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INTRODUCTION |
Ion channels are membrane proteins that interact closely with
permeant ions. Therefore, it is conceivable that the structures of
these proteins may assume different conformations during different functional states, such as in the opening and closing of ion channels. In inward rectifier K+ channels, the most studied gating
mechanism is the membrane voltage (Vm)1-dependent
channel block by internal Mg2+ and polyamines (1-4). This
Vm-dependent block results in inward
rectification, which contributes to the physiological functions of
these channels. However, little is known whether structural changes are
involved in the functions of inward rectifier K+ channels.
It has been shown that the gating of these channels depends on permeant
ions (5, 6), and most recent evidence supports that gating may be
attributed to conformational changes resulting from the interaction
between permeant ions and the backbone carbonyls in the selectivity
filter of the cloned Kir2.1 channels (7). Our previous study
demonstrated that external NH
induces the Kir2.1
channel into fast inactivation during hyperpolarization (8). We showed
that the NH
-induced inactivation is not because of
the NH
block of Kir2.1 channels. Furthermore, studies
in the R148Y mutant suggest that one or both of the two binding sites
located at the external pore mouth are involved in the
NH
-induced inactivation. Because the binding site is
located outside the electrical field (9), and yet the inactivation is
Vm-dependent, we propose that a
Vm-dependent process occurs within the
pore to effect channel closure.
Even though evidence supports the involvement of conformational changes
in the gating of the Kir2.1 channel, there remain doubts challenging
the hypothesis because of the lack of an intrinsic Vm sensor in these channels. In this study we
further analyzed the biophysical properties of the
NH
-induced inactivation. We found that its gating
shares several similarities with that of a cloned Cl
channel, ClC-0. The gating in both channel types depends on
Vm, concentrations of permeant ions, and is
described by a Boltzmann distribution with a non-zero offset (10-12).
It has also been demonstrated that an intrinsic Vm
sensor is not required in the gating of the ClC-0 channel. The
Vm dependence of gating arises from an intrinsically
Vm-dependent conformational change induced by the Vm-independent binding of
Cl
to the channel (12). Based on the model for the gating
in the ClC-0 channel, we propose a non-equilibrium kinetic scheme to account for the NH
-induced inactivation in the Kir2.1 channel.
Also, we further suggest that if global conformational changes are
indeed involved in the NH
-induced inactivation,
several amino acids lining the pore of a Kir2.1 channel should
participate in the Vm-dependent process preceding to channel closure. We tested this hypothesis using two
approaches. First, we examined whether the mutation of amino acids
located at different parts of the Kir2.1 channel influences the gating
of the NH
-induced inactivation. Second, we
investigated whether the kinetics of the NH
-induced inactivation is altered by MTSET modification of cysteine mutants whose
mutation is located at the internal pore. Our results show that the
mutation of several amino acids located at different part of a Kir2.1
channel indeed changed the gating of the NH
-induced inactivation. In addition, chemical modification at the internal pore
mouth reduced the kinetics of the NH
-induced inactivation.
Kinetic studies have provided us with bountiful information on how the
inward rectifier K+ channels operate to serve their
functions. However, we have very little information on their underlying
structures mainly because of technical limitations. The
NH
-induced inactivation provides us with a model to
study the structural-functional relationship within the Kir2.1 channels.
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EXPERIMENTAL PROCEDURES |
Molecular Biology and Preparation of Xenopus
Oocytes--
Site-directed mutations were generated in the wild-type
channel (IRK1 clone) using the Altered Sites II in vitro
mutagenesis systems (Promega, Madison, WI). The cysteine mutants were
constructed in the IRK1J clone. Purification of cDNA, and in
vitro T7 or SP6 transcription reactions (mMessage mMachine;
Ambion, Dallas, TX) were performed as described previously (13).
Xenopus oocytes were isolated by partial ovariectomy from
frogs anesthetized with 0.1% tricaine (3-aminobenzoic acid ethyl
ester). The incision was sutured, and the animal was monitored during
the recovery period before it was returned to its tank. Following the
last oocyte collection, frogs were anesthetized with 0.1% of tricaine and sacrificed by decapitation. All surgical and anesthetic procedures conformed to national ethics committee guidelines.
Electrophysiology Techniques--
Currents were recorded from
inside-out patches at room temperature using the giant and
single-channel patch-clamp techniques (14, 15) with an Axopatch 200A
amplifier (Axon Instruments, Foster City, CA). The resistance of the
electrode pipette ranged from 0.15 to 0.25 megohms for giant patch
recordings and from 1 to 3 megohms for single-channel recordings when
filled with the electrode solutions. The [NH
]
solutions contained the following (in mM):
NH4Cl (10-300),
N-methyl-D-glucamine (0-100), EDTA (5),
and HEPES (5), at pH 7.4. In the experiments of ionic strength, both
the external and internal solutions contained the following (in
mM): NH4OH (15), sucrose (200), EDTA (5), and
HEPES (5), at pH 7.4. The 100 mM [K+]
solution contained the following (in mM): KCl+KOH (100),
EDTA (5), and HEPES (5), at pH 7.4. Rundown of channel activity was
delayed by treating inside-out patches with
L-
-phosphatidylinositol-4,5-bisphosphate (Sigma)
(13, 16). MTSET (Toronto Research Chemicals, North York,
Ontario, Canada) was made as a stock in water each day, stored at
20 °C, and diluted in bath solution immediately before application.
The command Vm and data acquisition functions were
processed using a Pentium computer, a DigiData board, and pClamp6 software (Axon Instruments, Foster City, CA). Data sampling rates were
2.5-5 kHz, and the data were filtered at 0.5-1 kHz with an 8-pole low
pass filter (Frequency Devices, Rochester, NY). In the experiments of
Vm-dependent inactivation, the holding potential was 0 mV, prepulses ranged from
200 to +100 mV, and the
test Vm was
120 mV. Single-channel currents were recorded at
140 mV from a holding potential of 0 mV. The open and
closed events obtained from voltage steps have exactly the same
distributions as those obtained from the steady state (8, 17).
Capacitive currents were corrected using the built-in capacitance neutralization in the Axopatch 200A amplifier.
Data Analysis--
Instantaneous currents were determined by
fitting monoexponential functions to the currents at the test pulses
and by extrapolating them to their beginnings. Histograms of the
duration of time that channels remained open and closed were
constructed with square root-log ordinates (13). The histograms were
fit to monoexponential functions with the maximum log likelihood
method, and, in general, biexponential functions did not provide
significantly better fits (p > 0.05) than
monoexponential functions, as judged by the maximal likelihood ratio
test (4). Substates were observed, but they did not occur frequently.
Transitions between the open state and substate, as well as between the
closed state and substate, were not included in data analysis.
The time course of current inhibition of mutants by MTSET followed
single exponential decay. Time constants for MTSET modification were
obtained by fitting the time courses of current inhibition. The
apparent second-order rate constants for MTSET modification were then
calculated as the reciprocal of the respective time constants divided
by the concentration of MTSET. Results are presented as
mean ± S.E.
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RESULTS |
Biophysical Properties of the NH
-induced
Inactivation--
Previously, we have examined
NH
-induced inactivation with the voltage protocol
described in Fig. 1A. In this
study we analyzed further the gating properties of the
NH
-induced inactivation. Fig. 1B illustrates the representative currents recorded in 10 and 100 mM symmetrical [NH
], respectively. Inward currents inactivated during strong hyperpolarization and the
rate of the inactivation was higher in 100 mM
[NH
] than that in 10 mM
[NH
]. Note that some residual capacitive currents
were not corrected in the currents recorded in 10 mM
[NH
]. The degrees of inactivation, however, were
actually the same for both 10 and 100 mM
[NH
] (see Fig.
2A). Instantaneous tail
currents were recorded after prepulses to various test voltages. The
steady-state open probability was quantified by normalizing these tail
currents to the maximal one obtained following the most depolarizing
prepulse potential (normalized I). Fig. 2A shows that
normalized I was smaller at more negative Vm.
Changes in symmetrical [NH
] did not affect the
normalized I-Vm relationship. The normalized
I-Vm relationship was fitted with a Boltzmann
distribution containing a non-zero offset. The effective gating charge
is around 0.7, and the non-zero offset is about 0.2 for all
[NH
] tested. Because the time course of
inactivation could be fitted to a monoexponential function, the rate of
inactivation was calculated as the reciprocal of the time constant
(
). The rate of inactivation depended on Vm and
[NH
] (Fig. 2B).

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Fig. 1.
Inactivation of inward currents carried by
NH through Kir2.1 channels. A,
voltage protocol used to record the steady-state inactivation of
currents through Kir2.1 channels. The holding potential was 0 mV,
prepulses ranged from 200 to +100 mV, and the test
Vm was 120 mV. B, current traces
obtained from two different inside-out patches exposed to 10 and 100 mM symmetrical [NH ] as indicated. The
horizontal lines indicate zero current levels throughout
this study.
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Fig. 2.
Kinetics of the NH -induced
inactivation in various [NH ]. A and
B, the Vm dependence of normalized I and
rate of the inactivation in 10 ( , n = 5), 30 ( ,
n = 5), 50 ( , n = 5), 100 ( ,
n = 5), 200 ( , n = 4), and 300 mM [NH ] ( , n = 7).
Solid curves are the fit of data to a Boltzmann equation,
pmin + (1 pmin)/(1 + exp)( zF(V V0.5)/RT). C and E,
Vm dependence of kon and
koff. Values of kon and
koff were calculated from Equations 1 and 2 and are plotted against
Vm. The solid lines were the fit of the
data to a Boltzmann equation, kon = kon (0) × exp(zonFVm/RT),
where kon (0) is the on-rate at 0 mV,
zon is the gating charge, and F,
R, and T have their usual meanings. The
kon (0) and zon were as
follows: 2.04 s 1 and 0.39 in 10 mM
[NH ]), 2.31 s 1 and 0.45 in 30 mM [NH ], 3.09 s 1 and 0.43 in 50 mM, 5.3 s 1 and 0.39 in 100 mM, and 4.7 s 1 and 0.42 in 200 mM. D and F, dose dependence of
kon and koff. The
solid lines in D were the fit of the data to a
Hill equation in the form of ( /(1 + KO/[NH ]). and
KO were as follows: 135.0 s 1 and
22.3 at Vm = 200 mV ( ), 101.0 s 1
and 25.4 at Vm = 180 mV ( ), 77.2 s 1 and 27.5 at Vm = 160 mV ( ),
53.4 s 1 and 26.0 at V = 140 mV ( ), 39.5 s 1 and 28.9 at Vm = 120 mV ( ).
The solid lines in F were the fit of the data to
a Hill equation in the form of ( /(1 + KI/[NH ]). and
KI were as follows: 45.0 s 1 and
32.4 at Vm = 200 mV, 39.3 s 1 and
29.7 at Vm = 180 mV, 39.5 s 1 and
29.7 at Vm = 160 mV, 36.7 s 1 and
25.1 at Vm = 140 mV, 38.6 s 1 and
24.3 at Vm = 120 mV.
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The single Kir2.1 channel demonstrates one open and one closed state in
our previous study where we proposed that the
NH
-induced inactivation could be described by Scheme
1 (8). We then calculated kon and koff from
macroscopic currents recorded in various symmetrical [NH
] using Equations 1 and 2, shown below, where
f is the fraction of channels remaining open at steady state.
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(Eq. 1)
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(Eq. 2)
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Fig. 2C demonstrates that kon
increased exponentially with hyperpolarization with an effective gating
charge
0.4. The rate of inactivation increased with elevated
[NH
] and saturated in ~100 mM
[NH
], indicating that NH
binding
is involved in the inactivation. Fig. 2D shows that the dose
dependence of kon was similar at different
Vm. Because NH
is itself a
permanent ion through the Kir2.1 channel, the entering and leaving of
the ion at both sides of the membrane complicate the analysis of
kon. However, the Vm
dependence of the kon is the same at both low
and saturating [NH
] levels, suggesting that the
effective gating charge (0.4) is intrinsic to the on rate of the
NH
-induced inactivation. On the other hand,
koff did not show Vm
dependence (Fig. 2E). Increasing symmetrical
[NH
] also accelerated koff
to the same degree as it did to kon (Fig.
2F). Because steady-state open probability is equal to
koff/(kon + koff), the same [NH
]
dependence of kon and
koff accounts for the same normalized
I-Vm relationships in various symmetrical
[NH
] shown in Fig. 2A. Fig. 2,
D and F shows that the dependence of
kon and koff on
[NH
] are about the same, and both are not
Vm-dependent, suggesting again that the NH
binding site affecting inactivation is located
outside the electrical field (possibly at the external pore mouth
according to our previous study (8)).
In summary, our results show that the NH
binding site
affecting inactivation is located outside of the electrical field yet
the inactivating process is
Vm-dependent. Also,
kon and koff both depend
on [NH
]. Previously, we demonstrated that blocking
rate would be too slow to account for NH
acting as a
permeant blocker, and the inactivation is dependent on external rather than on internal NH
(8). Taken together our data
suggest that the external NH
-induced inactivation is
because of the conformational changes of the Kir2.1 channels.
NH
-induced Inactivation Is Affected by Mutation
of Amino Acids Ranging from the External to Internal Pore
Mouth--
To test whether conformational changes are involved in the
NH
-induced inactivation, we first examined whether
the NH
-induced inactivation is affected by mutation
of amino acids located at different parts of a Kir2.1 channel. We first
examined whether the amino acids (Glu-125, Ile-137, and Thr-141)
involved in Ba2+ binding within the Kir2.1 channel (18, 19)
may function in the NH
-induced inactivation. Fig.
3 shows that the
NH
-induced inactivation in the E125N and I137L
mutants is similar to the wild-type channels whereas it was greatly
reduced in the T141V mutant. We also included our previous recording in
the R148Y mutant (8), which shows little NH
-induced
inactivation.

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Fig. 3.
NH currents through the
wild-type channels and various mutants. Current traces were
obtained from inside-out patches exposed to 100 mM
symmetrical [NH ]. Iinst
indicates the instantaneous current.
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To test whether the NH
-induced inactivation involves
amino acids located at different parts of a Kir2.1 channel, we next
recorded currents through mutants whose mutation was located at the
internal pore mouth. Both Asp-172 and Glu-224 have been shown to be
accessible to internal polyamines and Mg2+ and thus are
reckoned to be located at the internal pore mouth (3, 4, 20). Fig. 3
shows that the rate of the NH
-induced inactivation in
the D172N mutant was increased (see also Fig. 4C). Both the rate and degree
of the NH
-induced inactivation were reduced in the
E224G mutant.

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Fig. 4.
Summaries of the kinetics of the
NH -induced inactivation in the wild type and
mutants. A and B, normalized
I-Vm and rate-Vm relationships
for the wild type and mutants. C and D, the
kon-Vm and
koff-Vm relationships for the
wild type and the mutants. The solid lines were the fit of
the data to a Boltzmann equation, kon = kon (0) × exp(zonFVm/RT).
Both kon and zon are
listed in Table I.
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Fig. 4 summarizes the normalized I-Vm relationship
(Fig. 4A) and the Vm dependence of the
kinetic parameters (Fig. 4, B-D) obtained in the
wild type and mutants. Except for the T141V mutant whose kinetics of
macroscopic currents was not analyzed, kon of
all mutants showed similar Vm dependence as the
wild-type channels. Fitting the
kon-Vm relationship with a
Boltzmann equation, we obtained kon and the
effective gating charge (zon), which are
listed in Table I. The
kon value at 0 mV, kon
(0), was decreased about 2-fold in the E125N and E224G mutants. Values
of zon ranged from 0.32 to 0.45 and do not seem to change dramatically although the change in the E125N mutant is
statistically significant compared with the wild-type channels.
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Table I
The gating and kinetic parameters of the NH4+-induced
inactivation in the wild type and mutants
In the T141 mutant, values could not be determined reliably and are
therefore marked by n.r.d. Footnotes indicate that groups were
significantly different from the wild-type (WT).
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Table I also lists the parameters obtained from fitting the normalized
I-Vm relationships to Boltzmann distributions. The
major findings shown in Fig. 4 and Table I are as follows. First, the
degree of the NH
-induced inactivation was
dramatically reduced in the T141V mutant. Second, the rate of
NH
-induced inactivation was accelerated in the mutant
D172N but was decreased in the E224G mutant. Third, the gating charge
was significantly decreased in the E224G mutant. Fourth,
V0.5 was shifted toward negative Vm in
the E125N and E224G mutants.
Table I shows that the gating charge in the E224G mutant was reduced
significantly. Recently, E224 has been shown to screen surface charge,
thereby affecting ion conduction (21). We next examined whether
surface-charge screening affects the gating properties of the
NH
-induced inactivation by comparing the inactivation
in different ionic strengths. Fig.
5A shows the currents recorded
from the wild types exposed to symmetrical 15 mM
[NH
] (the minimal [NH4OH] added to
keep the pH at 7.4 in the presence of 5 mM EDTA) and 200 mM sucrose. The normalized I-Vm (Fig.
5B) relationship was identical to those exposed to solutions
with larger ionic strength (Fig. 2A). These results suggest
that the decrease in gating charge in the E224G mutant is not because
of the reduced screening of surface charge. However, the rate of
inactivation (Fig. 5C), kon (Fig.
5D), and koff (Fig. 5E)
were all larger than those obtained in the wild types exposed to 10-50
mM [NH
] plus 100 mM
N-methyl-D-glucamine (Fig. 2), suggesting that
reducing ionic strength (less surface-charge screening) increases the
kinetics of the NH
-induced inactivation. In summary,
mutation of amino acids lining the channel pore but located at various
portions of the Kir2.1 channel affects the gating for the
NH
-induced inactivation.

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Fig. 5.
Effects of reduced ionic strength on the
NH -induced inactivation. A, currents
recorded in 15 mM [NH ] and 200 mM sucrose with the voltage protocol shown in Fig.
1A. B and C, the Vm
dependence of normalized I and rate of inactivation. The solid
curve is the fit of the data to Boltzmann equation described in
the legend for Fig. 2. D and E, the effects of
Vm on kon and
koff. The solid line is the fit of
data to a Boltzmann equation (n = 4).
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Single-channel Kinetics of NH
-induced
Inactivation--
The NH
-induced inactivation was
reduced to a large degree in the T141V mutant. Because the degree of
inactivation was very small the gating and kinetic values could not be
determined reliably using Equations 1 and 2. To further determine how
the kinetics of the NH
-induced inactivation is
affected in this mutant, we carried out single-channel recordings. Fig.
6 shows the single-channel traces and the
corresponding histograms of open and closed dwell times of the wild
type and T141V mutant recorded at
140 mV in 100 mM
symmetrical [NH
]. The distributions of open and
closed dwell times were well fitted to monoexponential functions. The
values of kon and koff
were then calculated as the reciprocals of fit time constants and are summarized in Table II. A 10-fold
increase in koff is the primary contributor to
the almost abolished NH
-induced inactivation in the
T141V mutant. These results suggest that several amino acids ranging
from the external to the internal pore mouth of the Kir2.1 channel are
involved in the NH
-induced inactivation, which is
thus likely because of global conformational changes of channel
structure.

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Fig. 6.
Effects of mutation on the single-channel
currents. Each panel shows a sample sweep at 140 mV
and the histograms for opening and closing time for the indicated
channel. Histograms are plotted with square root (sqrt)-log
ordinates. The distributions of the open and closed times were fitted
to monoexponential functions (continuous curves). Mean open
and closed times obtained from the fitted curve are given
above the histograms (n = 4-6).
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Table II
Effects of mutation on kon and koff for the
NH4+-induced inactivation
WT (cal) indicates that kon and
koff were calculated from macroscopic currents using
Equations 1 and 2. WT, wild-type.
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Chemical Modification of Substituted Cysteines in the Inner Pore
Reduces Current and Alters the Kinetics of NH
-induced
Inactivation--
To further confirm that the internal pore is
involved in the NH
-induced inactivation initiated by
external NH
binding, we next examined whether
chemical blocking in the internal pore changes the kinetics of the
NH
-induced inactivation. Lu et al. (22)
have shown that the wild-type Kir2.1 channel is sensitive to
modifications by MTS reagents. Thus, an MTS-insensitive channel, IRK1J
(C54V, C76V, C89I, C101L, C149F, and C169V), was constructed (22).
Several single-cysteine substitutions (constructed in IRK1J) in the M2
domain of a Kir2.1 channel are sensitive to internal MTSET modification
in 140 mM [K+], indicating that the
substituted residues are located at the internal pore mouth (22).
Therefore, we carried out experiments in these M2 cysteine mutants, as
well as in an E224C mutant. Fig. 7A shows that, in 100 mM symmetrical [NH
] MTSET did not
reduce the current through IRK1J nor did it affect the inactivation
process. Except for the I171C mutant, the MTSET modification increased
the steady-state open probability during the test pulse (
120 mV) in
all the other cysteine mutants. Also, MTSET modification significantly
decreased the rate of NH
-induced inactivation in the
Q164C, G168C, V169C, D172C, and I176C mutants.

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Fig. 7.
MTSET modification of IRK1J and the indicated
cysteine mutants. A, each panel shows two current
traces recorded from a voltage step from 0 to 120 mV under control
(solid lines) and complete modification (dotted
lines in IRK1J, Q168C, and V169C mutants) or 63% of peak current
block (dotted lines in the Q164C, I171C, D172C, I176C, and
E224C mutants). MTSET was 0.2 mM for the E224C mutant and 2 mM for all the other cysteine mutants. B and
C, bar graphs of the calculated
kon and koff for the
NH -induced inactivation under control and MTSET
modification (n = 4-6). In the cysteine mutants, in
which chemical modification produced complete current block (Q164C,
I171C, D172C, I176C, and E224C), kon and
koff were calculated from traces obtained at
63% of current block. Asterisks indicate that the MTSET
groups were significantly different from the control groups. *,
p < 0.05; **, p < 0.01; ***,
p < 0.001.
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Fig. 7, B and C summarize the effects of MTSET
modification on the kon and
koff (calculated using Equations 1 and 2) of the NH
-induced inactivation in the cysteine mutants.
MTSET modification reduced kon in all the
mutants whose NH
-induced inactivation is affected, i.e. in all but the I171C mutant. Also, MESET modification
reduced koff of the NH
-induced
inactivation in the Q164C, V169C, and D172C mutants but enhanced
koff in the I176C and E224C mutants. These
results suggest that MTSET modification at the internal pore mouth may
alter the flexibility of the Kir2.1 channel at the internal pore mouth
thereby changing the kinetics of the NH
-induced
inactivation. Also, our previous study provides evidence for the
involvement of the external pore mouth in the
NH
-induced inactivation. Together these results
suggest that the global changes of channel structure may be involved in
the NH
-induced inactivation.
State Dependence of MTSET Modification of Substituted Cysteines in
the Inner Pore--
Fig. 7, B and C shows that
the MTSET bound in the inner pore at 0 mV (at which there is no
NH
-induced inactivation) affects the kinetics of the
NH
-induced inactivation in some cysteine mutants.
However, it remains to be determined whether the structure of the inner
pore is changed during the inactivation such that the accessibility of
the MTSET to the substituted cysteines is state-dependent.
To further probe the structural changes of the inner pore during the
NH
inactivation, we next measured the
state-dependent rates of MTSET modification in different
conformational states of the Kir2.1 channel. Data shown in Fig. 7 were
obtained by measuring MTSET modification mainly during an open state
(at least 95% of time in the open state with a pulse frequency of 0.5 Hz and duration of 100 ms). Next the rates of MTSET modification during
the inactivated state were estimated by holding the patches at 0 mV and
stepping to
120 mV (100 ms) at 5 Hz. Using this protocol the channels were thus held at
120 mV for 50% of the total recording time. Shown
in Fig. 8 are these experiments, which
were carried out in the cysteine mutants whose kinetics of the
NH
-induced inactivation are greatly affected by MTSET
modification (Fig. 7, B and C).

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Fig. 8.
State dependence of the rates of the MTSET
modification in the cysteine mutants. A and
B, the time courses of MTSET modification obtained by
pulsing the patches to 120 mV at 0.5 and 5 Hz, respectively, in the
Q164C mutant exposed to 100 mM symmetrical
[NH ] and 100 mM symmetrical
[K+]. C, the averaged rates of MTSET
modification in 100 mM symmetrical
[NH ]. D, the averaged rates of MTSET
modification in 100 mM symmetrical [K+]
(n = 5-11). *, p < 0.05; **,
p < 0.01.
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Fig. 8A shows the time courses of MTSET modification
obtained with a pulse frequency of 0.5 and 5 Hz, respectively, in the Q164C mutant exposed to 100 mM symmetrical
[NH
]. The rate of MTSET modification was slightly
higher with a pulse frequency of 5 Hz. Because MTSET is positively
charged it is conceivable that its accessibility to the substituted
cysteine located in the pore is affected by Vm. As a
control, we also measured the MTSET modification in the cysteine
mutants exposed to 100 mM symmetrical [K+],
in which all the cysteine mutants do not show inactivation during
hyperpolarization (data not shown) (23). Fig. 8B illustrates the time courses of MTSET modification obtained with a pulse frequency of 0.5 and 5 Hz, respectively, in the Q164C mutant exposed to 100 mM symmetrical [K+]. The rate of MTSET
modification was slower with a pulse frequency of 5 Hz. Fig.
8C shows the averaged rates of MTSET modification obtained
in the Q164C, D172C, and I176C mutants in 100 mM
[NH
]. An increase of pulse frequency from 0.5 to 5 Hz did not significantly accelerate the rates of MTSET modification in
the Q164C mutant (p > 0.11) although the rate seemed
to be slightly higher at 5 Hz. The rates of MTSET modification in the
D172C and I176C mutants were not significantly changed
(p > 0.45) by an increase of pulse frequency. The rate
of MTSET modification was greatly accelerated in the D172C mutant
exposed to 100 mM [NH
] compared with
100 mM [K+]. The effect is not related to the
NH
-induced inactivation and is currently under
investigation in our laboratory. Fig. 8D shows the averaged
rates of MTSET modification in 100 mM symmetrical
[K+]. An increase of pulse frequency significantly
decreased the rates of MTSET modification in the Q164C, D172C, and
I176C mutants. MTSET modification in all the cysteine mutants used in
this study could not be reversed by washout in the control solutions
(100 mM [NH
] and 100 mM
[K+]). Considering the following factors, the rates of
MTSET modification during the NH
-induced inactivation may be increased in the Q164C, D172C, and I176C mutants. First, hyperpolarization significantly decreases the accessibility of MTSET to
the pore of the Kir2.1 channel (Fig. 8D). Second, the channels spend only 50% of the entire recording time at
120 mV where
the channels inactivate. Third, not all of the channels are in the
inactivated state at
120 mV (40% for Q164C, 60% for D172C, 70% for
I176C). Together these results suggest that MTSET modification may be
state-dependent to a certain degree in the Q164C, D172C,
and I176C mutants.
 |
DISCUSSION |
Refinement of the Kinetic Scheme for NH
-induced
Inactivation--
Scheme 1 is simplified from Scheme
2, where KO is the
dissociation constant for NH
binding in the open
state,
is the on rate, and
is the off rate between the open and
inactivated transition. The simplification was made by assuming that
the O and O·NH
states have the same conductance and
that the binding step is very rapid compared with any subsequent
conformational change. koff is also dependent on
[NH
], suggesting that an empty inactivated state
may exist. Scheme 2 is therefore modified as shown in Scheme
3, where KI is the
dissociation constant for NH
binding in the
inactivated state.
The fitting of the normalized I-Vm relationships to
Boltzmann distributions with non-zero offsets suggests that the system
is not an equilibrium one. Previous studies have demonstrated theoretically (24) and experimentally (10) that a non-equilibrium distribution of conformational states is created, if there exists a
coupling of ion translocation and conformational transitions. An
essential requirement for the coupling of ion translation and conformational transition is that transitions between the two states
can take place both in the empty and occupied state of the binding site
(24). In other words, it is essential to introduce a transition between
the O and I state. Therefore, Scheme 3 is further modified as shown in
Scheme 4. Scheme 4 predicts that the
observed kon and koff
have the following relationships with
1 and
2 and with
1 and
2,
respectively, as illustrated by Equations 3 and 4.
|
(Eq. 3)
|
|
(Eq. 4)
|
Fitting data shown in Fig. 2, D and F to
Equations 3 and 4, we obtained one
and one
(data not shown).
Because the gating is induced by NH
binding
2 should be much less than
1. Thus,
2 is equal to zero. On other hand, we propose that
1 =
2 to incorporate the coupling of ion
translocation and state transitions in Scheme 4.
Fig. 2A shows that the gating charge is around 0.7, and the
non-zero offset is about 0.2. Both values are similar to that of ClC0
channels (12). The effective gating charge is severalfold smaller than
that for voltage-gated cation channels (25). Similar to previous
discussion (12), we also suspect that the source of the gating charge
is the NH
ion itself, moving inwards during the
(O·NH
I·NH
) transition
rather than the movement of charge intrinsic to the protein. The reason
for this hypothesis is 2-fold. First, there are few charged amino acids lining the pore of a Kir2.1 channel. So far, D172 and E224 are the only
two charged residues that are known to be located in the electrical
field of the Kir2.1 channel. Neutralization of Asp-172 to a non-polar
residue did not affect the gating charge (Table I). The gating charge
in the E224G mutant is decreased but not completely eliminated. Second,
the gating depends on NH
ions, which are permeant
ions and thus efficient gating-charge carriers. Thus, we propose that
the conducting ions move through the Kir2.1 channel, entering and
leaving on both sides, and keeping the cycle under consideration above
out of equilibrium. Unlike the ClC0 channel, which shows a clear
time-asymmetric single-channel record arising from the double-barreled
nature of the channel (10), we could not actually observe a
time-asymmetric single-channel record in the Kir2.1, which is a
single-pore channel. However, we can reason that a channel may assume
two states. One state has the binding site accessible only from the
external side, and the other state has the binding site accessible only
from the internal side. In this case, in the presence of a large
electrochemical gradient, the channel exhibits a carrier-like behavior.
An NH
moves from high to low electrochemical
potential with each turn of the conformational cycle. Scheme
5 illustrates such an imaginary process.

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|
Scheme 5.
Illustration of how an NH
moves from high to low electrochemical potential with each turn of the
conformational cycle.
|
|
NH
(o) and NH
(i) denote the
NH
at the external and internal space, respectively.
The energy profile above the I1·NH
state indicates that the barrier to internal space is very high,
and thus the binding site is mainly accessible from the external side.
The energy profile above the I2·NH
shows the opposite energy-barrier profile for the NH
binding site.
Scheme 5 is evolved from the accumulated biophysical data of the
NH
-induced inactivation. Further experiments need to
be designed to test its validity. Also, we should emphasize that the
NH
-induced inactivation is a non-equilibrium system.
The parameters obtained by fitting data to Boltzmann equations thus do
not have physical meanings. However, these parameters are useful as a
first approximation for understanding the mechanism underlying the
gating in the Kir2.1 channel.
Global Conformational Changes Are Involved in
NH
-induced Inactivation--
In this study, we
provide two new pieces of evidence for conformational changes involved
in the NH
-induced inactivation in Kir2.1 channels.
The first line of evidence is that the NH
-induced
inactivation during hyperpolarization is affected by mutation of amino
acids located at various parts of the channel protein. These results
suggest that global conformational changes are involved in the
Vm-dependent closure of Kir2.1 channels.
In the following, we discuss the mutants whose gating for the
NH
-induced inactivation differs dramatically from the
wild types.
Glu-125 has been shown as a Ba2+ binding site that is
presumed to be located at the external pore mouth (19). Neutralization of this site indeed decreases both kon and
koff to the same degree (~30%) at all
Vm. The dissociation constants for
kon and koff are about
the same in the wild-type channels (Fig. 2, D and
F). According to Scheme 5, koff
depends on the ion translocation of NH
, which in turn
depends on NH
binding affinity
(KO for kon). It is
therefore likely that NH
dependence of
koff is affiliated with the NH
dependence of kon. Thus, the effects of E125N on
the kinetics of the NH
-induced inactivation may result from its effect on the NH
binding to the channel.
In the T141V mutant, which is located within the pore of the Kir2.1
channel (18), kon is increased slightly, but
koff is enhanced by 10-fold. Also, the degree of
the NH
-induced inactivation (see Fig. 3 and Fig.
4A) is greatly reduced. T141 is located close to the
K+ selectivity filter GYG, which, according to the
structure of KcsA channel, constitutes to the narrowest part to the
channel (26). Therefore, it is possible that Thr-141 may also be part of the narrow pore filter and thus stabilizes the inactivated state in
the Kir2.1 channel. Furthermore, the single-channel current of the
T141V is larger than that of the wild type (Fig. 6). As shown in Scheme
5, the NH
-induced inactivation may be gated by
NH
itself. The conducting ions move through the
Kir2.1 channel, entering and leaving on both sides, and keeping the
cycle under consideration out of equilibrium. An increase in
single-channel conductance may further drive the cycle out of
equilibrium in the direction of prompting the exit of the T141V mutants
from the inactivated state. In other words, koff
depends on the ion translocation of NH
. Therefore,
the increase in koff may result from the
increase of single-channel conductance in the T141V mutant.
Of all the mutants tested, E224G is the only one that shows a
significant decrease in the gating charge. Recently, Glu-224 has been
shown to screen surface charge, thereby affecting ion conduction (21).
However, Fig. 5 shows that the gating properties are the same for the
wild-type channels exposed to 15 mM
[NH
] and 200 mM sucrose, as well as for
those exposed to 10-50 mM [NH
] plus
100 mM N-methyl-D-glucamine.
Therefore, the effect of E224G mutant on gating charge cannot be
attributed to a decrease in surface charge screening. It is possible
that Glu-224 contributes directly to the gating charge in the
NH
-induced inactivation. On the other hand, the
permeability for K+ in the E224G mutant has been shown to
decrease (20). Therefore, it is also possible that the effect of
Glu-224 on the gating charge is because of the change in conductance
for NH
, which may be the gating charge itself.
The mutation at Glu-224 (E224G) decreases both the degree and
kon but does not affect
koff of the NH
-induced inactivation. Fig. 5, D and E shows that a
decrease in ionic strength increases both kon
and koff. Therefore, the decrease of
kon cannot be attributed to a reduction in the
screening of surface charge in the E224G mutant. Because
NH
binding is located at the external pore, the
effects of mutation at position 224 are likely to be because of a
decrease in the transition rate from the open to inactivated state
instead of a decrease in NH
binding.
The second line of evidence for the conformational changes hypothesis
is that the MTSET modification decreases kon and
koff in several cysteine mutants whose mutation
is constructed at the internal pore. Our results are consistent with
the hypothesis stating that the NH
-induced
inactivation is because of conformational changes of Kir2.1 channels.
MTSET modification changes the flexibility of the Kir2.1 channels,
which then close and reopen in a rate that is different from the
unmodified channels. Is it possible that the changes of
kon and koff are because
of the interaction of MTSET and NH
in the pore? For
example, an effect of kon can be because of
competition of the MTSET with the NH
bound at the external site. However, we consider this an unseemly possibility for
the following reasons. First, we have shown previously that the
NH
-induced inactivation is inconsistent with the
permeant ion block mechanism. Second, the NH
binding
site is located at the external pore mouth, yet the cysteine mutation
is positioned within the internal pore. Thus, it is unlikely that MTSET
would compete with NH
within the pore to decrease
kon. Third, in all the cysteine mutants where koff are decreased, kon
values are also decreased. Thus, our results are inconsistent with a
direct competition (koff should not be affected)
or knock-off (in that case, koff should
increased by MTSET modification).
According to Scheme 5, kon can be because of
variations in NH
binding affinity or the on-rate for inactivation or both. However, the cysteine-replacement is at the
internal pore mouth, and the NH
binding site is at
the external pore mouth, so the effect of kon observed in the cysteine mutants seems to indicate changes of the
on-rate for the NH
-induced channel closure.
State-dependent modification of ion channels by MTS
reagents has been used previously to probe the conformational changes of proteins in different states (27-29). We showed here that the MTSET
modification may also been state-dependent to a certain degree in the cysteine mutants located in the inner pore of the Kir2.1
channel. However, the rates do not seem to be affected dramatically
during the NH
-induced inactivation, indicating that
the major structural changes of Kir2.1 channels during the
NH
-induced inactivation may be located further
externally to site 164, e.g. close to the selectivity filter
(7) and site 141. The effects then of MTSET modification during the
open state (Fig. 7) on the NH
-induced inactivation
may be propagated to the narrow pore whose closure is restricted,
because the wider inner vestibule is held in a fixed place.
 |
CONCLUSIONS |
In this study, we performed further biophysical analyses of
NH
-induced inactivation. We find that the
NH
-induced inactivation is a non-equilibrium system.
The gating properties are similar to those of the
Cl
-dependent activation for the ClC0 channel.
Also, we provide further evidence that conformational changes are
probably proceeding to the closure of the Kir2.1 channels during the
NH
-induced inactivation based on the following
results. First, the mutation of several amino acids located at
different parts of a Kir2.1 channel changes the gating of the
NH
-induced inactivation. Second, chemical
modification at the internal pore mouth reduces the rate of the
NH
-induced inactivation, which is initiated by the
binding of NH
at the external pore mouth.
Although we have provided additional evidence supporting
the relationship between structural changes and gating mechanism, more
conclusive evidence still awaits a direct probe of the conformational changes in the Kir2.1 channel during inactivation. Furthermore, it
remains to be defined how the amino acids, which are involved in the
NH
-induced inactivation, move to effect changes of
the kinetics of the inactivation.