carried out in inside-out patches (except for Fig. 7;
see Fig. 7 legend), the pipette and internal solutions were always kept symmetrical and both contained x mM KCl + KOH, 2 mM EDTA, where x = 10-1000. For
x = 10-100, 90 mM of
N-methyl-D-glucamine (NMG) was also added.
Because the single-channel conductances obtained with 100 mM [K+] and 100 mM
[K+] + 90 mM [NMG] are similar, NMG was not
added for solutions containing [K+] > 100 mM. Because it has been shown that channels formed from coexpression of the WT and R148H subunits are sensitive to external divalent cations (9), in this study we used pipette solutions containing no divalent cations. The patch electrode solution was pH-buffered by the following buffers: 5 mM MES for
pHo < 7.0; 5 mM HEPES for 7.0
pHo
8.0; 5 mM CHES for pHo > 8.0. The rundown of
channel activity was avoided by treating the inside-out patches with 25 µM of
L-
-phosphatidylinositol-4,5-bisphosphate (Sigma) (11,
13).
Data Analysis--
Single-channel current amplitude was measured
by manually placing a cursor at the center of the closed or the open
state levels and from amplitude histograms. To compare the degree of
open channel noise in the WT channel and R148H mutant under various
recording conditions, current variance (
) of single-channel
recordings was calculated for the open and closed states using the
Clampfit basic statistics:
|
(Eq. 1)
|
where
is the mean of the samples, and
N is the sample number.
was calculated
from a segment in the open or closed level current of the sample sweeps
(187-506 sample points). The open channel current variance from a
sweep was normalized to the closed level current variance of the same
sweep, and the normalized
(O)/
(C) values were then used to
compare the magnitude of open channel noise among different
experimental conditions. Averaged data are presented as the means ± S.E. Student's independent t test was used to assess
statistical significance.
 |
RESULTS |
Expression of the R148H Mutant in Xenopus Oocytes--
Fig.
1A shows a series of sample
current traces recorded at testing pulses in a cell-attached patch
containing one single R148H mutant channel at pHo 7.0. Fig.
1B shows the single-channel current amplitude histogram
demonstrating a single predominant conducting state. The averaged
single-channel current-voltage (i-Vm)
relationship is plotted in Fig. 1C. The
i-Vm relationship of the R148H mutant
remains inwardly rectifying, and no outward single-channel current is
recorded.

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Fig. 1.
The
i-Vm relationship of the
R148H mutant at pHo 7.0. A, single-channel
currents were recorded at different Vm in a
cell-attached patch exposed to a 100 mM
[K+]o solution at pHo 7.0. Throughout
this report, the solid lines indicate the closed state; the
downward arrows identify the beginning of the voltage step,
the upward arrows indicate the end of the voltage step (500 ms) from a holding potential of 0 mV, and capacitance transients were
subtracted off-line using null traces. B, an amplitude
histogram from single-channel currents recorded at
Vm = 140 mV. C, the
i-Vm curves were obtained by
measuring single-channel current at various voltages from 4-11
patches. Throughout this study, single-channel conductances were
obtained by linear fits to the i-Vm
data between Vm = 140 and 60 mV. The
solid line is the best fit to data with a slope of 20 pS.
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|
Monotonously Decreased Single-channel Conductance of the R148H
Mutant with Increasing pHo--
After successful expression of
the R148H mutant in Xenopus oocytes, we explored the role of
148
in K+ permeation. First we examined whether the
histidine residue is accessible to external H+ and thus the
charge at position 148 can be controlled by varying pHo. Fig.
2A shows the single-channel
currents recorded at Vm =
140 mV from a
holding potential of 0 mV at various pHo in the WT channel and
R148H mutant. Changes of pHo have little effect on the
single-channel current in the WT channel, yet the single-channel
current amplitude becomes smaller at higher (more alkaline) pHo
in the R148H mutant. At low pHo, the single-channel current in
the mutant is almost comparable with (although still slightly smaller
than) the current in the WT channel. As pHo is increased, the
single-channel current of the R148H mutant decreases in a
pHo-dependent manner. The averaged single-channel
currents recorded from several patches similar to that shown in Fig.
2A are plotted against membrane potentials and are shown in
Fig. 2B. The i-Vm
relationships are very similar at pHo 5.0 to 6.5. Further
increase of pHo decreases single-channel conductance in a
pHo-dependent manner. Fig. 2C shows that
the conductance of the WT channel is little affected by changes in
pHo, which is consistent with previous studies (9, 14). In
contrast, the single-channel conductance of the R148H mutant is
sensitive to pHo. The
-pHo relationship can be
described by a simple one-to-one binding curve with a
pKa of 7.3, and
varies between 26 and 7 pS.
These findings strongly support that the side chain of histidine at
position 148 can be protonated by external H+, and 148
seems to enhance K+ ion permeation through the Kir2.1
channel pore.

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Fig. 2.
The effect of pHo on the
i-Vm relationship of the
WT channel and R148H mutant. A, representative
single-channel current traces of the WT channel and R148H mutant were
recorded at Vm = 140 mV, and the indicated
pHo from cell-attached patches exposed to the 100 mM [K+]o solution. B, the
i-Vm curves were obtained at the
indicated pHo (n = 2-15). C, the
single-channel conductances of the WT channel and R148H mutant are
plotted against the corresponding pHo values. The
curve superimposed on the R148H mutant data is the best fit
to the data of the form: max × [ [H+]/([H+]+Ka)]+
min with max = 26 pS, min = 7 pS, and pKa = logKa = 7.3. The dotted lines indicate max and
min.
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pHi Has No Effect on the Single-channel Conductance of
R148H Mutant--
The effect of pHi on the R148H mutant was
also examined. Fig. 3 shows that changes
of pHi from 6.0 to 9.0 do not produce any change of the
single-channel conductance of the R148H mutant. Thus the histidine at
position 148 seems to be accessible only to external H+ but
not to internal H+.

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Fig. 3.
Effects of pHi on the
i-Vm relationships of the
R148H mutant. Single-channel currents were measured in inside-out
patches (n = 2-5) exposed to symmetrical 100 mM [K+] at pHo = 7.4 and the
indicated pHi.
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Lack of Vm Dependence of the pKa of R148H
Mutant--
To determine whether the amino acid at position 148 is
located within the electrical field, Vm
dependence of the effects of pHo on the single-channel current
of the R148H mutant was examined. At each Vm,
the averaged single-channel currents recorded at different pHo
were normalized to those at pHo 6.0 (Fig.
4A). The normalized
i-pHo relationships are similar at different
Vm. Fig. 4B shows that the
pKa values determined from the
i-pHo relationships in Fig. 4A remain
very similar at different Vm. The lack of
Vm dependence of pKa suggests
that the amino acid at position 148 is probably located outside the
electrical field.

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Fig. 4.
The Vm
dependence of the effect of pHo on single-channel
currents. A, the fractional currents (averaged
single-channel currents recorded at various pHo normalized to
that at pHo 6.0), i/ipH 6.0,
are plotted against pHo. The curves are the best
fits to data of the form:
[H+]/([H+]+Ka) + C where
C = 0.15 ( 140 mV), 0.08 ( 120 mV), 0.11 ( 100 mV),
0.09 ( 80 mV), or 0.08 ( 60 mV). B, pKa
values obtained from panel A are very similar at
Vm = 60 to 140 mV.
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Biphasic Changes of Open Channel Noise with pHo--
It is
interesting to note that other than single-channel conductances, the
open channel noises also vary with pHo in the R148H mutant. For
example, it is evident in Fig. 2A that the open channel
noise increases when pHo is increased from 5.0 to 7.4. However,
further increases of pHo result in a decrease in open channel
noise. To compare open channel noise we calculated single-channel
current variance at the open channel level and then normalized it to
that at the closed level in the same sweeps (for details see under
"Experimental Procedures"). Fig. 5
demonstrates the normalized variance of single-channel currents in the
WT channel (Fig. 5A) and in the R148H mutant (Fig. 5B) at different pHo. The open channel noise varies
with pHo in a biphasic manner and is peaked at pHo 7.4 in the R148H mutant, whereas the open channel noise remains small and
does not change with pHo in the WT channel. The biphasic change
of the open channel noise in the R148H mutant seems to indicate that
the unitary conductance of the mutant channel quickly fluctuates
between two levels. The two levels probably are correlated with
protonation and deprotonation of residue 148 of the channel, because
the fluctuation is smallest either at pHo 6.0 (where the
channel is mostly in the protonated and high conductance state) or at
pHo 9.0 (where the channel is mostly in the deprotonated and
low conductance state) yet is most prominent at pHo 7.4, the
pKa of the histidine residue at position 148 in the
R148H mutant (Fig. 4).

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Fig. 5.
Effects of pHo on open channel
noise. Single-channel currents were recorded (with a sampling rate
of 10 kHz and filtering rate of 1 kHz) in cell-attached patches exposed
to 100 mM [K+]o at different
pHo. Open channel noises were then quantified and expressed as
(O)/ (C) (see "Experimental Procedures") for the WT channel
(A) and R148H mutant (B). Data groups shown in
panel A are not statistically different from each other
(p > 0.05), whereas the particular data group
indicated by an asterisk in panel B is
statistically different from that of the R148H mutant at pHo
7.4 (p < 0.001; n = 18 in each data
group).
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Higher Affinity between Permeating K+ Ions and the
R148H Mutant Channel Pore at pHo 7.4 than at pHo
6.0--
We have demonstrated that histidine at position 148 of the
R148H mutant can be accessed by H+ from the external space
and that protonation of this site may have a significant effect on ion
permeation through the Kir2.1 channel. The biphasic effect of
pHo on the open channel noise furthermore argues for a direct
rather than an indirect or allosteric effect of 148
on
K+ permeation through the Kir2.1 channel (see
"Discussion" for details). A direct effect of a positive charge
(148
) on the permeating K+, a positively charged ion,
most likely would be an elevation in the free energy experienced by
K+. Because the single-channel conductance is higher in
channel with 148
, what is likely to be elevated is the minimum
rather than the maximum of the energy profile. Because an increase in energy minimum may be manifested as a decrease in binding affinity for
K+ in the pore, we investigated the effects of varying
[K+] on the single-channel conductance of the WT channel
and R148H mutant. The i-Vm
relationships were measured in symmetrical [K+] ranging
from 10 to 1000 mM in inside-out patches exposed to internal Mg2+- and polyamine-free solutions. Fig.
6 shows the
i-Vm curves for the WT channel at
pHo 6.0, R148H mutant at pHo 6.0, and R148H mutant at
pHo 7.4 (panels A-C, respectively). Fig.
6D plots the single-channel conductance against
[K+] (
-[K+] plot) for the WT channel at
pHo 6.0, R148H mutant at pHo 6.0, and R148H mutant at
pHo 7.4. For the WT channel at pHo 6.0, the
single-channel conductance steeply increases as [K+] is
increased and reaches a plateau of ~53 pS with a
Kd = 49 mM. The [K+]
dependence of the single-channel conductance of the R148H mutant at
pHo 6.0 is similar to the WT channel except that the single-channel conductance saturates at 35 pS with a
Kd = 30 mM. In contrast, the
single-channel conductance of the R148H mutant at pHo 7.4 saturates at ~14 pS. Because the single-channel conductance is
already ~10 pS in 10 mM [K+], it is
plausible that the Kd for K+ binding in
the R148H mutant is smaller than 10 mM (an accurate estimate of Kd is difficult in this condition
because it is hard to accurately measure the single-channel conductance with [K+] < 10 mM). Thus the affinity
between K+ and the R148H mutant channel pore seems to be
higher at pHo 7.4 than at pHo 6.0. This is consistent
with the foregoing prediction that 148
elevates the energy minimum
experienced by the permeating K+ in the pore.

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Fig. 6.
Effects of [K+] on the
single-channel conductances of the WT channel and R148H channel.
A-C, i-Vm relationships
for the WT channel at pHo 6.0, R148H mutant at pHo 6.0 and R148H mutant at pHo 7.4, respectively. Single-channel
currents were recorded from inside-out patches exposed to the indicated
[K+] (n = 2-6). D,
single-channel conductances are shown as a function of
[K+]. Solid lines are the best fits to data in
the form: = max/(1+Kd/[K+]) with
Kd = 49 mM and max = 53 pS for the WT channel at pHo 6.0, Kd = 30 mM, and max = 35 pS for the R148H mutant at
pHo 6.0 and Kd = 4 mM and
max = 14 pS. E, effects of
[K+]o on open channel noise. Normalized open
channel noise values ( (O)/ (C)) in 10 mM
[K+] were obtained from single-channel currents recorded
in inside-out patches perfused with symmetrical [K+]
(n = 18). The open channel noises in 100 mM
[K+]o are taken from Fig. 5 for comparison.
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It is also interesting to note in Fig. 6D that the
single-channel conductances for the three experimental conditions are
the same in [K+] = 10 mM. If the increased
open channel noise in 100 mM [K+] (Fig. 5) is
indeed ascribed to the fluctuations of the single-channel conductance
between two conducting levels and thus results in the decreased
conductance of the R148H mutant in 100 mM
[K+] at pHo 7.4, it would be desirable to check
whether the open channel noises would also be the same in 10 mM [K+]o in different experimental
conditions, just like the single-channel conductance. Fig.
6E shows this is indeed the case. The open channel noises
are about the same for the WT channel at pHo 6.0, R148H mutant
at pHo 6.0 and R148H mutant at pHo 7.4 in 10 mM [K+]. Moreover, the open channel noises in
these conditions are all small and similar to the noises in the WT
channel and R148H mutant at pHo 6.0 in 100 mM
[K+]o. Thus the effect of protonation at position
148 in the R148H mutant, or 148
, on K+ conductance seems
to be different in different bulk K+ concentration,
suggesting that the free energy profile of the permeating
K+ ion in the pore actually varies with bulk K+ concentration.
Internal [K+] Has No Effect on the
pHo-dependent Changes of the Single-channel
Conductance in the R148H Mutant--
We have demonstrated that 148
has a significant effect on ion permeation through Kir2.1 channel pore,
and the effect is closely correlated with bulk K+
concentration. Because the results in Fig. 6 are obtained in symmetrical [K+] and the results in Figs. 1-4 suggest
that the residue at position 148 is located in the external pore mouth,
it is desirable to further examine whether it is external
K+ or internal K+ that is crucial for the
effect of 148
on K+ permeation. Fig.
7 shows the effects of asymmetrically
varying [K+] on the single-channel conductance of the
R148H mutant at pHo 7.4. In these experiments, the pipette
solution contained either 10 or 100 mM [K+],
and the inward currents were recorded at various
Vm in inside-out patches perfused with either 10 mM [K+] or 100 mM
[K+]. The results indicate that the single-channel
conductance of the R148H mutant is dependent on
[K+]o but not internal [K+]
([K+]i). The Vm
independence of the effect of pHo (Fig. 4) and the lack of
effect of pHi (Fig. 3) as well as [K+]i
(Fig. 7) on the single-channel conductance of the R148H mutant all
suggest that position 148 is located very close to the external pore
mouth.

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Fig. 7.
The pHo-dependent changes
of the single-channel conductance of the R148H mutant is sensitive to
[K+]o but not [K+]i.
Inward single-channel currents from inside-out patches exposed to four
different combinations of external/internal solutions were recorded at
pHo = 7.4. The junction potential change produced by changing
solutions between 10 mM [K+]i + 90 mM [NMG+]i and 100 mM
[K+]i was less than 2 mV and was not
corrected.
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 |
DISCUSSION |
In this study, we report successful expression of the Kir2.1
mutant R148H in Xenopus oocytes. We find that to obtain the
same level of functional channel expression, the amounts of the R148H cRNA injected into the Xenopus oocytes have to be about
10-100 times more than those of the WT cRNA. The fact that the R148H mutant retains inward rectification (because of
Vm-dependent block of the channels
by intracellular Mg2+ and polyamines), sensitivity to
extracellular Ba2+ block (data not shown), and
K+ selectivity (9) suggests the absence of major
conformational changes caused by the mutation. Our results show that
the side chain of the amino acid residue at position 148 is accessible to external H+ and that this amino acid is probably located
outside the electrical field. Furthermore, protonation of this position
is essential for a faster transportation of permeating K+
ions. Because changes of pHo from 5.0 to 9.0 do not have any
significant effect on the single-channel conductance of the WT channel
(Fig. 2), external H+ (or OH
) binding to a
part of the channel other than at position 148 probably can be
neglected as a first approximation in the discussion of the findings
here. Basically there are two possible mechanisms underlying the effect
of pHo at position 148: protonation-deprotonation at this
position may have an indirect or allosteric effect on channel protein
and thus on K+ permeation, or it may have a direct effect
on K+ permeation through the Kir2.1 channel pore.
If pHo modulated K+ permeation through the pore by
an allosteric effect, the single-channel conductance in the R148H mutant at pHo 6.0 presumably should have a more constant relationship with that at pHo 7.4 in any given
[K+]. It is therefore difficult to envisage why the
single-channel conductances would be the same for the R148H mutant at
pHo 6.0 and 7.4 in 10 mM [K+] yet are
quite different in 100 mM or higher
[K+]o (Fig. 6D). Moreover, in Figs.
2A and 5 we have noted that pHo has an effect on
both the open channel noise and the single-channel conductance. Most
interestingly, the open channel noise has a biphasic change. When
pHo is increased from 6.0 to 7.4 the open channel noise
increases. However, further increasing pHo results in a
decrease in the open channel noise. With an allosteric model, it is
hard to envisage the biphasic effect on the open channel noise by
pHo. Also, with a sampling interval of 100 µs, we still could
not resolve the open channel noise into discrete conducting states,
indicating that the effect of protonating the channel has very fast
kinetics. Because protein conformational change usually is a much
slower process, it seems unlikely that an allosteric effect involving large scale protein conformational changes is responsible for the
pHo effect in the R148H mutant.
In contrast to the indirect or allosteric model, a direct effect of
external H+ on K+ ion conduction seems much
more plausible. A direct effect of protonation-deprotonation in the
pore will certainly fit the very fast kinetics of the modulatory effect
of external H+ (revealed by the increased open channel
noise). In view of higher single-channel conductance in channels with
148
(Fig. 2), we have argued that the direct effect of 148
may
involve elevation of some energy minimum rather than energy maximum of
the permeating K+ ion. The findings in Fig. 6 are also
consistent with the notion that direct protonation of the pore at
residue 148 elevates some free energy minimum of the K+ ion
in the pore. It is very interesting, then, that the effect of
protonation of position 148 in the R148H mutant (the positive charge at
position 148, 148
) on K+ ion conductance or the free
energy profile of permeating K+ may be so different in
different [K+]o (Figs. 6 and 7). Because changes of
free energy minimum of permeating K+ ion by
protonation-deprotonation of the R148H mutant channel pore (manifested
as increased open channel noise at pHo 7.4) are evident only in
high (100 mM) but not in low (10 mM) [K+]o, it seems that 148
would have a
significant effect on K+ ion permeation only when the pore
is "crowded" with permeating K+ ions. In other words,
ion-ion interaction is very likely to be involved in the effect of
148
on K+ ion permeation, which would in turn imply
simultaneous existence of at least two permeating K+ ions
around position 148. We therefore propose that there is a set of ligand
groups around position 148 in the external pore mouth to accommodate
two permeating K+ ions (Fig.
8). When the ligand at position 148 has
an uncharged histidine (Fig. 8B), the ligand may participate
in coordinating the partly dehydrated permeating K+, and
the set of ligands represents two "complete" binding sites for
K+ ions. Because the energy minimum is always low, the
Kd and maximal single-channel conductance are both
small. However, when the ligand at position 148 is positively charged
(Fig. 8C), it can no longer participate in coordinating
K+ ions. The sites are now somewhat insufficient to
accommodate two K+ ions, and the second K+
would have to adopt a position (site b in Fig.
8A) competing for some coordinating ligands with the first
K+ (at site d in Fig. 8A). Both the
Kd for loading the second K+ to
site b in Fig. 8A and the maximal single-channel
conductance would therefore be larger. When [K+]o
is low (e.g.
10 mM) there would be no
significant occupancy by the second K+ of site b (whose
apparent Kd with site d already occupied by a
K+ presumably is ~49 mM, Fig. 6D).
The permeation of K+ thus would always happen on an
"unenhanced" basis whether position 148 is charged or not (if there
is little occupancy of site b by the other K+ ion there
would be no significant ion-ion interaction to enhance the exit of the
K+ at site d). The unitary conductance and open channel
noise level would therefore remain very similar in 10 mM
[K+]o, no matter whether it is WT channel or
R148H mutant, and no matter whether pHo is 6.0 or 9.0.

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|
Fig. 8.
Schematic illustration of the effect of
148 on K+ permeation through the Kir2.1 channel
pore. A, several ligand groups around position 148 form
a set of K+ binding sites in the external pore mouth. If
the set contains only one K+ ion (the single occupancy
state), the free energy level would be similar for the K+
ion at site a, b, c, or d, and thus the K+ ion may move
among these sites freely. B, if the residue at position 148 is an uncharged histidine, the set would have sufficient ligand to
"comfortably" accommodate two K+ ions at sites a and d
(the double occupancy state). There is little interaction between the
two ions at these sites, and the free energy level of each of the
K+ ions would not be significantly higher than the
K+ ion in the single occupancy state. Although it does not
take high [K+]o to achieve the double occupancy
state (the dissociation constant of the second K+ ion to
the set is small), the exit rates of the K+ ion at site d
(exiting inwardly to make inward K+ current) are not
significantly different whether the set is doubly or singly occupied.
C, when the residue at position 148 is charged (an arginine
or a protonated histidine), site a no longer represents a low energy
configuration to accommodate a K+ ion. The set of binding
ligands can still accommodate two K+ ions, but now the two
ions would be forced to take sites b and d. Because there would be
competition for a common ligand (black oval) between the two
ions, the free energy level of the K+ ions in such a double
occupancy state is significantly higher than the K+ ion in
the single occupancy state. It would therefore take higher
[K+]o to achieve the double occupancy state (the
dissociation constant of the second K+ ion to the set is
larger), but the exit rate of the K+ ion at site d would be
significantly enhanced when the set is doubly occupied. Note that the
relative position of residue 148 to the other binding ligands is
arbitrarily assigned. Also, we are not sure whether the uncharged
histidine at position 148 is directly involved in K+ ion
binding. However, all major points would remain the same as long as the
positive charge at position 148 serves to decrease the ability of other
nearby ligands to coordinate permeating K+ ions in the set
of K+ binding sites.
|
|
The multi-ion nature of inward rectifier K+ channels has
been supported by several lines of evidence including anomalous greater fraction effect (15), a flux-ratio exponent of 2.2 (16), steep Vm-dependent block by monovalent
ions (17), and Vm- and
[K+]o-dependent inward rectification
(1). However, it is unclear whether and how these multiple
K+ ions in the pore interact with each other. In this
study, we add a new line of evidence for the multi-ion nature of the
Kir2.1 channel pore. Furthermore, we suggest that the interaction
between the permeating K+ ions near position 148 is
essential for rapid transportation of the K+ ion through
the pore and that position 148 probably is directly involved in or
close to a set of two K+ binding sites in the external pore
mouth. The arginine residue at position 148 is highly conserved in the
Kir family, and it is likely that this highly conserved arginine at
position 148 will also influence K+ permeation through
other inward rectifier K+ channels in a way similar to what
is described in this study. The existence of a set of two closely
associated K+ binding sites in the external mouth of the
Kir2.1 channel is intriguing, because it is very similar to what is
described for the L-type Ca2+ channels
(18-20). It would be interesting to see whether this is a more general
attribute in the molecular design of these cationic channels in the
future. In addition to the set of K+ binding sites located
near the external mouth of the pore, it has recently been suggested
that K+ and Ba2+ may compete for a binding site
located in the electrical field in the Kir2.1 channels (11). Further
characterization of how K+ ions in these different binding
sites interact with each other or with the channel may provide us a
more complete picture of the ion permeation process through the pore of
the Kir2.1 channel.
 |
ACKNOWLEDGEMENTS |
We thank Drs. Jorge Arreola, Chyuan-Yih Lee,
and Kenneth K.-Y. Wu for helpful discussions.
 |
FOOTNOTES |
*
This work was supported by Academia Sinica and National
Science Council Grants 87-2314-B-001-039, 88-2314-B-001-042, and
88-2314-B-002-212 in Taiwan, R.O.C.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.
§
To whom correspondence may be addressed: Inst. of Biomedical
Sciences, Academia Sinica, 128 Yen-Chiu Yuan Rd., Section 2, Taipei
11529, Taiwan, R.O.C. Tel.: 886-2-2789-9024; Fax: 886-2-2785-3569; E-mail: ruchi{at}novell.ibms.sinica.edu.tw.
To whom correspondence may be addressed: Dept. of Physiology,
National Taiwan University, College of Medicine, 1 Jen-Ai Rd., Section
1, Taipei 100, Taiwan, R. O. C. Tel.: 886-2-2312-3456, Ext. 8236; Fax:
886-2-2396-4350; E-mail: cckuo{at}ha.mc.ntu.edu.tw.
 |
ABBREVIATIONS |
The abbreviations used are:
WT, wild type;
148
, the positive charge at position 148;
NMG, N-methyl-D-glucamine;
MES, 2-morpholinoethanesulfonic acid;
CHES, 2-(cyclohexylamino)ethanesulfonic acid;
pS, picosiemens.
 |
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.