From the University Laboratory of Physiology, Oxford OX1 3PT, United Kingdom
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ABSTRACT |
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The cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four
regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2
C26), which expresses independently of SUR1,
shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to
serine (C166S) increases the open probability of Kir6.2
C26 approximately sevenfold by reducing the time the
channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2
C26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 µM to 2.8 mM. Substitution
of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP
and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly
by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.
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INTRODUCTION |
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The cell KATP channel is an octameric complex of two
structurally unrelated subunits that assemble with 4:4
stoichiometry (Inagaki et al., 1995
, 1997
; Sakura et al.,
1995
; Clement et al., 1997
; Shyng and Nichols, 1997
).
Kir6.2 acts as an ATP-sensitive pore while SUR1 is a regulatory subunit that endows Kir6.2 with sensitivity to
sulfonylureas, K-channel openers, and the potentiatory effects of MgADP and MgGDP (Nichols et al., 1996
;
Gribble et al., 1997a
; Shyng et al., 1997a
; Trapp et al.,
1997
; Tucker et al., 1997
). Although both subunits are
normally required for functional expression of the KATP
channel, a truncated form of Kir6.2, which lacks the
last 26 amino acids (Kir6.2
C26), is able to express independently of SUR1 (Tucker et al., 1997
). This truncated isoform of Kir6.2 is inhibited by ATP, suggesting
that ATP mediates channel inhibition by interacting
with the Kir6.2 rather than the SUR1 subunit of the
KATP channel. In support of this idea, mutations in
Kir6.2 render the channel less sensitive to ATP (Tucker
et al., 1997
, 1998
; Shyng et al., 1997b
). While exploring
the effect of sulfhydryl reagents on KATP channel activity, we observed that mutation of the cysteine residue at
position 166 to serine dramatically decreases the sensitivity of Kir6.2
C26 currents to ATP (see accompanying paper, Trapp et al., 1998
). In this paper, we have
characterized the properties of this mutant channel in
detail. We show that the C166S mutation markedly increases the channel open probability by reducing the
percentage of time the channel spends in a long closed
state. By substituting different amino acids for C166, we
show that a more hydrophilic or bulkier residue at this
position simultaneously modifies the intrinsic gating
properties of the channel and reduces its sensitivity to
ATP. A correlation between ATP sensitivity and channel open probability has also been reported for mutations at N160, within the second transmembrane domain of Kir6.2, although the shift in ATP sensitivity was
much less dramatic (Shyng et al., 1997b
). We discuss
our results in terms of a model in which C166 influences entry of the channel into the long closed state,
and the reduced ATP sensitivity produced by mutation
of this residue is largely a consequence of the change in
the channel gating kinetics, rather than in the affinity
of ATP for its binding site.
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MATERIALS AND METHODS |
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Molecular Biology
Mouse Kir6.2 (Genbank D50581; Inagaki et al., 1995; Sakura et
al., 1995
) and rat SUR1 (Genbank L40624, kindly supplied by Dr.
G. Bell, University of Chicago, Chicago, IL; Aguilar-Bryan et al.,
1995
) were used in this study. A 26 amino acid COOH-terminal deletion of mouse Kir6.2 (Kir6.2
C26) was made by introduction of a stop codon at the appropriate residue using site-directed mutagenesis (Tucker et al., 1997
). Site-directed mutagenesis of Kir6.2
C26 was carried out by subcloning the appropriate fragments into the pALTER vector (Promega Corp., Madison, WI).
Mutations are indicated by the single amino acid letter code. For
oocyte expression studies, constructs were subcloned into the
pBF expression vector, which provides the 5' and 3' untranslated
regions of the Xenopus
globin gene. Synthesis of capped mRNA
was carried out using the mMessage mMachine large scale in
vitro transcription kit (Ambion Inc., Austin, TX).
Oocyte Handling
Female Xenopus laevis were anaesthetized with MS222 (2 g/liter
added to the water). One ovary was removed via a mini-laparotomy, the incision was sutured, and the animal was allowed to recover. Once the wound had completely healed, the second ovary
was removed in a similar operation and the animal was then
killed by decapitation while under anaesthesia. Immature stage
V-VI Xenopus oocytes were incubated for 75 min with 1.0 mg/ml
collagenase (type A; Boehringer Mannheim, Mannheim, Germany) and manually defolliculated. In most experiments, oocytes were injected with ~2 ng of mRNA encoding Kir6.2C26.
For coexpression experiments, ~0.04 ng Kir6.2 or Kir6.2
C26 was coinjected with ~2 ng SUR1 (giving a 1:50 ratio). The final injection volume was ~50 nl/oocyte. Control oocytes were injected with water. Isolated oocytes were maintained in tissue culture and studied 1-4 d after injection (Gribble et al., 1997b
).
Electrophysiology
Macroscopic currents were recorded from giant excised inside-out patches at a holding potential of 0 mV and at 20-24°C (Gribble et al., 1997b). Patch electrodes were pulled from thick-walled borosilicate glass (GC150; Clark Electromedical Instruments,
Reading, UK) and had resistances of 250-500 k
when filled
with pipette solution. Currents were evoked by repetitive 3-s voltage ramps from
110 to +100 mV and recorded using an EPC7
patch-clamp amplifier (List Electronik, Darmstadt, Germany).
They were filtered at 0.2 kHz, digitized at 0.5 kHz using a Digidata 1200 Interface, and analyzed using pClamp software (Axon
Instruments, Burlingame, CA). Single-channel currents were recorded from small inside-out membrane patches. They were filtered
at 5 kHz using an eight-pole Bessel filter and sampled at 10 kHz.
The pipette solution contained (mM): 140 KCl, 1.2 MgCl2, 2.6 CaCl2, 10 HEPES, pH 7.4 with KOH, and the internal (bath) solution contained (mM): 110 KCl, 2 MgCl2, 1 CaCl2, 30 KOH, 10 EGTA, 10 HEPES, pH 7.2 with KOH, and nucleotides as indicated. Tolbutamide was made up as a 0.05 M stock solution, and diazoxide as a 0.02 M stock solution, in 0.1 M KOH. Solutions containing ATP were made up fresh each day and the pH was readjusted after addition of the nucleotide. Rapid exchange of solutions was achieved by positioning the patch in the mouth of one of a series of adjacent inflow pipes placed in the bath.
Data Analysis
All data are given as mean ± SEM. The symbols in the figures indicate the mean and the vertical bars indicate 1 SEM (where this is larger than the symbol). Statistical significance was tested using an unpaired Student's t test, a paired t test, or by analysis of variance, as appropriate.
Macroscopic currents.
The slope conductance was measured by
fitting a straight line to the current-voltage relation between 20
and
100 mV; an average of five consecutive ramps was calculated in each solution. ATP dose-response relationships were
measured by alternating the control solution with a test ATP solution of decreasing concentration. The extent of inhibition by
ATP was then expressed as a fraction of the mean of the value obtained in the control solution before and after ATP application.
ATP dose-response curves were fit to the Hill equation:
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(1) |
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(2) |
Single-channel currents.
Single channel currents were analyzed
using a combination of pClamp and in-house software written by
Dr. P.A. Smith (Oxford University, Oxford, UK). Single-channel
current amplitudes were calculated from an all-points amplitude
histogram. Channel activity (nPo) was measured as the mean
patch current (I) divided by the single channel current amplitude (i), for segments of the current records of ~1 min duration.
Open probability (Po) was calculated from nPo/n, where n is
the number of channels in the patch and was estimated from the
maximum number of superimposed events. In the case of the
C166S mutation, we analyzed patches that showed no superimposed events, and we can be confident that only a single channel
was present because of the high channel open probability. For
analysis of channel kinetics, events were detected using a 50%
threshold level method. Burst analysis was carried out as described by Jackson et al. (1983).
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Kinetic Model
We fitted our data to the kinetic scheme given in Fig. 9, where O is the open state, C1 represents the short closed state observed within a burst of openings, and C2 represents the long closed state observed in the absence of ATP, which governs the interburst duration. In the presence of ATP, two additional closed states were observed: C2(ATP) and C3(ATP). The rate constants for this kinetic scheme were calculated directly from the data, using the following equations:
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(3) |
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(4) |
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(5) |
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(6) |
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(7) |
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(8) |
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(9) |
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(10) |
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where o is the mean open time,
C1 is the mean short closed
time,
C2 is the mean long closed time,
C3 is an additional long
closed state observed only in the presence of ATP, NC1, NC2, and
NC3 are the number of C1, C2, and C3 events, respectively, Po[ATP]
is the open probability in the presence of ATP, and Ki is the ATP
concentration that produces half-maximal inhibition.
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RESULTS |
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The C166S Mutation Alters the Macroscopic Current Properties
Currents recorded from cell-attached patches on oocytes expressing wild-type (wt)1 Kir6.2C26 were very
small, but increased ~20-fold when patches were excised into nucleotide-free solution (Fig. 1 A, a), reflecting the relief of the blocking action of ATP present in
the oocyte cytoplasm. In contrast, when the cysteine at
position 166 was mutated to serine (C166S; Fig. 1 B),
much larger currents were recorded in the cell-attached
configuration, and a smaller increase in conductance
was observed on patch excision (Fig. 1 A, b). After patch
excision, the current at
100 mV increased by 19 ± 2-fold (n = 17) for wtKir6.2
C26 currents and by 1.5 ± 0.2-fold (n = 13) for Kir6.2
C26-C166S currents (Fig.
1 C). This is consistent with our previous observation
that the C166S mutation renders the channel less sensitive to ATP (Trapp et al., 1998
; Tucker et al., 1998
).
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Kir6.2C26-C166S currents were also approximately
sevenfold larger than wtKir6.2
C26 currents in excised
patches. The mean current amplitudes at
100 mV after patch excision were
0.73 ± 0.18 nA (n = 17) for
wtKir6.2
C26 and
4.99 ± 1.17 nA (n = 13) for
Kir6.2
C26-C166S (P < 0.001, t test). Thus, the C166S
mutation may enhance the functional expression of the
channel, and/or augment the open probability, and/
or increase the single-channel conductance. Both native
KATP currents and wtKir6.2
C26 currents exhibit a time-dependent decline in amplitude in excised patches
(Fig. 1 A, a). By contrast, little or no rundown of
Kir6.2
C26-C166S currents was observed (Fig. 1 A, b),
even over a 15-min period. Likewise, while the amplitude of wtKir6.2
C26 currents is often increased above
control levels after removal of MgATP (Tucker et al.,
1997
), a similar "refreshment" was not observed for
Kir6.2
C26-C166S currents (Fig. 2 A). These differences suggest that the C166S mutation impairs channel
rundown: the inability of MgATP to refresh the current
may be a consequence of the fact that channel activity is already maximal, or reflect an additional defect in
the reactivation process itself.
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Fig. 1 D illustrates representative macroscopic current-
voltage relations for wtKir6.2C26 and Kir6.2
C26-C166S
currents. The rectification at positive potentials is not
much altered by the C166S mutation, but at hyperpolarized potentials the current-voltage relation deviates
from linearity.
ATP Sensitivity
The ATP sensitivity of wtKir6.2C26 and Kir6.2
C26-C166S currents is compared in Fig. 2. Kir6.2
C26-C166S currents were always significantly less ATP sensitive, as previously reported (see Trapp et al., 1998
). Although we observed considerable variability in ATP
sensitivity between patches initially, we subsequently discovered that this arises, in part, because the first application of a high concentration of ATP renders the
mutant channel more sensitive to subsequent ATP applications (Fig. 2 A, compare responses to 1 mM ATP).
For this reason, we applied 10 mM ATP for ~30 s to
"sensitize" Kir6.2
C26-C166S channels before measuring the ATP dose-response relationship.
Fig. 2 B shows that wtKir6.2C26 currents were half-maximally blocked (Ki) by 175 ± 27 µM ATP (n = 7): the
Hill coefficient was 0.96 ± 0.12 (n = 7). In contrast, even
as much as 10 mM ATP was not sufficient to inhibit
Kir6.2
C26-C166S currents completely. Consequently,
it was not clear whether ATP is able to block the mutant
channel fully, or if inhibition plateaus out at some saturating level. If it is assumed that the current is completely
blocked by ATP, and the data are fit to the Hill equation, we obtain a Ki of 2.82 ± 0.34 mM and a Hill coefficient of 0.69 ± 0.07 (n = 10) for the Kir6.2
C26-C166S
data. If, instead, we do not assume that the current is
completely blocked at saturating ATP concentrations, the data are best fit with a modified form of the Hill
equation with a Ki of 1.26 ± 0.23 mM, a Hill coefficient
of 0.97 ± 0.13, and a residual conductance of 23 ± 5%
(n = 10) at saturating ATP concentrations. With both
methods of analysis, however, it is clear that the ATP
sensitivity of Kir6.2
C26-C166S channels is significantly
less than that of wtKir6.2
C26 channels.
A mutation may reduce the ATP sensitivity of
Kir6.2C26 in one or more of the following ways: it
may (a) impair the ability of the channel to close, (b)
interfere with the transduction mechanism by which
ATP binding induces channel closure, or (c) reduce
the affinity of the ATP binding site. To distinguish between these possibilities, we examined the single-channel kinetics of wild-type and mutant channels.
Single-Channel Current Analysis
Fig. 3, A and B, shows single-channel currents and
mean single-channel current-voltage relations recorded from inside-out patches excised from oocytes
expressing wtKir6.2C26 and Kir6.2
C26-C166S. The
single-channel conductance, measured between
20
and
80 mV, was 71.7 ± 1.9 pS (n = 3) for
wtKir6.2
C26 and 67.2 ± 1.9 pS (n = 3) for
Kir6.2
C26-C166S, respectively. These values are not significantly different, which indicates that the cysteine
mutation does not alter the single-channel conductance. The larger macroscopic currents observed for
the C166S mutation (Fig. 1) must therefore reflect an
enhanced channel density and/or open probability.
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In contrast to the single-channel conductance, the kinetics of Kir6.2C26-C166S channels were markedly
different from those of wtKir6.2
C26 channels (Fig. 3
A). At negative membrane potentials, both native KATP
channel openings and those of wtKir6.2
C26 occur in
bursts that are separated by relatively long closed intervals (Ashcroft et al., 1988
; Sakura et al., 1995
; Tucker et
al., 1997
; Proks and Ashcroft, 1997
). The duration of
these bursts is prolonged, and the frequency of the interburst intervals is reduced when C166 is mutated to
serine. As a consequence, the open probability at
60
mV was increased approximately sevenfold, from 0.11 ± 0.03 for wtKir6.2
C26 currents (n = 3) to 0.79 ± 0.02 for Kir6.2
C26-C166S currents (n = 6; Table I). This
effect can entirely account for the larger macroscopic
currents observed for Kir6.2
C26-C166S, which were
also approximately sevenfold larger than wtKir6.2
C26 currents.
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Table II compares the kinetic parameters of
wtKir6.2C26 and Kir6.2
C26-C166S currents measured at
60 mV in the absence of ATP. The open time
distribution of wtKir6.2
C26 currents was best fit by a
single exponential, with a mean open time (
o) of 0.79 ± 0.06 ms (n = 3), while the closed time distribution was
fit by the sum of two exponentials, with mean time constants of 0.31 ± 0.03 and 12.6 ± 2.9 ms (n = 3), respectively. The short closed time (
C1) represents the closures that occur within a burst of openings, while the
long closed time (
C2) represents those that occur between bursts. The value of
C1 is similar to that previously reported for wtKir6.2
C26 currents (Proks and
Ashcroft, 1997
) and for both native and wild-type KATP
channels (Table II; Ashcroft et al., 1988
; Proks and Ashcroft, 1997
). The mean open time of wtKir6.2
C26 currents is significantly shorter than that of wtKATP currents (Kir6.2/SUR1; Table II), consistent with earlier
observations that the sulfonylurea receptor influences
channel gating (Proks and Ashcroft, 1997
).
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One effect of mutating C166 to serine on the kinetics of Kir6.2C26 was a dramatic reduction in the frequency with which the channel entered the long
closed state (C2). As Table II shows, the percentage of
closed times accounted for by this state decreased ~80-fold. In addition, the mean open time and the
number of openings per burst were markedly increased, resulting in prolongation of the mean burst
duration by >100-fold (Table II). These effects account for the increase in open probability. Both
C1
and
C2 were unaffected.
Fig. 3 C illustrates the effect of membrane potential on
the open probability of wild-type and mutant Kir6.2C26
channels. While the open probability of wtKir6.2
C26
showed no significant voltage dependence, that of
Kir6.2
C26-C166S decreased slightly with hyperpolarization. The latter effect accounts for the small rectification of the macroscopic Kir6.2
C26-C166S currents
observed over the same voltage range (Fig. 1 D, b).
Since Kir6.2C26-C166S currents do not run down, it
was possible to analyze their kinetics accurately at a
number of voltages in the same patch. We found that
o
slightly decreased, and
C1 slightly increased, on hyperpolarization (Fig. 4). A similar behavior was observed
for the intraburst kinetics of wild-type KATP channels in
ventricular myocytes (Zilberter et al., 1988
). The observed changes in
o and
C1 are not sufficient to account for the decrease in the channel open probability,
suggesting that an increase in
C2 or in the frequency of
the C2 state must also occur. Because of the rarity of the
long closed state in Kir6.2
C26-C166S channels, it was
not possible to measure with sufficient accuracy whether these parameters were changed, but as discussed later
this may be estimated from a kinetic model.
Finally, we explored the effect of ATP on wtKir6.2C26
and Kir6.2
C26-C166S single-channel kinetics (Fig. 5).
Application of ATP to the intracellular solution reduced the open probability of both types of channels,
~40% inhibition being produced by 100 µM and 10 mM ATP in the case of wtKir6.2
C26 and Kir6.2
C26-C166S channels, respectively. As observed for the macroscopic currents, the ATP sensitivity of Kir6.2
C26-C166S single-channel currents was quite variable. It was
not possible to sensitize the channels in the same way as
described for the macroscopic patches because many
single-channel patches did not recover full activity
upon washout of ATP.
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Kinetic analysis of wtKir6.2C26 currents (Table III)
revealed that ATP reduced
o, but was without effect on
C1. Similar results have previously been described for
native KATP channels (Nichols et al., 1991
; Kakei et al.,
1985
). Because of the presence of multiple channels in
the patch, the low open probability, and rundown of
channel activity, it is not possible to give an accurate estimate of the effect of ATP on the long closed state for
wtKir6.2
C26 currents. The high open probability and
lack of rundown of Kir6.2
C26-C166S channels, however, enables a more detailed kinetic analysis. This shows (Table III) that, in addition to decreasing the
open time, ATP reduces the burst duration, increases
the percentage of the long closed state (C2), and causes
the appearance of a further long closed state (C3).
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We also analyzed Kir6.2C26-C166S single-channel
currents recorded in the cell-attached configuration
(Table III). In cell-attached patches, the open probability was higher than that observed for inside-out patches
exposed to 10 mM ATP, and the mean open time was
slightly smaller. Surprisingly, although it is unaffected by ATP in excised patches,
C1 was somewhat longer in
cell-attached patches. Furthermore, we only observed a
single long closed state in the cell-attached configuration, in contrast to the two long closed states found
when inside-out patches were exposed to 10 mM ATP.
These differences in gating between the cell-attached patch and the inside-out patch exposed to ATP argue
that in the intact cell channel activity may be regulated
by cytosolic substances other than ATP, and/or that
disruption of the cytoskeleton on patch excision alters
channel gating.
Effects of Mutating C166 to Other Residues
To further explore how mutation of cysteine 166 affects
the channel gating and ATP sensitivity, we engineered
a range of mutations at this site. The effect of these mutations on the single-channel kinetics, open probability,
and ATP sensitivity is shown in Fig. 6 and Tables I and
II. Replacement of cysteine 166 with valine had little effect on any of these parameters, indicating that a cysteine at position 166 is not critical for channel function and arguing that C166 is probably not involved in disulfide bond formation. Threonine, alanine, methionine,
and phenylalanine at position 166 caused a marked increase in channel open probability and a parallel reduction in ATP sensitivity, similar to that observed with
the serine mutation. As is the case for the C166S mutation, the increase in open probability reflects a marked
reduction in the frequency of the long closed times. Indeed, there was a strong correlation between the effect
of the mutation on the single-channel kinetics and on
the ATP sensitivity (Tables I and II). Currents recorded
for Kir6.2C26-C166L channels were unusual in that
they exhibited three different modes of gating (Fig. 6
B). On rare occasions, the channel kinetics were similar
to those observed for wtKir6.2
C26, while on other,
more frequent, occasions the channel entered a highly
active state resembling that found for Kir6.2
C26-C166S currents. Most of the time, however, the channel exhibited an intermediate form of kinetic behavior and
the data given in Table II was calculated for this mode
of gating. As expected from its intermediate effect on
the single-channel kinetics, leucine produced a smaller
shift in both ATP sensitivity and open probability than
the C166S mutation (Fig. 6 C, Table I).
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Coupling to SUR
We next examined the effect of SUR1 on the open
probability and single-channel kinetics of wild-type and
mutant channels. When SUR1 and wtKir6.2 were coexpressed, the channel open probability was approximately double that of wtKir6.2C26 (Table I). This effect reflects an increase in the mean open time and a
marked reduction in the percentage of long closed
times (Table II). The number of openings per burst
was also increased so that the burst duration of wtKATP
currents was longer than that of Kir6.2
C26 currents.
These effects account for the higher open probability of Kir6.2 currents when coexpressed with SUR1. Similar results have previously been reported for coexpression of Kir6.2
C26 and SUR1 (Proks and Ashcroft,
1997
). In contrast, SUR1 produced only a slight increase in the mean open time of Kir6.2
C26-C166S
channels and did not affect the burst duration (Table
II). Although there is a marked reduction in the percentage of long closed times, this does not produce a
significant increase in the open probability, largely because the long closed state occurs so rarely in the mutant channel. The ability of SUR1 to enhance the ATP
sensitivity of Kir6.2 was also absent when C166 was mutated to serine; indeed, SUR1 appeared to decrease the
ATP sensitivity (Table I). A possible explanation for the
apparent reduction in ATP sensitivity produced by
SUR1 is that, like MgADP, MgATP interacts with the
NBDs of SUR1 to promote KATP channel activity (Gribble et al., 1998
). This effect would be more apparent
for Kir6.2 mutants that show reduced ATP sensitivity.
We also explored whether the C166S mutation influences the ability of SUR1 to endow Kir6.2 with sensitivity to MgADP, diazoxide, and tolbutamide. To ensure
that the KATP current we record only reflects current
flow through channels comprising both Kir6.2 and
SUR1 subunits, we used full length Kir6.2 containing
the C166S mutation and coexpressed it with SUR1.
Like Kir6.2 (Inagaki et al., 1995; Sakura et al., 1995
),
Kir6.2-C166S does not express functional channels independently of SUR1, as only background currents
(similar to those recorded in uninjected oocytes) were observed in patches excised from oocytes expressing
Kir6.2-C166S (not shown). Thus, all channels recorded
from oocytes coinjected with Kir6.2-C166S and SUR1
must be composed of both types of subunit. Fig. 7 A
confirms that SUR1 endows Kir6.2 with sensitivity to the blocking effect of tolbutamide and the potentiatory
actions of MgADP and diazoxide. Diazoxide was tested
in the presence of MgATP because its stimulatory action is dependent upon the presence of intracellular
hydrolyzable nucleotides (Dunne, 1989
; Kozlowski et
al., 1989
). In contrast to wild-type KATP currents, 100 µM MgADP, 200 µM diazoxide (tested in the presence
of 100 µM ATP), and 100 µM tolbutamide did not influence Kir6.2-C166S/SUR1 currents (Fig. 7, B and C).
One possible explanation for the lack of effect of
agents that potentiate channel activity, such as MgADP
and diazoxide, is that Kir6.2-C166S/SUR1 channels are
already fully activated in control solution. This idea is
supported by the fact that diazoxide was able to enhance Kir6.2-C166S/SUR1 currents if the current amplitude was first reduced by 5 mM ATP (Fig. 7, B and
C). Taken together, these results suggest that the coupling of Kir6.2 to SUR1 is partially, but not completely,
impaired by the C166S mutation.
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Fig. 7 C also shows that mutation of C166 to valine
(rather than serine) partially restored the effects of
agents that mediate their effects through SUR1. There
was no significant difference in tolbutamide block of
Kir6.2C26-C166V/SUR1 and wild-type KATP currents (t test), while the difference in the stimulatory effect of
MgADP was barely significant (P = 0.05). The ability of
diazoxide to stimulate channels inhibited by ATP was
also largely restored (compare data in the presence of
100 µM ATP with those in the presence of 100 µM ATP
plus diazoxide; P < 0.001). The inhibitory effect of 100 µM ATP was also enhanced by SUR1 when C166 was
mutated to valine: 100 µM ATP blocked Kir6.2
C26-C166V currents by 40 ± 3% (n = 11), compared with
94 ± 1% (n = 8) when the mutant channel was coexpressed with SUR1 (compare also Figs. 6 C and 7 C).
These results indicate that the coupling of Kir6.2 to
SUR1 is not markedly impaired, if at all, by the C166V
mutation. A disulfide bond between the cysteine residue at position 166 of Kir6.2 and SUR1 is therefore not
essential for subunit interaction.
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DISCUSSION |
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C166 Is Involved in Channel Gating
Our results suggest that the cysteine residue at position
166 plays an important role in the gating of the KATP
channel pore. Mutation of this residue to serine, threonine, alanine, methionine, or phenylalanine causes a
marked increase in the channel open probability, principally by causing a dramatic decrease in the frequency
of the long closed state. Thus, C166 may lie within, or
close to, a gate that is involved in closing the KATP channel. Its location at the cytoplasmic end of the putative
second transmembrane domain (TM2) is consistent
with this idea. Further support is provided by the observation that mutation of the adjacent residue (I167M)
also had a profound effect on channel gating (Tucker
et al., 1998). The fact that the C166S mutation does not
affect the short closed time indicates that the conformational change associated with this state is distinct
from that of the long closed state and suggests that
there may be two distinct gating mechanisms.
Mutation of N160 in TM2 of Kir6.2 to aspartate alters
the rectification properties of the Kir6.2/SUR1 channel (Shyng et al., 1997b), as is expected by analogy with
other Kir channels (Lopatin et al., 1994
), and indicates
that this residue lies within the channel pore. Surprisingly, this mutation also enhanced the channel open
probability, leading Shyng et al. (1997b)
to propose
that N160 may be involved in channel gating. However,
while mutations in C166 can increase Po up to sevenfold, those in N160 produced rather moderate increases in open probability (up to 1.2-fold; Shyng et al.,
1997b
). An alternative hypothesis, therefore, is that mutation of N160 has an allosteric effect on a gate located at the end of TM2. However, since we observed that the
presence of SUR1 affects gating, the possibility that the
N160 mutations affect the link between SUR1 and
Kir6.2, and thereby the channel open probability, cannot be excluded.
A gate located at the intracellular mouth of the pore
has also been postulated for both voltage-gated K+
channels and cyclic nucleotide-gated (CNG) channels
on the basis of mutagenesis studies within, or close to,
the cytosolic end of the sixth transmembrane domain
(S6), which is equivalent to the region of Kir6.2 in
which C166 is located (Liu et al., 1997; Loukin et al.,
1997
; Zong et al., 1998
). Mutations in the S6 domain of
the yeast voltage-gated K+ channel have a similar effect
on the gating kinetics to that of mutating C166 to
serine in Kir6.2: they markedly decrease a long closed
state, and thereby prolong the burst duration and increase the channel open probability (Loukin et al.,
1997
). Likewise, in CNG channels, three amino acids
within the cytosolic domain connecting S6 to the cyclic
nucleotide-binding domain in the COOH terminus
profoundly affect the gating kinetics (Zong et al.,
1998
). Further support for the idea that the cytosolic
end of S6 is involved in channel gating comes from experiments in which cysteines were introduced into this
region of the Shaker K+ channel. Thiol-reactive reagents were able to block the modified channel in the
open state but not in the closed state, arguing that access to the pore is regulated by an intracellular gate (Liu et al., 1997
). This gate may operate as an intracellular lid to the pore, which leaves an internal cavity between the gate and the selectivity filter when it closes,
because large molecules, such as quaternary ammonium ions, can be trapped inside the pore of the Shaker
K+ channel when the gate is closed (Holmgren et al.,
1997
). Whether a similar mechanism operates for the
pore of Kir6.2 remains a matter for speculation.
Kinetic Model of the KATP Channel
To understand the effect of the C166S mutation on the
channel open probability, it is helpful to consider a
model of channel gating. Fig. 8 A shows the simplest
kinetic scheme capable of explaining our data: O is
the open state, C1 represents the short closed state
observed within a burst of openings, and C2 represents the long closed state observed in the absence of
ATP, which governs the interburst duration. The values of the rate constants at 60 mV calculated for
wtKir6.2
C26, Kir6.2
C26-C166S, wtKir6.2/SUR1, and
Kir6.2
C26-C166S/SUR1 currents are given in Table
IV. The major effect of the C166S mutation was a profound decrease in the rate constant k2, which was reduced to <1% of that of the wild-type channel. This indicates that, although the mutation decreases the rate
of entry into the long closed state (C2), it does not influence the exit rate. Thus the higher open probability of Kir6.2
C26-C166S is explained by the fact that this
channel rarely enters the long closed state. The presence of the sulfonylurea receptor also affected the rate
constants, markedly decreasing k2 and slightly reducing
k1, for both wild-type and mutant channels.
|
|
It was not possible to examine the voltage dependence of the rate constants for wtKir6.2C26 currents
because of the presence of channel rundown. We were
able to do so, however, for channels containing the
C166S mutation, which does not run down. The voltage dependence of the rate constants k1, k
1, k2, and k
2 in the absence of ATP, calculated for Kir6.2
C26-C166S
currents, is given in Fig. 8 B. These values were calculated directly from the measured data and provide a
unique solution to the kinetic scheme. Whereas k1 decreases with depolarization, the three other rate constants increase as the membrane potential is made
more positive. The fact that k
2 decreases with hyperpolarization means that
C2 must increase with hyperpolarization and will therefore contribute to the observed decrease in open probability (Fig. 3 C) and rectification of the current-voltage relation (Fig. 1 D, b) at
negative potentials. The values for z
, which reflect the
voltage dependence of gating, were greater than unity
for k2 and k
2, suggesting that more than one charge
must move to gate the channel. Interestingly, there appears to be a small voltage-independent component to
k
1, k2, and k
2, which suggests that part of the gating
mechanism is located outside the membrane voltage
field. Although we cannot measure whether the rate
constants for Kir6.2
C26 channels show a similar voltage dependence, it seems likely that this is the case for
k1, k
1, and k2 since the voltage dependence of the open
and closed times of the native KATP channel (Kir6.2/
SUR1) resembles that of Kir6.2
C26-C166S (Zilberter
et al., 1988
).
Mechanism of Action of C166 Mutations
The residue at C166 is critical for the gating of
Kir6.2C26, since most mutations at this position substantially affected the single-channel kinetics. Two
main modes of gating were observed: the wild-type
mode and a high Po mode found when C166 was mutated to S, T, A, or M. An intermediate mode of gating
was observed with the C166L mutation, but this appeared to be thermodynamically less favorable as it was
less stable and at times the channel switched to either
the wild-type or the high Po mode. The data therefore
suggest that the channel prefers to adopt either the wild-type or high Po mode of gating.
Analysis of a range of mutations at residue 166 of
Kir6.2C26 suggests that for the channel to enter the
long closed state (C2) this residue must be both small
and hydrophobic. Replacement of cysteine by serine is
a relatively conservative substitution, yet it had a dramatic effect on channel gating. The major difference in these two amino acids is their hydrophobicity (2.5 and
0.8, respectively, Table I), which suggests that hydrophobicity may be an important factor. This may also
explain why alanine (which is smaller, but less hydrophobic [1.8], than cysteine) markedly alters the channel kinetics. Size also appears to be a critical factor
since phenylalanine, which has a similar hydrophobicity to cysteine but is much larger, also caused a dramatic increase in the channel open probability.
Effect of Mutations on Channel ATP Sensitivity
The results obtained for channels carrying mutations at position 166 shed some light on the mechanisms by which ATP closes the KATP channel. In particular, they clearly show that ATP does not inhibit the KATP channel by acting as an open channel blocker; if this were the case, an increase in channel open probability should result in an enhanced ATP sensitivity, whereas a reduction in the inhibitory potency of ATP is actually observed. The conclusion that ATP does not block the channel pore is also consistent with the fact that the block by ATP is not voltage dependent.
Our results demonstrate a striking correlation between the effect of mutations at C166 on the channel
open probability and ATP sensitivity. A similar correlation has been observed for mutations at N160, although the absolute changes in both parameters were much less dramatic: Ki shifted to 46 µM and Po increased 1.2-fold with the N160D mutation (Shyng et al.,
1997b). Shyng et al. (1997b)
hypothesized that the apparent decrease in ATP sensitivity resulted from changes
in the channel gating, rather than alteration of ATP
binding, although they did not examine the single-channel kinetics.
To explain how mutation of C166 may alter the ATP
sensitivity, we once again turn to a kinetic model of the
channel (Fig. 9, see Appendix ). In wtKir6.2C26 channels, ATP was without effect on the short closed time
(C1), but reduced the mean open time (Table III). A
reduction in the mean open time with ATP (Nichols et
al., 1991
; Kakei et al., 1985
), and no measurable effect
of ATP on the short closed time (Qin et al., 1989), has
also been reported for native KATP channels. Thus, ATP
either does not bind to the short closed state or, if it
does, this binding does not cause a transition to the
long closed state. By contrast, ATP must interact with
the open state (by interaction we mean that the effect
of bound ATP is manifest when the channel is in a
given state, but do not imply that ATP binding is state
dependent). We introduce a new closed state C2(ATP) to
describe the closed state in which ATP is bound (Fig.
9). As discussed in more detail in the Appendix , it
seems very likely that C2 and C2(ATP) represent equivalent conformational states that differ only in that in
one case ATP is bound to the channel and in the other
case it is not. The effects of ATP on the long closed
times could not be accurately quantified for
wtKir6.2
C26 currents because of the presence of channel rundown and the difficulty of determining whether
the patch contained more than one channel. But it is
clear from simple observation of the current records
that ATP enhances the frequency of the long closed
state (C2), as previously suggested for native KATP channels (Nichols et al., 1991
; Kakei et al., 1985
).
We now consider the effect of mutating C166 to
serine. As is the case for wtKir6.2C26 channels, ATP
was without effect on the short closed time, but reduced the mean open time, of Kir6.2
C26-C166S channels. Because of their high open probability and reduced rundown, we were able to analyze the effect of
ATP on the long closed times of Kir6.2
C26-C166S
channels. We observed that ATP increased the percentage of long closed times and reduced their lifetime
(Table III). It also introduced an additional closed
state, C3. These data suggest that in addition to interacting with the open state, ATP interacts with the long
closed state, C2. In our model, this additional closed
state is designated C3(ATP) (Fig. 9; see Appendix ). Further support for the idea that C2 and C2(ATP) represent
similar conformational states is provided by the fact
that their mean lifetimes must be very similar, as we observe only three exponentials in the closed time distribution. As is clear from Fig. 9, we have assumed that
only a single ATP molecule binds to the channel at a
time; thus, when ATP is bound, the channel will be either in state C2(ATP) or C3(ATP). Consequently, our model
gives a Hill coefficient of 1 for ATP binding, as is observed experimentally.
As described in detail in the Appendix , kinetic analysis of our results favors the idea that the rate constant k2a, like k2, is affected by the C166S mutation. As a consequence, the transition from the open state to the long closed state C2(ATP) is impaired. The reduction in k2a is sufficient to fully account for the observed effect of the C166S mutation on the apparent ATP sensitivity and it is not necessary to assume changes in other rate constants. However, access to the C3(ATP) state is reduced indirectly, due to the diminished occupancy of the state C2, which results from the decrease in k2.
The rate constant k2a contains both ATP-dependent and ATP-independent components as it includes gating of the pore, ATP-binding, and the transduction mechanism by which ATP binding results in channel closure. Our kinetic analysis suggests that the C166S mutation affects only the ATP-independent part of the rate constant k2a (see Appendix ). Thus, it is not necessary to invoke a change in either the affinity ATP binding or the transduction process to explain our results.
Shyng et al. (1997b) proposed that the modest shifts
in ATP sensitivity produced by mutation of N160 are
secondary to changes in the intrinsic gating of the channel. While we also favor this idea, the shift in ATP sensitivity they observed was not large enough to exclude the
possibility that it results from a partial functional uncoupling of Kir6.2 from SUR1, since SUR1 enhances the ATP sensitivity of Kir6.2. When SUR1 is absent, the
Ki for ATP inhibition increases from 10 to 100 µM
(Tucker et al., 1997
), a shift that is greater than that observed for the N160 mutations. This explanation cannot account for our results, however, since we observed
marked shifts in the ATP sensitivity of Kir6.2
C26 in
the absence of SUR1.
Why Do Mutant Channels Not Show Rundown and Reactivation?
Wild-type KATP currents and Kir6.2C26 currents run
down with time after patch excision, but are reactivated
after exposure to MgATP (Tucker et al., 1997
). A similar phenomenon is observed for native KATP channels
(Findlay and Dunne, 1986
; Ohno-Shosaku et al., 1987
).
The mechanism of rundown remains unknown, but
since Kir6.2
C26 currents run down when expressed in
the absence of SUR1, the rundown state cannot represent a dissociation from the sulfonylurea receptor.
Both rundown and reactivation are almost completely
abolished when cysteine 166 is mutated to serine. The simplest explanation for the lack of reactivation is that
the open probability of the mutant channel is already
maximal and thus cannot be further increased. The
reason why rundown does not occur is less clear. One
possibility is that the rate constant for entering the rundown state is dramatically reduced by the cysteine mutation. Another possibility is that the rundown state is
accessed from the long closed state, which is only rarely
entered by the mutant channel. We favor the latter explanation because, in wild-type channels, rundown is
associated with a gradual increase in the frequency and
duration of the long closed state. Furthermore, when
C166 is mutated to valine, which does not affect the
channel kinetics, the currents also run down.
Effect of Mutation of C166 on Coupling to SUR1
When C166 was mutated to serine in the full-length
form of Kir6.2, and the mutant channel was coexpressed with SUR1, the resulting currents were no
longer sensitive to tolbutamide. The simplest explanation of this finding is that SUR1 is unable to couple to Kir6.2-C166S. We do not think this is the case, however,
for two reasons. First, Kir6.2-C166S did not express by
itself, but only when coexpressed with SUR1. This argues that Kir6.2-C166S requires the presence of SUR1
for functional expression, as is the case for wild-type
Kir6.2, and that the mechanism by which SUR1 enables the functional expression of Kir6.2 remains intact in
this mutant. Secondly, diazoxide was able to increase
Kir6.2-C166S/SUR1 currents, providing they were first
partially blocked by high ATP concentrations. It is now
well established that the potentiatory effect of diazoxide is endowed by SUR1 (Inagaki et al., 1996; Gribble
et al., 1997a
; Shyng et al., 1997a
). Thus, this result implies that SUR1 is able to confer diazoxide sensitivity on
Kir6.2-C166S as well as on wild-type Kir6.2. Given that
SUR1 is able to couple to Kir6.2, why is an inhibitory effect of tolbutamide not observed? One possibility is that
it may be a consequence of the change in the gating kinetics since tolbutamide, like ATP, stabilizes the long
closed state (C2) (Gillis et al. 1989
), which is only rarely
accessed in Kir6.2-C166S channels. The fact that tolbutamide inhibition is not compromised when C166 is
mutated to valine, which also does not affect the single-channel kinetics, is consistent with this idea.
Conclusions
In conclusion, our results suggest that the cytosolic end of the second transmembrane domain of Kir6.2 may contribute to an intracellular gate that regulates access to the channel pore and governs the long closed state of the channel. Mutation of C166 to a more hydrophilic or bulkier residue modifies the intrinsic gating properties, such that the channel enters the long closed state more rarely. Because ATP acts by enhancing entry into this state, mutation of C166 to serine (or to threonine, alanine, methionine, or phenylalanine) reduces the ability of ATP to cause channel inhibition. This effect appears to be primarily a consequence of the altered intrinsic gating kinetics rather than any change in the affinity of the ATP-binding site, or in the mechanism by which ATP-binding is linked to gating. The inability of tolbutamide, which also enhances the long closed state, to block Kir6.2-C166S/SUR1 channels is also consistent with this idea. Mutations that alter the apparent ATP sensitivity of the channel cannot therefore be taken to suggest a change in the ATP-binding site unless it can be shown that the intrinsic gating of the channel is unaffected.
![]() |
FOOTNOTES |
---|
We thank Drs. G. Yellen and J. Röper for critical discussion and advice.
This study was supported by the Medical Research Council, the Wellcome Trust, and the British Diabetic Association. S. Trapp holds a fellowship from the Deutsche Forschungsgemeinschaft. S.J. Tucker is a Wellcome Trust Fellow.
Address correspondence to Frances M. Ashcroft, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. Fax: 44-1865-272469; E-mail: frances.ashcroft{at}physiol.ox.ac.uk
Original version received 18 March 1998 and accepted version received 22 May 1998.
Stefan Trapp and Peter Proks contributed equally to this work and should be considered co-first authors. ![]() |
APPENDIX |
---|
The simplest kinetic scheme capable of explaining our data is given in Fig. 9. Here we discuss the major implications of the kinetic analysis of this model.
Effects of the Mutation C166S on the Transition between the Open State and Long Closed States C2, C2(ATP)
Our results favor the idea that the rate constant k2a, like
k2, is affected by the C166S mutation. We have estimated whether this is the case in the following way. We
can calculate k2a for both wtKir6.2C26 and Kir6.2
C26-C166S from the reduction in the mean open time in
the presence of ATP (using Eq. 3), which gives a value
of 1.4 × 106 and 2.9 × 103 s
1 M
1, respectively. It is
also possible to derive k2a independently for the C166S
mutation by simultaneously solving Eqs. 4-9 in the presence and absence of ATP: this gives a value of 4.1 ± 1.7 × 103 s
1 M
1. In our experiments, the mean inhibitory effect of 10 mM ATP on single Kir6.2
C26-C166S
channel currents was less than that measured for the
macroscopic currents, and the Ki for the single-channel currents derived from the kinetic model shown in Fig.
9 was 18 mM, compared with a value of 2.8 mM measured for the macroscopic currents. In contrast, the inhibitory effect of 100 µM ATP on the single-channel
Kir6.2
C26 currents was comparable to that measured
for the macroscopic currents (196 and 175 µM, respectively). To calculate k2a for the C166S mutation, we
therefore used data obtained from the single-channel
patch in which the block by 10 mM ATP was comparable to that observed for the macroscopic currents (62 vs. 68%, respectively). This yields a value for k2a of 9.7 × 103 s
1 M
1. Thus, regardless of how k2a is calculated, its
value is at least two orders of magnitude smaller than
that obtained for the wild-type channel. This indicates
that the C166S mutation impairs the ability of the channel to enter the long closed state C2(ATP).
The rate constant k2a is influenced not only by the intrinsic closing rate of the channel, but also by the affinity of ATP for its binding site and by the mechanism
that transduces ATP binding into changes in channel
gating. A key question, therefore, is whether the C166S
mutation produces a change in the affinity with which
ATP binds to the channel, or if it influences the transduction mechanism. The ratio of the rate constants k2a
for wild-type Kir6.2C26 (wt) and Kir6.2
C26-C166S
(C/S) currents is similar to that obtained for the ratio
of the rate constants k2:
![]() |
(11) |
This suggests that the effect of the C166S mutation on the rate constant k2a, which governs channel closing in the presence of ATP, is similar to that found for the intrinsic closing rate constant k2 (Fig. 9).
We may express the rate constants in terms of Eyring rate theory as follows
![]() |
![]() |
![]() |
![]() |
where is the vibration frequency (6.2 × 1012 s
1 at T = 298°K), and the energy barriers correspond to that for
wild-type Kir6.2
C26 (
GB), an additional barrier introduced by the C166S mutation (
G(C/S)), and a reduction in the energy barrier produced by ATP, which
is
GATP for wild-type Kir6.2
26 and
GATP(C/S) for
Kir6.2
26-C166S. Eq. 11 implies that the reduction of
the energy barrier in the presence of ATP is identical
for Kir6.2
C26 and Kir6.2
C26-C166S channels (i.e.,
GATP =
GATP(C/S)). Therefore, it is unlikely that the
C166S mutation has an effect on the affinity of the
ATP-binding site or the transduction mechanism by
which ATP binding influences gating. This suggests
that the mutation principally affects the transition rate
between the open state and the second closed state C2(ATP). Further,
![]() |
(12) |
Using values calculated for k2(C/S) and k2(wt) in the absence of ATP, G(C/S) = 11.8 kJ/mol. We can also estimate
GATP and
GB, which are
19.4 and 79.4 kJ/
mol, respectively, for wild-type Kir6.2
C26.
Effects of the Mutation C166S on Other Rate Constants
We now consider the effect of the C166S mutation on
the other ATP-dependent rate constants (k2a, k3a, k
3a). From the model shown in Fig. 9, the ratio of the Ki's for
wild-type Kir6.2
C26 and for Kir6.2
C26 carrying the
C166S mutation can be expressed (from Eq. 10) as:
![]() |
![]() |
where Po is the open probability in the absence of ATP. Using the values measured for Po, k2, and Ki (obtained from the macroscopic currents) it can be shown that
![]() |
![]() |
The ratio k2a/k2 is the same for wtKir6.2C26 and for
Kir6.2
C26-C166S (see above). Thus, the apparent
shift in the ATP sensitivity caused by the C166S mutation can be explained by a change in the rate constants
determining the transition from the open state to the
second closed state (k2, k2a), and it is not necessary to
postulate additional effects on other ATP-dependent
rate constants. We cannot completely exclude the possibility that effects on other rate constants do, in fact,
occur, but in this case they must be balanced by a compensating change in another rate constant. It is worth
noting that the above condition for the ratio of ATP-dependent terms also holds for the alternative model
in which the C3(ATP) state is accessed from C2(ATP) rather
than from C2.
Consideration of Other Kinetic Schemes
A variation on the model given in Fig. 9 would be to assume that C3(ATP) is accessed from C2(ATP), rather than
from C2, via an ATP-independent transition. Although
the rate constant k3 will be somewhat smaller in this
case, the model is qualitatively the same as that shown
in Fig. 9. We also considered models in which more
than one ATP molecule interacts with the channel. It is
known from previous studies of the effect of ATP on
the macroscopic current kinetics that KATP channel closure by ATP is a first order process (Qin et al., 1989;
Nichols et al., 1991); that is, the closed state is produced by binding of a single molecule of ATP. However, it remains possible that additional ATP molecules
may bind to the closed state(s). The Hill coefficient of
ATP inhibition will be 1, provided that all states with
more than one ATP molecule bound make a negligible
contribution to the channel open probability. If this is
not the case, and states with two or more ATP molecules bound contribute significantly to the channel open
probability, then the Hill coefficient will be >1 (for a discussion of models with more than one ATP molecule
bound and Hill coefficients >1, see Nichols et al., 1991
).
If we assume that an additional ATP molecule can bind
to the C2(ATP) state of our kinetic scheme to produce
C3(ATP)2 rather than binding to C2 (Fig. 9), then
![]() |
![]() |
Channel activity at different ATP concentrations was calculated from this equation, using the rate constants calculated from measured values determined from the single channel recordings. The relationship between channel activity and [ATP] was the fit with the Hill equation to determine the Hill coefficient of the model; a Hill coefficient close to 2 was obtained. This is greater than that observed experimentally (h = ~1 in all cases) and leads us to favor the scheme shown in Fig. 9. We cannot exclude the possibility that further ATP molecules do, in fact, bind (for example, to C2(ATP) or C3(ATP)), but if this is the case, the closed state(s) they produce must occur very rarely as they are not observed experimentally for the C166S mutation.
![]() |
Abbreviation used in this paper |
---|
wt, wild type.
![]() |
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