From the Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110
KATP channels are a functional complex of sulphonylurea receptor (SUR1, SUR2) and inward rectifier K+ (Kir6.1, Kir6.2) channel subunits. We have studied the role of the putative pore forming subunit (Kir6.2) in regulation of rectification and gating of KATP channels generated by transfection of SUR1 and Kir6.2 cDNAs in COSm6 cells. In the absence of internal polyvalent cations, the current-voltage relationship is sigmoidal. Mg2+ or spermine4+ (spm) each induces a mild inward rectification. Mutation of the asparagine at position 160 in Kir6.2 to aspartate (N160D) or glutamate (N160E) increases the degree of rectification induced by Mg2+ or spermine4+, whereas wild-type rectification is still observed after mutation to other neutral residues (alanine-N160A, glutamine-N160Q). These results are consistent with this residue lining the pore of the channel and contributing to the binding of these cations, as demonstrated for the equivalent site in homomeric ROMK1 (Kir1.1) channels. Since Kir6.2 contains no consensus ATP binding site, whereas SUR1 does, inhibition by ATP has been assumed to depend on interactions with SUR1. However, we found that the [ATP] causing half-maximal inhibition of current (Ki) was affected by mutation of N160. Channels formed from N160D or N160Q mutant subunits had lower apparent sensitivity to ATP (Ki,N160D = 46.1 µM; Ki,N160Q = 62.9 µM) than wild-type, N160E, or N160A channels (Ki = 10.4, 17.7, 6.4 µM, respectively). This might suggest that ATP binding to the channel complex was altered, although examination of channel open probabilities indicates instead that the residue at position 160 alters the ATP-independent open probability, i.e., it controls the free energy of the open state, thereby affecting the "coupling" of ATP binding to channel inhibition. The results can be interpreted in terms of a kinetic scheme whereby the residue at Kir6.2 position 160 controls the rate constants governing transitions to and from the open state, without directly affecting ATP binding or unbinding transitions.
Key words: sulphonylurea receptor; Kir6.2; ATP; gating; rectificationKATP channels are present in most, if not all, excitable
tissues, and share the property of being inhibited by intracellular nucleotide triphosphates (Ashcroft, 1988).
Structurally unique amongst K channels, these KATP
channels are formed by coexpression of an ABC protein (SUR1, or SUR2; Aguilar-Bryan et al., 1995
; Inagaki et al., 1996
) and an inward rectifier K channel subunit (Kir6.1 or Kir6.2; Inagaki et al., 1995a
, b
; Inagaki et
al., 1996
). Expression of Kir6.2 alone does not result in
functional ion channels, suggesting an intimate and
requisite interaction between these two subunits. The
nature of the inhibitory action of ATP is unknown, but
the recent expression of functional KATP channels from
cloned subunits (Inagaki et al., 1995b
; 1996
; Nichols et
al., 1996
) provides the necessary tools to begin to address this important question. Given the presence of
nucleotide binding fold in SUR, and the homology between Kir6.2 and other Kir subunits that form homomeric inward rectifier channels (Nichols and Lopatin,
1997
), it seems likely that nucleotide sensitivity of the
channel resides in the SUR protein and that the ion
channel is formed predominantly by the Kir6.2 subunit. We have recently demonstrated that at least part
of the nucleotide sensitivity of KATP channels is conferred by the SUR subunit (Nichols et al., 1996
; Gribble
et al., 1997
). The present study utilizes mutations in a
putative pore lining residue in Kir6.2 to demonstrate
that Kir6.2 does indeed form the ion-conducting pathway, since mutations at this residue cause changes in
the rectification induced by Mg2+ ions or polyamines
that can be predicted from the effects of similar mutations in homomeric inward rectifying K channels (Fakler et al., 1994
; Ficker et al., 1994
; Lopatin et al., 1994
;
Lu and MacKinnon, 1994; Stanfield et al., 1994
; Wible
et al., 1994
). Significantly, we find that this same pore-lining residue controls the apparent sensitivity of the
channel to inhibition by ATP. Analysis of channel open
probability in the absence of ATP indicates that this effect results from an alteration of the stability of the
open state of the channel, not from a modification in
ATP binding affinity. These results have important implications for the interpretation of experiments assessing nucleotide sensitivity of KATP channels.
Expression of Recombinant KATP Channels in COSm6 Cells
COSm6 cells were plated on glass coverslips at a density of 2.5 × 105 cells per well (30-mm six-well dishes) and cultured in Dulbecco's Modified Eagle Medium plus 10 mM glucose (DMEM-HG), supplemented with FCS (10%). The following day, pCMV-Kir6.2 (5 µg) and pECE-haSUR cDNA (5 µg) were cotransfected into the COSm6 cells with diethylaminoethyl-dextran (0.5 mg/ml). Cells were incubated for 2 min in HEPES-buffered salt solution containing DMSO (10%), and then for 4 h in DMEM-HG plus 2% FCS and chloroquine (100 µM), and then returned to DMEM-HG plus 10% FCS.
Generation of Kir6.2 Mutations
Mutant constructs were prepared by overlap extension at the junctions of the relevant domains by sequential PCR. Resulting PCR products were subcloned into pCMV vector and sequenced to verify the correct mutation before transfection.
86Rb+ Efflux Measurements
For 86Rb+ flux experiments, 86RbCl (1 µCi/ml) was added in fresh DMEM-HG containing FCS (10%) 24 h after transfection. Cells were incubated for 12-24 h before measurement of Rb-efflux. For efflux measurements cells were incubated for 30 min at 25°C in Krebs' Ringer solution, with or without metabolic inhibitors (2.5 µg/ml oligomycin plus 1 mM 2-deoxy-D-glucose), glibenclamide, or diazoxide. At selected time points, the solution was aspirated from the cells and replaced with fresh solution. The 86Rb+ in the aspirated solution was counted.
Patch-clamp Measurements
Patch-clamp experiments were made at room temperature in an
oil-gate chamber which allowed the solution bathing the exposed surface of the isolated patch to be changed in less than 50 ms (Lederer and Nichols, 1989). Shards of glass were removed from the culture dishes and placed in the experimental chamber. Micropipettes were pulled from thin-walled glass (WPI Inc., New
Haven, CT) on a horizontal puller (Sutter Instrument Co., Novato, CA), fire polished, and the tips coated with a 1:1 mixture of
light mineral oil and Parafilm (American National Can Co.,
Greenwich, CT) to reduce capacitative currents. Electrode resistance was typically 0.5-1 M
when filled with K-INT solution (see
below). Microelectrodes were "sealed" onto cells by applying light
suction to the rear of the pipette. Inside-out patches were obtained by lifting the electrode and then passing the electrode tip
through the oil-gate. Membrane patches were voltage-clamped
with an Axopatch 1B patch-clamp (Axon Instruments, Foster City,
CA). PClamp software and a Labmaster TL125 D/A converter
(Axon Instruments) were used to generate voltage pulses. Data
were normally filtered at 0.5-3 kHz; signals were digitized at 22 kHz (Neurocorder; Neurodata, New York, NY) and stored on video
tape. Experiments were replayed onto a chart recorder or digitized into a microcomputer using Axotape software (Axon Instruments). The standard bath (intracellular) and pipette (extracellular) solution used throughout these experiments (K-INT) had
the following composition: 140 mM KCl, 10 mM K-HEPES, 1 mM
K-EGTA, with additions as described. The solution pH was 7.3. Calculations of free [Mg2+] were made with a program written by
M. Kurzmak (Department of Biological Chemistry, University of
Maryland, Baltimore, MD), which was based on the formulations
of Fabiato and Fabiato (1979)
.
Data Analysis
Off-line analysis was performed using ClampFit and Microsoft Excel programs. Stationary fluctuation analysis of macroscopic currents (Neher and Stevens, 1977; Sigworth, 1980
) was performed on short (<1 s) recordings of currents at +50 mV following a
step to zero [ATP] (over which time mean channel activity run-down, or inactivation, was negligible) and in 5 mM [ATP]. Currents (50 pA < mean current < 1 nA, corresponding to ~25-500
channels) were filtered at fc = 3 kHz and digitized at 10 kHz with
12-bit amplitude resolution. Mean channel current (I), and variance (
2) in the absence of ATP were obtained by subtraction of
the mean patch current and variance of patch current in 5 mM
ATP (all channels closed) from mean current and variance estimated in zero ATP. Single channel current (i) was assumed to be
constant at 3.75 pA, corresponding to single channel conductance of 75 pS (see Fig. 1). The mean open probability (PO) was
then calculated by fitting the Eq. 1:
![]() |
(1) |
Model simulations of steady-state [ATP] dose response relationships were generated using Microsoft Excel. Simulations of current relaxations were generated with an adaptive Runge-Kutta method using MathCad5.0+ (Mathsoft Inc.). Wherever possible, data are presented as mean ± SE (standard error). Microsoft Solver was used to fit data by a least-square algorithm.
Asparagine 160 in Kir6.2 Is a Critical Pore Lining Residue in KATP Channels
KATP channels in native tissues do not show strong inward rectification, and neither do exogenous KATP channels generated by coexpression of wild-type Kir6.2 and
SUR1 subunits (Figs. 1 and 2). In the absence of Mg2+
or polyamines the current-voltage relationship through
channels is essentially linear up to about +100 mV. Addition of 20-100 µM spermine, spermidine, or putrescine
induces a very weak inward rectification, detectable
above about +20 mV (Figs. 1 and 2). Work on cloned
inward rectifiers has demonstrated that strong inward
rectification is controlled by a pore lining residue in the M2 transmembrane domain (Stanfield et al., 1994;
Lu and MacKinnon, 1994) and that the presence of a
negative charge at this "rectification controller" position confers strong inward rectification by generation of a high affinity site for the voltage-dependent binding
of cytoplasmic polyamines or Mg2+ (Fakler et al., 1994
;
Ficker et al., 1994
; Lopatin et al., 1994
). Kir6.2 subunits
are homologous to Kir1.1 (ROMK1, Ho et al., 1993
) channels in this region of the M2 segment
both subunits contain an asparagine (N160 in Kir6.2) at the
"rectification controller" position. Mutation of this residue to a negatively charged aspartate (N160D) or
glutamate (N160E) residue results in the expression of
KATP channels that rectify strongly in the presence of cytoplasmic polyamines or Mg2+ ions (Figs. 2 and 3). As
shown in Fig. 1, single channel conductance is unaltered, and, as shown in Fig. 2, channels are still sensitive to inhibition by high concentration (5 mM) of
ATP. Substitution of a neutral glutamine (N160Q, Fig.
2) or alanine (N160A) results in channels that still do
not rectify strongly in the presence of spermine. These
results suggest that KATP channel pores are lined by
Kir6.2 subunits in a directly analogous way to those formed by homologous combinations of Kir1 and Kir2
subfamily subunits.
N160D and N160E mutant channels show identical,
very slow "activation" kinetics resulting from unblock of
spermine (Fig. 2). It has previously been demonstrated
that neutralization of the naturally occurring negative
charge at this position (D172) in Kir2.1 is insufficient
to completely remove polyamine sensitivity (Yang et al.,
1995) and that a second region in the COOH terminus (Taglialatela et al., 1994
), specifically involving E224
(Yang et al., 1995
), is also a contributor to polyamine
binding within the channel pore. It seems that within
the Kir6.2 pore, a single negative charge at position 160 can introduce an extremely high affinity polyamine
binding site, with very slow blocker off-rate (Fig. 2).
Rapid assessment of the blocking ability of other
polyamines and Mg2+ was obtained for wild-type (WT)
and N160D mutant channels using voltage ramps between 100 and +100 mV (Fig. 3). The slow kinetics of
spermine unblock and the slow kinetics of channel deactivation at very negative voltages (see Fig. 2) give rise
to different shaped current-voltage (I-V) relationships
depending on the direction and speed (V/s
1) of the
ramp. In Fig. 3, quasi-steady-state I-Vs were obtained using 1-s ramps. The mutation N160D increases sensitivity of the channels to each of the polyamines and to
Mg2+. As shown in Fig. 1, the single channel conductance of each mutant channel is the same, and as shown
in Fig. 4 B, the density of current through N160D,
N160E, and N160Q mutant channels in patches was
comparable to that through wild-type channels. However, although for the neutral substitution N160Q
there was a similar rate of 86Rb+ efflux when cells were
metabolically poisoned and channels maximally activated (Fig. 4 A), the efflux was reduced to about 40% of
wild type for the N160D mutant channels. This reduction of macroscopic conductance of N160D channels
can then be explained by suggesting that, at the resting
potential of transfected cells (i.e., ~EK), rectification of
the N160D mutant channels is ~60% complete due to
intrinsic polyamine and Mg2+ levels (Shyng et al., 1996
;
Bianchi et al., 1996
). In Rb+ efflux experiments, the
flux through N160A mutant channels was also considerably lower than that through wild-type channels. However, the hydrophobic N160A substitution does not induce strong rectification. In this case, the observed
density of channels formed by the N160A mutant subunits in excised patches was also considerably lower
than that observed with wild type and the other mutant
subunits (Fig. 4 B), suggesting channel formation is impaired by this mutation such that the reduced Rb+ efflux is explained by a lower number of channels. Substitution of positively charged arginine (N160R) or histidine
(N160H) did not result in measurable KATP conductance in excised membrane patches, although 86Rb+ efflux was slightly increased above the untransfected cell
level (Fig. 4 A).
Apparent Nucleotide Sensitivity Is Altered by N160 Mutations
We have recently demonstrated that mutations in the
second nucleotide binding fold of SUR1 abolish the
channel activating effects of MgADP (Nichols et al.,
1996). Mutations at N160 do not abolish the ability of
MgADP to activate recombinant channels (not shown),
but, unexpectedly, they affect the apparent sensitivity to inhibition by ATP itself. Fig. 5 shows the currents recorded from patches expressing wild-type and mutant
channels in response to different [ATP] in the absence
of Mg2+. In each case, the patches were exposed to zero
ATP before and after the test solutions in order to assess the degree of run-down during the maneuver. Fig.
7 A shows [ATP]-response relationships averaged for all
patches in which there was less than 20% run-down during exposure to test solutions. It is clear that certain mutations (N160D, N160Q, and N160E in particular) reduce the apparent sensitivity to inhibition by
ATP (shifting the Ki from 10.4 µM [N160, wild type] to
46.1, 62.9, and 17.7 µM, respectively). Conversely, the N160A mutation causes a slight shift in Ki to lower
[ATP] (6.4 µM).
Mutation N160Q Induces a Prominent Inactivated State
Novel and complicating behavior was observed with
N160Q mutant channels (Figs. 1 D and 4-6). Current
through N160Q channels declines slowly in zero ATP.
However, this current is transiently and repeatedly recovered after returning to ATP-containing solution (Fig. 6). This phenomenon is observed in the complete
absence of Mg2+, indicating that it represents transition
to a reversibly "inactivated" state in zero ATP and distinguishing it from the "run-down" that is observed for
wild-type and mutant channels in zero ATP which can
only be recovered by exposure to hydrolyzable ATP analogues (Findlay and Dunne, 1986; Ohno-Shosaku et
al., 1987
; Nichols and Lederer, 1991
). The rate of this
inactivation was generally well fit by a single exponential in any given patch, and the exponential did not
change with time after patch excision (see Fig. 6 B and C). However, there was considerable variation of the
time course between patches (see Fig. 6 B) from a time
constant of about 1 s to about 18 s (see Fig. 6 C),1 consistent with this process being regulated by the cellular environment such that the rate of inactivation in any
given patch is a reflection of the status of the cell from
which it was isolated. While we have no immediate
physical explanation for the inactivation process, it is
curious that N160Q channels also showed prominent subconductance states. As shown in Fig. 1 D, when a
patch is moved from high [ATP] to zero [ATP], an initial burst of activity rapidly declines to a steady level
where the channel spends a lot of time in subconductance states, the predominant one being about half of
the full open state. It is conceivable that the inactivation process actually represents a switch from the full
conducting to the partially conducting state.
N160 Mutations Affect the ATP-independent Open Probability
Are N160 mutations affecting nucleotide binding? Given
the homology between Kir6.2 and other ATP-insensitive
Kir channel subunits, the lack of reports of permeant
ion effects on ATP inhibition, and the lack of consensus nucleotide binding sites on Kir6.2 subunits, it seems
a reasonable hypothesis that ATP inhibition of channel activity results from ATP interaction with a site on another protein. Observation of the records in Fig. 1 provides a clue to an alternative explanation for the shifts
in Ki. N160D mutant channels have a noticeably higher
open probability in the absence of ATP and polyamines than do wild-type or N160A channels. If the open state
of the channel is stabilized, then the apparent ATP-sensitivity will be shifted for any coupled process, as is the
case for cyclic nucleotide sensitivity of cyclic nucleotide-gated channels (Goulding et al., 1994; Gordon
and Zagotta, 1995
; Varnum et al., 1995
). Given the variable density of expressed channels, very few patches contained only one or a few channels, as necessary to
directly assess channel open probability. To obtain a
quantitative estimate of the channel open probability,
we therefore performed noise analysis of short sections
of recordings from patches containing typically 10-200
channels (see METHODS). Samples of currents were recorded immediately after patch excision in zero ATP
and after exposure to 5 or 10 mM ATP. As shown in
Fig. 7 B, there is a correlation between channel open
probability and apparent Ki for ATP inhibition when
compared between different mutant channels.
A Change in Open Probability Can Account for Shifts in Apparent ATP Sensitivity
We considered the consequences of such changes in
open probability on apparent ATP sensitivity by examining a simplified version of a model for the native KATP
channel (Nichols et al., 1991). This model and other kinetic models for the KATP channel (Qin et al., 1989
) are
based on detailed analysis of the kinetic response of endogenous KATP channels to changes of ATP concentration. They assume that multiple, sequential, ATP binding steps move the channel into closed states that are
increasingly distant from the open state. In a comprehensive version of the model (Nichols et al., 1991
), a
total of four sequential binding steps were required to
give full agreement with kinetic data. In the present
simulations, we have simplified the simulations by reducing the number of ATP binding steps to two, which
adequately predicts steady-state [ATP] dependence (Fig.
8). Previous analysis of single channel events (e.g., Qin
et al., 1989
) have suggested that within bursts there is
no dependence of the open duration on [ATP], and
hence in the present simulations we placed an additional closed state between the first ATP binding step
and the open state (Fig. 8), such that open duration
will be independent of [ATP]. The steady-state [ATP] dependence and peak open probability of the wild-type
channel are well described by this model (Fig. 8), with
the equilibrium constant (KCO) for the allosteric transition between the ATP unbound state (C) and the open
state (O) = 5. The steady-state [ATP]-response curves
and peak open probability of other mutations can be
predicted solely by varying KCO (to values for each mutant of N160D, 80; N160E, 10; N160A, 5). The ability to
reproduce the effects of mutations on both Ki and peak
PO by varying only KCO is consistent with the effect of
mutations at position 160 being to alter the free energy
of the open state relative to the free energy of closed states, with WT and N160A channel open states being
the least stable and N160D being the most stable.
By assigning specific values to individual rate constants (Fig. 9) we can simulate the time dependence of
currents in response to step changes in [ATP]. The
model qualitatively reproduces all of the essential behavior of patch currents, solely by changing KCO, corresponding to alterations in the free energy of the open state. As discussed above, the behavior of N160Q mutant channels differs qualitatively from other channels
in exhibiting a marked inactivation following channel
opening upon exposure to zero ATP (Fig. 6 B). This inactivation is clearly distinguishable from MgATP-dependent run-down (Ohno-Shosaku et al., 1987; Ashcroft, 1988
; Kozlowski and Ashford, 1990
; Takano et al., 1990
;
Nichols and Lederer, 1991
; Tung and Kurachi, 1991
;
Furukawa et al., 1994
; Hussain and Wareham, 1994
; Furukawa et al., 1996
) in being recoverable without exposure to Mg2+-containing solutions. We propose that this
inactivation results again from alteration in the stability
of ATP-independent states. In order to simulate the
N160Q mutant channel behavior, we make the additional assumption that KCO for N160Q channels is the
same as that for N160D channels, but that the KIO is
now also reduced 10-fold from 1.0 to 0.1.
Asparagine 160 of Kir6.2 Is a Critical Pore Lining Residue in KATP Channels
KATP channels in native tissues do not show strong inward rectification (Ashcroft, 1988; Nichols and Lederer, 1991
), and neither do exogenous KATP channels
generated by coexpression of Kir6.2 and SUR1 subunits
(Figs. 1 and 2). Work on cloned inward rectifiers has
demonstrated that strong inward rectification is controlled by a pore lining residue in the M2 transmembrane domain (Lu and MacKinnon, 1994; Stanfield et
al., 1994
) and that the presence of a negative charge at
this "rectification controller" position confers strong
inward rectification by generation of a high affinity site for the voltage-dependent binding of cytoplasmic
polyamines or Mg2+ (Fakler et al., 1994
; Ficker et al.,
1994
; Lopatin et al., 1994
). Kir6.2 subunits are homologous to Kir1.1 (ROMK1, Ho et al., 1993
) channels in
this region of the M2 segment, both subunits contain an asparagine (N160 in Kir6.2) at the rectification controller position. We have introduced mutations at position 160 in order to determine whether this residue
also lines the KATP channel pore and acts as a rectification controller in Kir6.2. Several mutations resulted in
expression of functional KATP channels (Figs. 1-5). In
each of the four expressed mutations, the single channel conductance of fully open channels was unaffected
(Fig. 1), and channels were still sensitive to inhibition
by ATP (Fig. 5). Substitution of a neutral glutamine
(N160Q) or alanine (N160A) resulted in channels with
unchanged rectification properties, but as shown in
Fig. 2, mutation of this residue to a negatively charged
aspartate (N160D) or glutamate (N160E) residue resulted in the expression of KATP channels that rectify
strongly in the presence of cytoplasmic polyamines or
Mg2+ ions. These results suggest that KATP channel
pores are lined by Kir6.2 subunits in a directly analogous way to those that are formed by Kir subunits without an additional SUR-like subunit. Substitution of positively charged arginine (N160R) or histidine (N160H)
did not result in measurable KATP conductance in excised membrane patches, but in Rb efflux experiments
we did observe a slight increase in efflux rate compared
to untransfected cells (Fig. 4 A), indicating that a small
conductance was expressed. Lu and MacKinnon (1994)
have previously demonstrated that the equivalent mutation in ROMK1 results in low level expression of permanently rectified channels. It is possible that such is
the case here. If so, however, expression may be too low
to be detected in patch experiments.
In ROMK1 channels, the mutation N171D (Lu and
MacKinnon, 1994) increases sensitivity to block by spermine by ~3 orders of magnitude. Mutation N160D in
Kir6.2 increases spermine sensitivity ~5 orders of magnitude and leads to a very tight binding of spermine (Fig. 2). The off-rate of spermine from N160D or N160E
mutant channels is 10- to 100-fold slower than in other
Kir channels containing a negative charge at the rectification controller position. This implies that at least one
other, as yet unrecognized, residue must also contribute to stabilizing the binding of spermine in the channel. In all Kir channel subunits examined to date, introduction or removal of a negative charge at this position
leads to a significant increase or decrease in the degree
of rectification induced by Mg2+ or polyamines (Stanfield et al., 1994; Lu and MacKinnon, 1994; Wible et al.,
1994
; Lopatin et al., 1994
; Ficker et al., 1994
; Fakler et
al., 1994
; Yang et al., 1995
), and Reuveny et al. (1996)
have further shown that mutation of D172 to asparagine increases the selectivity of Kir2.1 (IRK1) channels
to Rb+ compared to K+. Previous investigations have
not examined the effects of such mutations on single
channel current. The relatively large single channel
conductance (~75 pS, Fig. 1) facilitates such measurements in channels formed by Kir6.2 subunits. It is apparent that even though large changes in rectification
result from N160 mutations, single channel conductance is unaltered. This result is consistent with earlier
work on voltage-gated K+ channels, which demonstrated
that the H5, or P-loop between the 5th and 6th transmembrane regions (S5 and S6, corresponding to M1
and M2 transmembrane regions in Kir subunits) was
the major determinant of both K+ selectivity and single
channel conductance (Hartmann et al., 1991
; Heginbotham et al., 1992
; Heginbotham and MacKinnon,
1992
; MacKinnon and Yellen, 1990
; Yellen et al., 1991
;
Yool and Schwartz, 1991
). Thus, although position 160 in the Kir6.2 M2 segment contributes to the internal
entrance to the pore, K+ passage through this region is
apparently not the rate limiting step in permeation.
Pore Lining Residues Control Gating of the KATP Channel
Since the Kir6.2 subunit contains no consensus ATP-binding site, it is initially surprising that N160 mutations (that alter the pore structure) should alter the
ATP sensitivity of channel activity (Figs. 4 and 5). It is
clear that certain mutations (N160D, N160Q, and N160E
in particular) reduce the apparent sensitivity to inhibition by ATP, shifting the Ki (ATP concentration causing inhibition to half that in zero [ATP]) from 10.4 µM
(N160, wild type) to 46.1, 62.9, and 17.7 µM, respectively. However, the correlation between apparent Ki
and peak open probability in the absence of nucleotides (Fig. 7) is consistent with the idea that the effect of N160 mutations is predominantly to alter the stability of the ATP-independent open state, and thus indirectly affect the efficacy of ATP inhibition, rather than
altering ATP binding. Such an effect is thus analogous
to those of mutations in cyclic nucleotide-activated ion
channels that alter the activating efficacy, but not the
binding affinity, of cyclic nucleotides for these channels (Goulding et al., 1994; Gordon and Zagotta, 1995
;
Varnum et al., 1995
; Tibbs et al., 1997
), although this
coupling of open probability to apparent ligand sensitivity is a previously unconsidered notion with regards to KATP channels, wherein many studies have demonstrated apparent shifts in [ATP] sensitivity without considering the possibility that open probability might be
the affected parameter (e.g., Cameron et al., 1988
; Findlay and Faivre, 1991
). N160D and N160E mutant effects
are well explained by assuming that the mutations stabilize the open state, and as shown in Figs. 8 and 9, can
be simulated by increasing the equilibrium constant
(KCO) of the allosteric transition between the last closed
and the open state.
The N160Q mutation was unique in uncovering a
marked inactivation following channel opening after
removal of ATP. This result can be explained by assuming that the mutation additionally stabilizes an inactivated state that is reached from the open state. The behavior can be simulated by an additional change in the
KOI equilibrium constant, with KCO shifted to be approximately equal to that resulting from the N160D
mutation (Fig. 8). The physical reality of such alterations
is presumably alterations in the stability of the open
pore that result from changes in the pore structure.
Unfortunately, we are not yet able to construct a consistent picture of the effects of residue substitutions based
on the residue side-chain structure. The stabilization of
the open state by N160D and N160Q mutations might
be consistent with a weak long range electrostatic interaction between N160 and another residue, such that
this interaction is strengthened by substitution of a permanent negative charge (N160D) or by elongating the
side chain (N160Q). The loss of electrostatic interaction by substitution of a hydrophobic residue (N160A)
would lead to destabilization and decreased PO and
hence Ki, as is observed. The substitution of both an
elongated side-chain and a permanent negative charge
(N160E) might then be expected to have an even more
significant effect. However, although by comparison to
wild-type channels the trend towards higher Ki and increased PO is continued, the N160E mutant phenotype
is not quantitatively as strong as predicted. N160Q mutant channels demonstrate a state-dependent inactivation mechanism that is not seen with native KATP channels or with the other pore mutants examined in this
study. Although we presently have no mechanistic explanation for this inactivation, it provides evidence for
a change in the pore structure (or at least of some portion of the channel involving Q160 in the Kir6.2 pore)
before ATP-gating, such that transition to the inactivated state cannot occur when the channel is in the
ATP-bound state. Although there is a present bias towards the assumption that ATP-gating of the channel is
essentially due to interaction with the SUR subunit (Inagaki et al., 1995a, 1996
; Nichols and Lopatin, 1997
), the lack of effect of SUR1 mutations on sensitivity to
ATP inhibition (Nichols et al., 1996
; Gribble et al., 1997
)
combined with this interaction between Kir6.2 pore
structure and ATP gating might indicate that ATP inhibition is more intimately involved with the Kir6.2 subunit than supposed.
In conclusion, the present results establish that the
KATP channel pore is formed from Kir6.2 subunits in an
analogous way to the pores formed by other Kir subunits that do not require a SUR-like subunit for channel activity. The results demonstrate an intimate relationship between pore structure and nucleotide gating
of the channel, and demonstrate how the stability of
the open channel can be critical in determining apparent inhibitory affinity of gating nucleotides. The results
have further implications for the interpretation of studies examining the ATP-sensitivity of KATP channels. The
simulations demonstrate how it is possible to generate
changes in the Ki for nucleotide inhibition of current, without altering nucleotide binding itself. Thus, for instance, the difference in ATP sensitivity reported for
KATP channels formed from SUR1 or SUR2 subunits
(Inagaki et al., 1996) may not necessarily reflect differences in nucleotide binding to the two subunits. Indeed, it is apparent from published records comparing
channels formed by coexpression of Kir6.2 with SUR1
and with SUR2 (Inagaki et al., 1996
), that intra-burst
open probability is higher when channels are formed
with the latter sulfonylurea receptor subunit.
Original version received 6 March 1997 and accepted version received 20 May 1997.
We are grateful to Dr. S. Seino for providing us with the Kir6.2 clone, and to Dr. Joe Bryan and Jack Clement IV (Baylor College of Medicine) for discussion during the course of this work and for providing us with the N160D mutant of Kir6.2.
This work was supported by grants HL451231 and HL54171 from the National Institutes of Health (C.G. Nichols) and an Established Investigatorship from the American Heart Association (C.G. Nichols).