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The Journal of Neuroscience, August 1, 2002, 22(15):6499-6506
Localization of the Activation Gate for Small Conductance
Ca2+-activated K+ Channels
Andrew
Bruening-Wright1,
Maria A.
Schumacher2,
John
P.
Adelman1, and
James
Maylie3
1 Vollum Institute and Departments of
2 Biochemistry and Molecular Biology and
3 Obstetrics and Gynecology, Oregon Health and Sciences
University, Portland, Oregon 97201
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ABSTRACT |
Small conductance Ca2+-activated
K+ (SK) channels open in response to increased
cytosolic Ca2+ and contribute to the
afterhyperpolarization in many excitable cell types. Opening of SK
channels is initiated by Ca2+ binding to calmodulin
that is bound to the C terminus of the channel. Based on structural
information, a chemomechanical gating model has been proposed in which
the chemical energy derived from Ca2+ binding is
transduced into a mechanical force that restructures the protein to
allow K+ ion conduction through the pore. However,
the residues that comprise the physical gate of the SK channels have
not been identified. In voltage-gated K+ (Kv)
channels, access to the inner vestibule is controlled by a bundle
crossing formed by the intracellular end of the sixth transmembrane
domain (S6) of each of the four channel subunits. Probing SK channels
with internally applied quaternary amines suggests that the inner
vestibules of Kv and SK channels share structural similarity. Using
substituted cysteine accessibility mutagenesis, the relatively large
molecule [2-(trimethylammonium)] methanethiosulfonate accessed
positions near the putative bundle crossing more rapidly in the open
than the closed state but did not modify S6 positions closer to the
selectivity filter. In contrast, the smaller compound, 2-(aminoethyl)
methanethiosulfonate (MTSEA), modified a position predicted to lie in
the lumen immediately intracellular to the selectivity filter
equivalently in the open and closed states. The pore blocker
tetrabutylammonium impeded MTSEA access to this position in both open
and closed channels. The results suggest that the SK channel gate is
not formed by the cytoplasmic end of S6 but resides deep in the channel
pore in or near the selectivity filter.
Key words:
SK channel; pore; activation gate; cysteine scanning; gating; inner vestibule
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INTRODUCTION |
Three highly homologous small
conductance Ca2+-activated
K+ (SK) channel subunits have been cloned
(SK1, SK2, and SK3), each containing six putative transmembrane
segments with predicted topologies similar to voltage-gated
K+ (Kv) channels (Kohler et al., 1996 ).
The fourth transmembrane domain, S4, contains positively charged
residues, but SK channels are not gated by voltage. Rather, they are
gated solely by intracellular Ca2+ ions.
SK channels function as heteromeric complexes with calmodulin (CaM)
that is constitutively attached to a binding domain, the CaMBD, in the
membrane-proximal region of the intracellular C terminus of the
channel. CaM functions as the Ca2+ sensor
for SK channels, transducing the
Ca2+-gating signal through the CaMBD to a
yet unidentified activation gate (Xia et al., 1998; Keen et al., 1999;
Schumacher et al., 2001).
Crystal structure and electron paramagnetic resonance (EPR)
studies of the bacterial K+ channel, KcsA,
and structure-function studies of Kv and cyclic nucleotide-gated (CNG)
cation channels have given rise to mechanistic models for the coupling
of the gating cue to channel opening (Perozo et al., 1999; del Camino
et al., 2000; Flynn et al., 2001). Within the KcsA channel, the four M2
segments form a vestibule in the shape of an inverted teepee (Doyle et
al., 1998). The bundle crossing near the membrane interface with the
cytoplasm forms a structure like the "smokehole" of a teepee
that constitutes the physical gate of the channel. Rearrangement of the
helices in response to the gating cue opens or closes the smokehole.
This model is supported by EPR measurements that detected relatively
large movements near this region of KcsA during gating and smaller
movements on the intracellular side of the selectivity filter (Perozo
et al., 1999). Further investigation of KcsA has led to the hypothesis that selectivity filter conformational changes during gating are coupled to the rearrangement of the channel activation gate (Perozo et
al., 1999; Liu et al., 2001; Zhou et al., 2001b).
For Shaker Kv channels, the substituted cysteine accessibility method
(SCAM) (Liu et al., 1997; del Camino et al., 2000, 2001; Yellen, 2001)
and cross-linking experiments (Holmgren et al., 1998) strongly suggest
that the S6 bundle crossing forms the activation gate. Thus, for both
KcsA and Kv channels, the residues that form the activation gate are
believed to reside at the cytoplasmic end of the inner vestibule. In
contrast, studies of the CNG channel suggest that the selectivity
filter of the CNG1 channel functions as the channel gate (Sun et al.,
1996; Becchetti et al., 1999; Flynn et al., 2001). This is particularly
relevant to SK channels, because both CNG and SK channels are voltage
independent, relying on the binding of an intracellular ligand to a
C-terminal domain for channel gating (Zagotta and Siegelbaum, 1996; Xia
et al., 1998; Keen et al., 1999).
In this study, block by intracellular quaternary amines and SCAM were
used to probe the topology of the SK2 channel inner vestibule, examine
conformational changes during SK channel gating, and test whether the
distal domain of S6 forms an SK activation gate.
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MATERIALS AND METHODS |
Molecular biology. Constructs were subcloned either
into pJPA5 for expression in COSm6 cells or into the oocyte expression vector pBF. In vitro mRNA was synthesized from pBF
constructs using SP6 polymerase (Invitrogen, Gaithersburg, MD).
Site-directed mutagenesis was performed using pfu polymerase
(Stratagene, La Jolla, CA) and the overlap PCR technique (Ho et al.,
1989). The complete nucleotide sequences of the coding regions were
verified before expression studies.
Electrophysiology. For COSm6 experiments, cells were
transfected using Polyfect reagent (Qiagen, Valencia, CA) as per
the manufacturer's instructions and plated on Fisherbrand Growth
coverslips (Fisher Scientific, Houston, TX); currents were recorded
24-48 hr after transfection. For oocyte experiments, 50 nl of mRNA
(20-50 ng) per oocyte was microinjected as described previously, and currents were recorded 2-7 d after injection. Bath solution contained either 150 mM (COSm6) or 120 mM (oocyte) potassium methanesulfonate, 10 mM HEPES, 1 mM EGTA, and
CaOH to yield the desired free Ca2+
concentration and was adjusted to a pH of 7.2 with methanesulfonic acid
(Fabiato and Fabiato, 1979). Free Ca2+ for
all open-state experiments was adjusted to 1 µM
to maximally activate the channels, except for A392C, which was
adjusted to 10 µM. For closed-state
experiments, no Ca2+ was added (0 Ca2+), yielding a free
Ca2+ level of <2
nM. Inside-out patches were pulled using
borosilicate glass patch electrodes filled with 10 µM Ca2+ solution
and pulled to resistances between 1 and 3 M . Leak and background
currents were measured by changing the bath solution on the inside face
of the patch to 0 Ca2+ to close SK2
channels. Rapid solution changes were performed using an RSC-200
(Molecular Kinetics, Pullman, WA). Currents were measured and
digitized with an EPC9 (Heka Elektronik, Lambrecht/Pfalz, Germany),
currents were sampled and filtered at 1 kHz, and analysis was performed
using Pulse (Heka Elektronik) and Igor (Wavemetrics, Lake Oswego, OR)
software. No differences in Ca2+
sensitivity, gating kinetics, or methanethiosulfonate (MTS)
reactivity were observed between patches from oocytes or COSm6 cells,
and in some cases, results obtained from both expression systems were combined. All reagents were diluted from concentrated stocks prepared the day of the experiment, and MTS compounds were used within 30 min of
mixing into solution.
Data analysis. All values are reported as the mean ± SEM of n experiments. Statistical significance was evaluated
using a Student's t test, and a p value of
 0.05 was considered significant. Modification rates were related to
the time constant ( ) of single exponential fits to plots of current
amplitude versus MTS exposure duration by the reciprocal of × MTS, where MTS was the concentration of [2-(trimethylammonium)]
methanethiosulfonate (MTSET) or 2-(aminoethyl) methanethiosulfonate
(MTSEA) used in the experiment. Dose-response relationships for
tetraethylammonium (TEA), triethylhexylammonium (C6-TEA),
tetrabutylammonium (TBuA), and tetrahexylammonium (THexA) were fit with
a single binding isotherm: Icontrol × X/(X + IC50), where
X is the concentration of blocker,
Icontrol is the current amplitude
before application of blocker, and IC50
represents the concentration at which macroscopic current is half blocked.
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RESULTS |
At least four gating models may be postulated for SK channels
(Fig. 1A). Channel
opening and closing could occur by: (1) a smokehole at the S6 bundle
crossing similar to the KcsA and Kv channel-gating models (model a),
(2) an expansion or collapse of the inner vestibule (model b), (3) a
"ball-and-chain" mechanism analogous to that which underlies N-type
inactivation of Kv channels, perhaps with CaM as the blocking particle
(model c), and (4) a rearrangement of the selectivity filter (model d).
The different models were tested by examining the topology and
gating-dependent conformational changes in the inner vestibule using
excised membrane patches containing heterologously expressed SK2
channels.
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Figure 1.
Possible SK channel-gating mechanisms.
A, SK channel-gating models. In each model, two of four
subunits are depicted in cross section embedded in the membrane with
CaM (dumbbells) associated with the intracellular
C-terminal domain. For the open state, the SK-CaM complex is modeled
as a dimer-of-dimers, as indicated by the
CaMBD/Ca2-CaM crystal structure. In the four
closed-state models (a-d), the CaMBD/CaM complex is
monomeric (Schumacher et al., 2001). a, The S6 bundle
crossing acts as the gate preventing access to the lumen and
selectivity filter, as suggested for Kv and KcsA channels.
b, Collapse of the inner vestibule, including the
aqueous lumen, closes SK channels. c, CaM may act as a
blocking particle in a ball-and-chain type mechanism. d,
Selectivity filter rearrangement prevents ion permeation in closed SK
channels, as suggested for ligand-gated CNG channels. B,
Sequence alignments of S6 and proximal cytoplasmic residues of Shaker
B, a cyclic nucleotide-gated channel (CNG1), and the
proton-gated bacterial K+ channel KcsA. Boxed
region, Amino acids 386-403 examined with the SCAM technique
in this study. Numbered arrows, Residues of
particular importance (see Results). Shaded residues,
Residues conserved in all SK family members.
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A conserved inner vestibule topology between SK and
Kv channels
Small quaternary amines, such as TEA, have been used to probe the
architecture of the inner pore region of Kv channels, providing evidence for an inner vestibule within the membrane field that is
accessed only in the open state (Armstrong and Hille, 1972; Choi et
al., 1993; Holmgren et al., 1997; Liu et al., 1997). Furthermore, these
experiments showed that increasing the length of one or more of the
four hydrophobic side chains of TEA increases the potency of block,
suggesting that a hydrophobic pocket stabilizes the interaction between
channel and pore blocker (Choi et al., 1991, 1993). Despite limited
primary sequence homology between SK and Kv channels in the inner
vestibule region, both channel types have highly hydrophobic S6 domains
(Fig. 1B).
Dose-response relationships for block of SK2 by TEA and three of its
derivatives, TBuA, THexA, and C6-TEA, were obtained by applying
compounds to the intracellular face of inside-out patches (data not
shown). TEA only weakly blocked SK2 (IC50 = 46.8 ± 2.2 mM; n = 8). However,
increasing one of the four side chains from two to six carbons (C6-TEA)
decreased the IC50 more than fivefold (8.6 ± 0.4 mM; n = 3). When all four
side chains were increased from two to four carbons (TBuA), the
IC50 decreased ~26-fold (1.8 ± 0.2 mM; n = 6). Increasing all of the
side chains to six carbons (THexA) increased blocking potency
>3000-fold (0.014 ± 0.002 mM; n = 5). Therefore, the rank order of potency for block
of SK2 channels by these compounds, THexA > TBuA > C6-TEA > TEA, is similar to that determined for Kv channels,
although in general, the IC50 was lower for SK2
than for Shaker channels (e.g., ~120 times lower for TEA and 86 times
lower for C6-TEA) (Choi et al., 1993). For both channel types, the
potency of block increased with increased side-chain length. Moreover,
as for Kv channels, SK channel block by all of the compounds was
voltage dependent. Ratios of mean IC50 at 80 mV
to mean IC50 at  80 mV were 0.16, 0.04, 0.08, and 0.17 for TEA, C6-TEA, TBuA, and THexA, respectively. The results suggest open-state access by TEA derivatives to an inner vestibule within the SK2 membrane field.
State-dependent conformational changes in the S6 helix
State-dependent accessibility to the inner vestibule of SK2
channels was explored using SCAM and cysteine-reactive reagents. Each
of nine residues in S6 and eight residues C-terminal to S6 were
individually mutated to cysteine (Fig. 1B) and
assayed for reactivity with MTS compounds when the channels were either
open or closed. If the side chain of the introduced cysteine residue resides in the conduction pathway and is available to MTS compounds, then disulfide bond formation may irreversibly reduce
K+ permeation either through electrostatic
or steric hindrance (Karlin and Akabas, 1998). If the cysteine residue
points away from the pore, buried in the lipid bilayer or surrounding
protein, or if MTS reagents do not attach or attach only very slowly,
little if any current reduction will be observed. Thus, the
availability and orientation of specific residues can be tested in the
open and closed state.
Inside-out patches containing heterologously expressed wild-type (WT)
or cysteine-substituted SK2 channels were exposed to 1 mM
MTSET for 8 sec in either the open or closed state. This concentration
and duration of exposure were sufficient to reveal sites that reacted
quickly with MTSET and to reveal strong state dependence while avoiding
the complications of channel rundown observed in some of the mutant
channels. Representative traces showing the effects of MTSET exposure
in the open and closed states are presented in Figure
2 for WT and two different
cysteine-substituted sites in SK2, A392C, and R396C. For open-state
experiments, channels were first closed and opened, confirming rapid
solution exchange (typical time constants of 20 and 50 msec for
activation and deactivation using 1 µM and 0 Ca2+ solutions, respectively). Channels
were then exposed to 1 mM MTSET for 8 sec, and the fraction
of irreversibly blocked current was measured after MTSET washout (Fig.
2A, left). WT SK2 contains nine native
cysteine residues, including one at position 386 in S6 (Fig.
1B). As shown in Figure 2A
(left), WT SK2 channels were rapidly and reversibly blocked
by ~14% in the open state, as current recovered to ~97% of the
control level after MTSET washout. This suggests that MTSET does not
react with endogenous cysteines to block ion permeation but rather
behaves as a reversible pore blocker. To evaluate reactivity in the
closed state, channels were closed and then exposed to MTSET for 8 sec,
washed in 0 Ca2+ solution for 2 sec, and
reopened to determine the amount of irreversible block. Figure
2A (right) shows that for WT channels, the
current after closed-state MTSET exposure was ~96% of the control
current. These results demonstrate that MTSET did not irreversibly
react with WT SK2 in either the open or closed states (open-state
current reduced by 3.6 ± 1.1%, n = 8;
closed-state current reduced by 3.8 ± 1.3%, n = 6). In contrast, R396C, which is located just inside the cytoplasm
beneath S6, demonstrated irreversible reactivity with MTSET in both
states (open-state current reduced by 44.8 ± 3.7%,
n = 5; closed-state current reduced by 49.7 ± 4.5%, n = 3) (Fig. 2C), suggesting that
this site is available to MTSET regardless of whether channels are open
or closed. A392C, which is located between the selectivity filter and
putative bundle crossing, showed state-dependent modification by MTSET,
reacting rapidly and irreversibly in the open state and not in the
closed state (Fig. 2B). The current was reduced by
69.4 ± 5.4% (n = 8) and 1.6 ± 6.6%
(n = 9) in the open and closed states, respectively, suggesting that conformational changes occur at or near position 392 during gating.
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Figure 2.
MTSET reactivity in WT and cysteine-substituted
SK2 channels. A-C, Representative traces showing open-
and closed-state MTSET reactivity for WT, A392C, and R396C,
respectively. For open-state experiments (left),
channels were first closed and then reopened using saturating
Ca2+ solutions to verify adequate exchange rates.
Next, MTSET (1 mM) was applied to open channels for 8 sec,
MTSET was washed out, and current amplitudes before and after MTSET
application were compared. For closed-state experiments
(right), MTSET (1 mM) was applied to closed
channels (0 Ca2+) for 8 sec and washed out with 0 Ca2+ solution for 2 sec, channels were reopened, and
current amplitudes were compared before and after closed-state
application. Bars above the traces indicate when MTSET
was applied, the dashed line indicates 0 current level,
and the solid line above the trace
indicates Ca2+ steps from saturating (1 µM; O, open state) to 0 Ca2+ (<2 nM; C, closed
state) solution.
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To obtain more information about accessibility of introduced cysteines,
the modification rates by MTSET were determined for both open and
closed states. The open-state modification rate was defined by the
inverse exponential constant of a single exponential fit to the decay
of the current amplitude plotted as a function of the product of
cumulative exposure time and MTSET concentration (Fig.
3B,D). For the representative
traces shown in Figure 3B,D, open-state exponential
constants were 0.2 and 0.1 mMsec and rates were
4.2 × 103 and 1.1 × 104
M 1sec1
for A392C and R396C, respectively. Closed-state modification rates were
determined by repeating the closed-state protocol (Fig. 2, right
panels) several times. Current amplitudes after MTSET washout and
channel reopening were measured after each 8 sec exposure (Fig.
3A,C) and plotted as a function of the product of cumulative exposure time and MTSET concentration (Fig. 3B,D,  ). The
data points were fitted by a single exponential, and the inverse of the
exponential constant defined the closed-state modification rate. For these A392C and R396C examples, closed-state exponential constants were 65.9 and 0.2 mMsec and rates were
15 and 5944 M1sec1,
respectively. For comparison, the open- and closed-state modification time courses were overlaid for A392C in Figure 3B and for
R396C in Figure 3D.
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Figure 3.
Determination of MTSET modification rates.
A, Repeated closed-state 1 mM MTSET
application to A392C channels using the protocol of Figure 2.
B, Open-state (O, black
trace) and closed-state (C, ) decay time
courses for A392C normalized to control current and plotted versus the
product of cumulative MTSET exposure and MTSET concentration
(mMsec). The open-state trace is taken from Figure
2B, and the closed-state decay is determined by
measuring current amplitude after each of the 10 applications of MTSET
shown in A. A single exponential was fit to the data
yielding an exponential constant of 0.2 and 65.9 mMsec for
the open and closed state, respectively. C, Closed-state
modification at position R396C. Channels were repeatedly exposed to 10 µM MTSET according to the closed-state protocol shown in
Figure 2. D, Open-state (O, solid
trace) and closed-state (C, ) decay time
courses of R396C normalized as in B. The open-state
trace is taken from Figure 2C, and the closed-state rate
was determined by measuring the current amplitude after each of the 10 applications of MTSET shown in A. A single exponential
was fit to the data yielding an exponential constant of 0.09 and 0.17 mM for open and closed states, respectively.
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A summary of the MTSET results for the different cysteine-substituted
channels is presented in Figure 4,
showing the percentage of inhibition after a single 8 sec exposure to
MTSET for each position in the open and closed states (Fig.
4A) and the modification rate for open and closed
channels (Fig. 4B). Positions without bars either did
not yield current, or current amplitudes were too small for reliable
measurements (Fig. 4A). MTSET modification rates were
determined as shown in Figure 3 for two positions deep in the inner
vestibule (386C and T387C) and four positions in the putative
bundle-crossing region (391, 392, 395, and 396). Current through WT or
T387C channels was not affected by MTSET exposure in either the open or
the closed state (modification rates < 2 M1sec1).
Positions V391C, A392C, and V393C within S6 showed significant state
dependence after a single MTSET exposure, and V391C and A392C were
modified significantly faster in open than in closed channels (V391C:
47.9 ± 11.4 M1sec1,
n = 4; 6.7 ± 2.1 M1sec1,
n = 5; p < 0.05; A392C: 1.5 ± 0.4 × 103
M1sec1,
n = 8; 33.6 ± 6.3 M1sec1,
n = 6; p < 0.05 for open and closed
states, respectively). Modification of position 395 resulted in
significant block in either state with channels in the closed state
being more rapidly modified than those in the open state (open state:
9.7 ± 1.6 × 102
M1sec1,
n = 4; closed state: 3.9 ± 2.4 × 103
M1sec1,
n = 5; p < 0.0001). State-independent
modification was clearly observed for position 396 that resides on the
S6-cytoplasm interface (open state: 7.4 ± 2.4 × 103
M1sec1,
n = 3; closed state: 5.9 ± 0.6 × 103
M1sec1,
n = 6; p > 0.5). Further into the
cytoplasmic domain, at positions 398 and 399, the percentage of
closed-state inhibition was greater than open-state inhibition (Fig.
4A). Significant MTSET reactivity was not detected at
positions N-terminal to 391 in the open or closed states. It is
important to note the differences between the percentage of inhibition
after an 8 sec exposure to MTSET and the modification rate. For
example, Figure 4A shows a much higher percentage
block for A392C in the open than in the closed state after an 8 sec
exposure. However, the steady-state percentage block by MTSET during
repeated application to closed A392C channels was similar to that in
the open state (Fig. 3A). This is because access to position
392 is strongly state dependent, and therefore the modification rate is
considerably slower in the closed state than in the open state,
although the block eventually reaches the same level. In contrast, an 8 sec MTSET application to R396C channels produced equivalent current
reductions, and the rates of modification in the open or closed states
were similar (Figs. 3D, 4B). Together, the
results suggest that the S6 region of SK2 channels undergoes
conformational changes during gating that alter MTS access to several
positions.
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Figure 4.
Summary of MTSET reactivity for WT and
cysteine-substituted channels. A, Reactivity was
measured for open (gray bars) or closed
(black bars) channels, and the dashed
line indicates the predicted boundary between the cytoplasm and
membrane. Data from n  3 patches were normalized
and averaged and plotted as the percentage of inhibition ±SEM after an
8 sec exposure to 1 mM MTSET. Note that positions 391, 392, and 393 show stronger open- than closed-state reactivity, whereas
positions at the predicted membrane/cytoplasm boundary (395 and 396)
show equal open- and closed-state reactivity. Cytoplasmic positions
(398 and 399) show stronger closed- than open-state reactivity.
*Statistically significant differences between open- and closed-state
reactivity (p < 0.05). Reactivity at
position 390 could not be reliably measured (<10% current reduction).
B, Open-state ( ) and closed-state () modification
rates for WT, T387C, and four residues in the putative bundle-crossing
region. Rates were determined from single exponential fits to current
decay (see Results) and are presented as mean ± SEM from
n 3 patches.
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Localization of the SK2 activation gate
The open-state-dependent access to positions 391-393 can be
interpreted in three ways: (1) these residues rotate away from the pore
lumen during channel closing, (2) a gate C-terminal to 393 occludes
MTSET access when channels are closed, and (3) the inner vestibule
narrows when channels close, and this constriction restricts access to
these positions.
Compared with a K+ ion (Pauling radius of
1.33 Å), MTSET is a relatively large molecule that may be modeled to
fit in a cylinder ~5.8 Å in diameter and 10 Å long (Karlin and
Akabas, 1998; Flynn and Zagotta, 2001) and so may be sterically
hindered from reporting the availability of residues that
K+ ions might access, such as those
N-terminal to position 391. MTSEA is a smaller, positively charged
compound (head group diameter of ~3.6 Å) that has been used to
reveal state-dependent access to sites in the inner vestibule of Shaker
channels and in the pore of CNG channels (Liu et al., 1997; Liu and
Siegelbaum, 2000). Modification by MTSEA of residues examined in Figure
4 yielded results similar to those for MTSET. One important difference
was that MTSEA could additionally access T387C, a site deeper in the SK2 inner vestibule that was not modified by MTSET (Fig.
5). Application of MTSEA (2.5 mM) to open or closed T387C channels resulted in complete
block within 8 sec (open-state inhibition, 99.9 ± 1.2%, n = 4; closed-state inhibition, 94.2 ± 7.2%,
n = 6). WT channels containing 386C were not
significantly altered by MTSEA in either the open state (inhibition of
2.5 ± 3.3%; n = 7) or closed state (increase of
1.3 ± 2.1%; n = 3), and the double mutation
C386S and T387C was completely blocked by MTSEA in both the open
(99.6 ± 6%; n = 5) and closed (97.9 ± 4%;
n = 3) states, demonstrating that MTSEA reacts
specifically at position T387C. The rate of modification of T387C by
MTSEA was determined as described for MTSET, except that the exposure
time during repeated MTSEA application in the closed state was
decreased from 8 to 1 sec to accurately record channel modification
(Fig. 5B). The open-state and closed-state modification
rates of T387C by MTSEA were not significantly different (open state,
379 ± 109 M1sec1,
n = 6; closed state, 286 ± 26 M1sec1,
n = 3; p > 0.4). Modification rates
for WT channels were <1 M1sec1 in
either the open or closed states.
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Figure 5.
MTSEA reactivity for WT, T387C, and T387C/C386S
channels. A, Current reduction was determined for the
three channels using 8 sec applications of 2.5 mM MTSEA, as
described for MTSET in Figure 2. *T387C, T387C in the
C386S background. B, Open-state (O,
black trace) and closed-state (C,  )
modification of T387C by MTSEA. Data are plotted and fitted as for
MTSET in Figure 3. Single exponential fits to the data yielded
exponential constants of 1.7 and 3.5 mMsec for open and
closed states, respectively.
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One concern when using MTSEA is that it may cross the membrane, in this
case, accessing T387C from the external vestibule and causing
trans modification. This possibility was addressed by
including 10 mM cysteine in the external patch
pipette solution (Holmgren et al., 1996), which had no effect on the
extent or rate of modification (data not shown). It is also possible
that MTSEA in an uncharged form might access position 387 by
partitioning into the membrane or hydrophobic portions of the protein.
If MTSEA accessed T387C only through the pore, then preapplication of
TBuA, a pore blocker, should restrict MTSEA access to the site.
Therefore, open or closed T387C channels were first exposed to 10 mM TBuA (~97% block of open-state current).
Then, in the continued presence of TBuA, 2.5 mM
MTSEA was applied for 8 sec. After washout of both compounds, current
amplitudes before and after exposure were compared (Fig.
6A, inset).
The protocol was repeated several times, and the data were plotted and
fitted as described for Figure 3A. As shown in Figure 6,
TBuA protected T387C from MTSEA modification in the open and closed
states. Rates were ~10-fold slower in the open state when channels
were preblocked with TBuA (36.7 ± 7.5 M1sec1;
n = 5) and ~18-fold slower in the closed state when
TBuA was present (16.2 ± 3.6 M1sec1;
n = 4). Together, the data suggest that MTSEA gains
access to T387C by fitting into the pore in either the open or closed
states.
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Figure 6.
TBuA protects T387C from MTSEA modification.
A, TBuA protects T387C from modification by MTSEA in the
open state. The open-state modification time course without
TBuA (black trace) is the same as in Figure
5B. The open-state modification time course in the
presence of 10 mM TBuA (~97% block,  ) was determined
by repeatedly applying 2.5 mM MTSEA for 8 sec to blocked
channels (inset) and measuring the fractional current
remaining after each exposure (inset shows an example of
a single exposure; dashed line represents zero current).
Calibration (inset): 50 pA, 2 sec; bars
indicate when TBuA and MTSEA were applied to open
(O) channels. Single exponential fits to the data
yielded exponential constants of 1.7 and 34.9 mMsec in the
absence and presence of blocker, respectively. B,
TBuA protects T387C from modification by MTSEA in the closed state. The
closed-state modification time course with no blocker present () is
taken from Figure 5B. The closed-state modification time
course in the presence of TBuA ( ) was determined by repeatedly
applying 2.5 mM MTSEA for 8 sec to closed channels in the
presence of TBuA and measuring the fractional current remaining after
each exposure. Single exponential fits to the data yielded exponential
constants of 3.7 and 77.5 mMsec in the absence and presence
of blocker, respectively. C, The WT channel MTSEA
modification rate in the open state ( ) and closed state () was
<1 M1sec1. The open-state
MTSEA modification rate of T387C without () and with () 10 mM TBuA present and the closed-state MTSEA modification
rate of T387C without () and with () 10 mM TBuA are
shown. Data are presented as mean ± SEM from
n 3 patches.
|
|
| |
DISCUSSION |
The molecular details of Ca2+ gating
of SK channels have begun to emerge from biochemical,
electrophysiological, and crystallographic studies (Xia et al., 1998;
Schumacher et al., 2001). The results suggest that SK channel opening
may involve a large-scale reorganization in which four monomeric
CaMBD/CaM complexes transition into two dimers of
CaMBD/Ca2+-CaM. This rearrangement of the
proximal C terminus may impose a conformational alteration on the
associated S6 helices that opens the SK channel gate (Schumacher et
al., 2001). The structural identity of the gate, the positions that
occlude ion permeation in the closed state, and the mechanism by which
the conformational change is transduced from the CaMBD-CaM complex to
the channel gate are not known.
SK2 channel block by TEA and longer side-chain derivatives suggests
that the inner vestibules of SK and Kv channels are conserved. SCAM
results with the largest cysteine-specific probe used in this study,
MTSET, revealed open-state-dependent access to three positions just
internal to the putative S6 bundle crossing (391-393) (Figs. 3, 4).
This was in contrast to more distal positions, such as A395C and R396C,
that rapidly reacted with MTSET in both the open and closed states of
the channel and to positions closer to the selectivity filter
(386-390) that were not affected by MTSET exposure in either the open
or closed state of the channel. These data are similar to the
reactivity profile observed at analogous positions in Shaker Kv
channels (Liu et al., 1997) and are consistent with a SK channel gate
formed at the S6 bundle crossing between positions 392 and 395 (Fig.
1A, model a).
The KcsA crystal structure reveals a wide aqueous lumen immediately
cytoplasmic to the selectivity filter that is believed to be essential
for rapid K+ conduction (Doyle et al.,
1998; Roux and MacKinnon, 1999; Zhou et al., 2001b). This lumen is
~10 Å wide and accommodates a fully hydrated
K+ ion, thereby maintaining
K+ in a low-energy state near the middle
of the cell membrane (Doyle et al., 1998). Functional data from Kv
channels and EPR data from KcsA support the maintenance of the lumen
even in closed channels. For KcsA, the width of the lumen as measured
by EPR does not markedly change when pH favors channel opening versus
channel closing (Perozo et al., 1999; Liu et al., 2001). For Kv
channels, a long-chain TEA derivative can be trapped between the
selectivity filter and the gate formed by S6, presumably in an
analogous aqueous lumen (Holmgren et al., 1997). Thus, in Kv and KcsA
channels, access to the lumen is regulated by an intracellular gate
that prevents ion exchange with cytoplasmic
K+ in the closed state.
Two experiments presented here are consistent with the existence of a
lumen, but not an intracellular gate, in closed SK channels. First,
position 387 was rapidly modified by MTSEA even in closed channels,
suggesting that there must be a lumen large enough to accommodate MTSEA
even in closed channels (Figs. 5, 6). Second, a pore blocker with a
fixed charge, TBuA, protected closed T387C channels from modification
by MTSEA. The protection afforded was even greater in the closed than
the open state, suggesting that the TBuA-binding site is
preserved in closed SK channels (Fig. 6). These data are not consistent
with a gate formed by the S6 bundle crossing between positions 392 and 395.
State-independent access to residue T387C deep in the vestibule raises
the question of why MTSET access to the more distal position 392 is
state dependent (Fig. 4A,B). The simplest
interpretation of this result is that during channel closing, position
392 rotates away from the conduction pathway, perhaps into the channel
protein surrounding the S6 helices. In this way, an unobstructed path to position 387 is maintained in the closed channel, and
state-dependent modification of position 392 is achieved. It is also
possible that 392 does not move but that surrounding protein moves to
protect the site in the closed state. Although this possibility cannot be ruled out, state-dependent MTSET access to V391C and V393C, immediately adjacent to A392C, and to the more distal L398C and E399C,
is also consistent with a rotation of the S6 helix during Ca2+ gating (Fig. 4).
To model the position of 387 and the other sites examined, SK2 residues
were substituted for their counterparts in the KcsA crystal structure
(Fig. 7). Despite the sequence divergence
between KcsA and SK2, the orientations predicted from the substituted structure are consistent with results from the SCAM experiments in the
inner vestibule. Residues that are predicted to reside in the inner
vestibule, facing the permeation pathway (387, 391, and 392), reacted
with MTS compounds in open SK channels to reduce current amplitude, and
residues that point away from the permeation pathway (386 and 390),
buried in surrounding protein or membrane, were not affected by MTS
exposure. Although this model adequately predicts the reactivity of
residues relatively deep in the inner vestibule (~386-392), more
cytoplasmic residues are less well depicted. For example, position 396 appears to point away from the permeation pathway, but MTSET reactivity
at this site significantly decreases current amplitude, and
state-dependent access to positions 398 and 399 would not necessarily
be predicted based on the model. This divergence from the data at the
cytoplasmic end of the model is not surprising; not only is the gating
mechanism fundamentally different between SK and KcsA channels, but
MTSEA access to T387C suggests that, at least in the closed state, the
bundle-crossing region of SK is wider than in KcsA.
View larger version (51K):
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|
Figure 7.
Model of SK2 residues substituted into the KcsA
crystal structure. Two of four subunits are shown (gray
ribbon). For each subunit, S5, the pore region, S6, and the S6
extension into the cytoplasm are shown. For reference, the selectivity
filter at the top (S) is ~3.3 Å wide.
L, The lumen immediately internal to the selectivity
filter. Amino acids in yellow (387,
395, 396, 401, and
402) show approximately equal open- and closed-state
reactivity, blue residues (391,
392, and 393) show more open-state than
closed-state reactivity, and red (398 and
399) shows more closed-state than open-state reactivity.
Reactivity to MTSET is shown for all residues except T387C, which
depicts MTSEA reactivity.
|
|
Using the model of the deep inner vestibule and pore region as an
approximation of the SK structure, position 387 projects into the
lumen, residing ~8 Å from the selectivity filter (Fig. 7). Because
MTSEA would fit in a cylinder ~6 Å wide by 10 Å long, with a head
group ~3.6 Å in diameter (Karlin and Akabas, 1998; Flynn and
Zagotta, 2001), a vestibule of at least these dimensions is predicted
near position 387 even in closed SK channels. The closed-state
vestibule may be even larger than this, particularly if TBuA protects
T387C from MTSEA by binding to its site close to the selectivity
filter, as demonstrated for Shaker and KcsA channels (Choi et al.,
1993; Zhou et al., 2001a), and suggested by the voltage dependence of
the open-state block in SK channels. If the smokehole (Fig.
1A, model a), the collapsing vestibule (Fig. 1A, model b), or the ball-and-chain
(Fig. 1A, model c) mechanism accounts for
SK2 channel gating, MTSEA access to T387C should be occluded in the
closed state. Because open- and closed-state modification rates do not
differ (Figs. 5B, 6C), these models cannot
accurately describe SK2 channel gating. Rather, rapid access by MTSEA
to the region immediately cytoplasmic to the selectivity filter even in
the closed state suggests that the SK channel activation gate lies at
or external to the selectivity filter (Fig. 1A,
model d). The difference between Kv and SK channels is
clearly reflected by the residue analogous to 387 in the Shaker Kv
channel (I470C) that is rapidly modified by MTSEA in the open but not
closed state (Liu et al., 1997).
Recent evidence in other K+ channels
suggests that structural changes may occur not only in the inner
vestibule but also in the selectivity filter during gating. When pH is
altered to favor gating of KcsA, EPR measurements detected small
movements at the intracellular end of the selectivity filter (Perozo et
al., 1999). Furthermore, KcsA crystal structures solved in high and low
concentrations of K+ show two distinct
selectivity filter conformations, one presumably conductive and the
other nonconductive (Zhou et al., 2001b). In an inward rectifier type
K+ channel, altering the electronegativity
of the pore alters gating at hyperpolarized potentials (Lu et al.,
2001). Finally, in Shaker Kv channels, some mutations in the
selectivity filter lead to subconductance states with ion selectivities
that differ from the selectivity profile of fully open channels (Zheng
and Sigworth, 1997, 1998).
The data presented here support a model for SK channel gating in which
Ca2+-induced rearrangements of the
CaMBD/CaM complex are transduced through S6 to the channel activation
gate, which lies in or near the channel selectivity filter. This is
similar to the gating model for CNG channels, in which cyclic
nucleotide binding to the channel C terminus is transduced to a gate
that presumably resides in the channel selectivity filter (Sun et al.,
1996; Flynn et al., 2001). Together, these studies suggest that there
may be a conserved selectivity filter-based gating mechanism for many channels gated by intracellular ligands that is distinctly different from the activation gating mechanism proposed for voltage-gated channels.
| |
FOOTNOTES |
Received April 3, 2002; revised May 16, 2002; accepted May 20, 2002.
This work was supported by National Institutes of Health grants (J.P.A.
and J.M.). We thank Chris Bond, Dr. Paco Herson, and Dr. Aaron Gerlach
for stimulating discussions.
Correspondence should be addressed to James Maylie, Department of
Obstetrics and Gynecology, Oregon Health and Sciences University, Portland, OR 97201. E-mail: mayliej{at}ohsu.edu.
| |
REFERENCES |
-
Armstrong CM,
Hille B
(1972)
The inner quaternary ammonium ion receptor in potassium channels of the node of Ranvier.
J Gen Physiol
59:388-400[Abstract/Free Full Text].
-
Becchetti A,
Gamel K,
Torre V
(1999)
Cyclic nucleotide-gated channels. Pore topology studied through the accessibility of reporter cysteines.
J Gen Physiol
114:377-392[Abstract/Free Full Text].
-
Choi KL,
Aldrich RW,
Yellen G
(1991)
Tetraethylammonium blockade distinguishes two inactivation mechanisms in voltage-activated K+ channels.
Proc Natl Acad Sci USA
88:5092-5095[Abstract/Free Full Text].
-
Choi KL,
Mossman C,
Aube J,
Yellen G
(1993)
The internal quaternary ammonium receptor site of Shaker potassium channels.
Neuron
10:533-541[Web of Science][Medline].
-
del Camino D,
Yellen G
(2001)
Tight steric closure at the intracellular activation gate of a voltage-gated K+ channel.
Neuron
32:649-656[Web of Science][Medline].
-
del Camino D,
Holmgren M,
Liu Y,
Yellen G
(2000)
Blocker protection in the pore of a voltage-gated K+ channel and its structural implications.
Nature
403:321-325[Medline].
-
Doyle DA,
Morais Cabral J,
Pfuetzner RA,
Kuo A,
Gulbis JM,
Cohen SL,
Chait BT,
MacKinnon R
(1998)
The structure of the potassium channel: molecular basis of K+ conduction and selectivity.
Science
280:69-77[Abstract/Free Full Text].
-
Fabiato A,
Fabiato F
(1979)
Calculator programs for computing the composition of the solutions containing multiple metals and ligands used for experiments in skinned muscle cells.
J Physiol (Lond)
75:463-505.
-
Flynn GE,
Zagotta WN
(2001)
Conformational changes in S6 coupled to the opening of cyclic nucleotide-gated channels.
Neuron
30:689-698[Web of Science][Medline].
-
Flynn GE,
Johnson Jr JP,
Zagotta WN
(2001)
Cyclic nucleotide-gated channels: shedding light on the opening of a channel pore.
Nat Rev Neurosci
2:643-651[Web of Science][Medline].
-
Ho SN,
Hunt HD,
Horton RM,
Pullen JK,
Pease LR
(1989)
Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
Gene
77:51-59[Web of Science][Medline].
-
Holmgren M,
Liu Y,
Xu Y,
Yellen G
(1996)
On the use of thiol-modifying agents to determine channel topology.
Neuropharmacology
35:797-804[Web of Science][Medline].
-
Holmgren M,
Smith PL,
Yellen G
(1997)
Trapping of organic blockers by closing of voltage-dependent K+ channels: evidence for a trap door mechanism of activation gating.
J Gen Physiol
109:527-535[Abstract/Free Full Text].
-
Holmgren M,
Shin KS,
Yellen G
(1998)
The activation gate of a voltage-gated K+ channel can be trapped in the open state by an intersubunit metal bridge.
Neuron
21:617-621[Web of Science][Medline].
-
Karlin A,
Akabas MH
(1998)
Substituted-cysteine accessibility method.
Methods Enzymol
293:123-145[Web of Science][Medline].
-
Keen JE,
Khawaled R,
Farrens DL,
Neelands T,
Rivard A,
Bond CT,
Janowsky A,
Fakler B,
Adelman JP,
Maylie J
(1999)
Domains responsible for constitutive and Ca2+-dependent interactions between calmodulin and small conductance Ca2+-activated potassium channels.
J Neurosci
19:8830-8838[Abstract/Free Full Text].
-
Kohler M,
Hirschberg B,
Bond CT,
Kinzie JM,
Marrion NV,
Maylie J,
Adelman JP
(1996)
Small-conductance, calcium-activated potassium channels from mammalian brain.
Science
273:1709-1714[Abstract/Free Full Text].
-
Liu J,
Siegelbaum SA
(2000)
Change of pore helix conformational state upon opening of cyclic nucleotide-gated channels.
Neuron
28:899-909[Web of Science][Medline].
-
Liu Y,
Holmgren M,
Jurman ME,
Yellen G
(1997)
Gated access to the pore of a voltage-dependent K+ channel.
Neuron
19:175-184[Web of Science][Medline].
-
Liu YS,
Sompornpisut P,
Perozo E
(2001)
Structure of the KcsA channel intracellular gate in the open state.
Nat Struct Biol
8:883-887[Web of Science][Medline].
-
Lu T,
Ting AY,
Mainland J,
Jan LY,
Schultz PG,
Yang J
(2001)
Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter.
Nat Neurosci
4:239-246[Web of Science][Medline].
-
Perozo E,
Cortes DM,
Cuello LG
(1999)
Structural rearrangements underlying K+-channel activation gating.
Science
285:73-78[Abstract/Free Full Text].
-
Roux B,
MacKinnon R
(1999)
The cavity and pore helices in the KcsA K+ channel: electrostatic stabilization of monovalent cations.
Science
285:100-102[Abstract/Free Full Text].
-
Schumacher MA,
Rivard AF,
Bachinger HP,
Adelman JP
(2001)
Structure of the gating domain of a Ca2+-activated K+ channel complexed with Ca2+/calmodulin.
Nature
410:1120-1124[Medline].
-
Sun ZP,
Akabas MH,
Goulding EH,
Karlin A,
Siegelbaum SA
(1996)
Exposure of residues in the cyclic nucleotide-gated channel pore: P region structure and function in gating.
Neuron
16:141-149[Web of Science][Medline].
-
Xia XM,
Fakler B,
Rivard A,
Wayman G,
Johnson-Pais T,
Keen JE,
Ishii T,
Hirschberg B,
Bond CT,
Lutsenko S,
Maylie J,
Adelman JP
(1998)
Mechanism of calcium gating in small-conductance calcium-activated potassium channels.
Nature
395:503-507[Medline].
-
Zagotta WN,
Siegelbaum SA
(1996)
Structure and function of cyclic nucleotide-gated channels.
Annu Rev Neurosci
19:235-263[Web of Science][Medline].
-
Zheng J,
Sigworth FJ
(1997)
Selectivity changes during activation of mutant Shaker potassium channels.
J Gen Physiol
110:101-117[Abstract/Free Full Text].
-
Zheng J,
Sigworth FJ
(1998)
Intermediate conductances during deactivation of heteromultimeric Shaker potassium channels.
J Gen Physiol
112:457-474[Abstract/Free Full Text].
-
Zhou M,
Morais-Cabral JH,
Mann S,
MacKinnon R
(2001a)
Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors.
Nature
411:657-661[Medline].
-
Zhou Y,
Morais-Cabral JH,
Kaufman A,
MacKinnon R
(2001b)
Chemistry of ion coordination and hydration revealed by a K+ channel-Fab complex at 2.0 A resolution.
Nature
414:43-48[Medline].
Copyright © 2002 Society for Neuroscience 0270-6474/02/22156499-08$05.00/0
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TOP
ABSTRACT
INTRODUCTION
