The Journal of General Physiology
J. Gen. Physiol. © The Rockefeller University Press $8.00
Volume 126Number 3September 2005
Terminus of RCK1 Domain Regulates Ca
and Jianmin Cui
Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106
Department of Biomedical Engineering, Cardiac Bioelectricity and Arrhythmia Center,
Washington University, St. Louis, MO 63130
Center for Computational Biology,
Large conductance, voltage- and Ca
and hearing owing to dual activation by membrane depolarization and intracellular Ca
-activated K channel, MthK, each of four
eight of which may form a gating ring. The structure of the MthK channel suggests that the RCK domains reorient
with one another upon Ca
binding to change the gating ring conformation and open the activation gate. Here
we report that the conformational changes of the NH
terminus of RCK1 (AC region) modulate BK
modulation depends on Ca
occupancy and activation states, but is not directly related to the Ca
These results demonstrate that AC region is important in the allosteric coupling between Ca
channel opening. Thus, the conformational changes of the AC region within each RCK domain is likely to be an
important step in addition to the reorientation of RCK domains leading to the opening of the BK
gate. Our observations are consistent with a mechanism for Ca
the AC region inhibits channel activation when the channel is at the closed state in the absence of Ca
binding and depolarization relieve this inhibition.
) channels regulate blood vessel tone, synaptic transmission,
. Similar to an archeon
subunits of BK
may contain two cytosolic RCK domains and
-dependent activation of BK
channels such that
I N T R O D U C T I O N
al., 1991; Adelman et al., 1992; Butler et al., 1993;
Tseng-Crank et al., 1994), are dually activated by voltage
and cytosolic Ca, allowing outward K
repolarize the membrane and prevent further entry of
Ca ions into the cell through voltage-dependent Ca
channels (Marty, 1981; Pallotta et al., 1981; Hudspeth
and Lewis, 1988b; Robitaille et al., 1993; Cui et al.,
1997; Yazejian et al., 1997). They play key regulatory
roles in diverse physiological functions involving cellular
, such as neurotransmitter release (Robitaille et
al., 1993; Yazejian et al., 1997), cochlear hair-cell tuning
(Hudspeth and Lewis, 1988a,b; Fettiplace and Fuchs,
1999; Duncan and Fuchs, 2003), arterial smooth muscle
tone regulation (Nelson et al., 1995; Brenner et al.,
2000), and immunity (Ahluwalia et al., 2004). Recently,
it was found that a mutation in the BK
linked to generalized epilepsy and paroxysmal dyskinesia
by altering Ca sensitivity of the channel (Du et al.,
possesses common structural features of ho-
motetrameric voltage-gated K
ion-selective pore formed by transmembrane segments
S5 and S6 and a selectivity filter from four
and a voltage-sensing module formed by transmembrane
segments S1–S4. In addition, the
long intracellular COOH terminus that may form a
channels, encoded by the Slo1 gene (Atkinson et
channels, including an
Slo1 protein has a
graphic structures of the K
and an archeon
as the homology of BK
proposed that the intracellular COOH terminus of
each Slo1 protein may contain two consecutive RCK
domains (RCK1 and RCK2), which form a gating ring
in the tetrameric channel (Jiang et al., 2001, 2002a).
Three possible divalent binding sites have been identi-
fied in the
Slo1 protein (Zeng et al., 2005) of which two
binding sites for the intracellular Ca
sensing module are proposed to be located in the
RCK1 domain (Bao et al., 2002; Xia et al., 2002) and
in Ca bowl, a COOH-terminal motif that contains
repeated Asp residues (Schreiber and Salkoff, 1997;
Schreiber et al., 1999; Bian et al., 2001; Bao et al.,
2004). Mutation of residues in these proposed Ca
binding sites reduces or abolishes Ca
channel activation (Schreiber and Salkoff, 1997; Bao et
al., 2002, 2004; Xia et al. 2002; Zeng et al., 2005).
While the identification of Ca
been a focus of recent research on BK
(Bian et al., 2001; Braun and Sy, 2001; Shi and Cui,
2001; Zhang et al., 2001; Bao et al., 2002, 2004; Pisko-
rowski and Aldrich, 2002; Shi et al., 2002; Xia et al.,
2002; Qian and Magleby, 2003; Zeng et al., 2005), the
-sensing module. Based on the X-ray crystallo-
with these channels, it is
channel, MthK, as well
binding sites has
Correspondence to Jianmin Cui: email@example.com
Abbreviations used in this paper: MWC, Monod-Wyman-Changeux;
228The AC Region Regulates BK
molecular process of how Ca
channel opening has been less studied. Nevertheless,
the proposed mechanism of MthK activation based on
its structure provides an excellent model for this aspect
channel activation. In the MthK channel, RCK
domains in the gating ring interact with one another at
two interfaces, a fixed interface and a flexible interface
(Jiang et al., 2002a). By comparing the structures of
MthK and the RCK domain of the
is proposed that Ca binding to the MthK channel
alters the orientation among RCK domains in the gat-
ing ring by rearranging the flexible interface, which
changes the conformation of the gating ring and opens
the channel by pulling the linker between the inner he-
lix and the gating ring (Jiang et al., 2001, 2002a). The
-dependent gating mechanism of BK
to be similar to this mechanism by a recent study dem-
onstrating that the gating properties of BK
on the length of the linker between S6 and gating ring
and which may be pulled by a conformational change
in the gating ring during Ca
(Niu et al., 2004). However, the nature of conforma-
tional changes in the BK
dependent activation may not be identical to that in
the MthK channels. This is evident based on many dif-
ferences between the function and structure of these
two channels, including the affinity and location of the
Ca binding sites (Moczydlowski and Latorre, 1983;
McManus and Magleby, 1991; Cox et al., 1997; Cui et
al., 1997; Schreiber et al., 1999; Bao et al., 2002; Jiang
et al., 2002a,b; Xia et al., 2002). In BK
structural relationship between Ca
the flexible interface is not clear. Whether a similar
conformational change occurs at the flexible interface
binding to the BK
Here, by comparing structure and function between
homologues mSlo1 (Butler et al., 1993) and dSlo1
(Adelman et al., 1992), we have identified a region in
terminus of RCK1, called AC region, that mod-
ulates Ca sensitivity of channel activation. We found
that the difference in Ca
and dSlo1 was not due to differences in metal binding
sites between the two channels. Instead, conforma-
tional differences in this AC region caused activation
energy to change. The effects of AC region on channel
gating depend on Ca occupancy and activation state
of the channel, suggesting that AC region is important
in the energetic coupling between Ca
the opening of the activation gate. In the crystal struc-
ture of the gating ring, the AC region of each RCK do-
main does not interact with any structure in other RCK
domains and is distant from the flexible interface
(Jiang et al., 2002a). The above observation, along with
our findings, indicate that conformational changes oc-
binding is coupled to
E. coli K
gating ring during Ca
binding sites and
channel needs to be
sensitivity between mSlo1
cur in each individual RCK domain during BK
tion upon Ca binding. Regardless of whether such
conformational changes within each RCK domain may
either be driven by or combine with the reorientation
among different RCK domains at the flexible inter-
faces, the conformational change in the AC region is a
very important step in the allosteric machinery linking
Ca binding to channel opening.
An abstract of this work has been presented in the
49th Annual Meeting of Biophysical Society.
M A T E R I A L S A N D M E T H O D S
Mutagenesis and Expression
The mbr5 splice variant of the mouse
al., 1993) and the
channel (Adelman et al., 1992) were used to make mutant
and chimeric channels. The wild-type (WT) mSlo1 and dSlo1 se-
quences included in each of the chimeras, given as the residue
numbers in the primary sequence of the respective proteins are
as follows: Chim1, dSlo1:1-611, mSlo1:631-1169; Chim2, dSlo1:1-
475, mSlo1:462-1169; Chim3, dSlo1:1-432, mSlo1:419-1169;
Chim4, dSlo1:1-389, mSlo1:376-1169; Chim5, dSlo1:1-336,
mSlo1:323-1169; Chim6, dSlo1:1-271, mSlo1:257-1169; Chim7,
dSlo1:71-127, mSlo1:1-43 and 114–1169; d[mAC], dSlo1:1-333
and 433–1164, mSlo1:320-418; m[dAC], dSlo1:334-432, mSlo1:1-
319 and 419–1169. All mutant and chimeric constructs were
made using overlap-extension PCR (Shi et al., 2002) with
polymerase (Stratagene). The PCR-amplified regions were veri-
fied by sequencing. RNA was transcribed in vitro with T3 poly-
merase for mSlo1 and T7 polymerase (Ambion) for dSlo1 con-
Xenopus laevis oocytes were each injected with 0.05–50 ng
of RNA and incubated in 18
C for 2–6 d before recording.
channel (Butler et
DrosophilaA1C2E1G3I0 splice variant of the
Macroscopic currents were recorded from inside-out patches us-
ing Axopatch 200-B patch-clamp amplifier (Axon Instruments)
and PULSE acquisition software (HEKA Electroniks), low-pass fil-
tered at 5 kHz with the amplifier’s built-in four-pole Bessel filter
and digitized at 20-
s intervals. Solution compositions were as
follows: external (extracellular), 140 mM K-methanesulphonic
acid, 20 mM Hepes, 2 mM KCl, 2 mM MgCl
ternal (intracellular), 140 mM K-methanesulphonic acid, 20 mM
Hepes, 2 mM KCl, 1 mM EGTA, pH
the same composition as basal internal solution except that it
contained 5 mM EGTA to give a free [Ca
too low to produce BK
channel activation (Cui et al., 1997). Re-
were obtained by adding appropriate
volumes of CaCl
solutions to basal internal solution.
The actual free [Ca
was measured using a Ca
trode (Orion). All currents were recorded at room temperature
7.20; basal in-
7.20. The “0 [Ca
0.5 nM, which is
Relative conductance was calculated from tail current amplitudes
?50 mV. Conductance–voltage (G-V) relations were fitted with
the Boltzmann distribution
where k is Boltzmann’s constant, T is absolute temperature, and
?GAct is the free energy of channel opening. ?GAct is the total
energy increase provided by voltage (?GV ? ?zeV, where e is
G/Gmax11 exp ∆GAct kT
Krishnamoorthy et al.229
elementary charge and z is the number of equivalent gating
charges) and Ca2? and Mg2? binding (?GCa and ?GMg). In the
absence of Ca2? and Mg2? at 0 mV, ?GAct ? ?G0 (Cui and Al-
drich, 2000). Hence,
The change in the contribution of Ca2? binding to ?GAct as a re-
sult of an increase in [Ca2?]i, ??GCa, was measured at a constant
[Mg2?]i and vice-versa. ??GCa and ??GMg were calculated as a
measure of the shift and change in shape of the G-V relation be-
tween two specified values of [Ca2?]i or [Mg2?]i as
where V1/2 is the voltage at half maximum of the G-V relation
(Figs. 1, 3, and 4). ??GCa and ??GMg represent the Ca2? sensitiv-
ity and Mg2? sensitivity, respectively, of a channel measured be-
tween two specified Ca2? or Mg2? concentrations. While in gen-
eral the Ca2? sensitivity and Mg2? sensitivity of the channel
should be evaluated between 0 and saturating concentrations of
metal ions, our results show that ??GCa of dSlo1 is consistently
larger than that of mSlo1 between any pair of [Ca2?]i used in our
experiments (see Figs. 1, 4, 6, and 10). Mean ??GCa of WT and
mutant channels was measured and calculated at 0 [Mg2?]i and
two [Ca2?]i values that allowed us to obtain a complete G-V rela-
tion. In Figs. 4 and 6, mean ??GCa of mutant channels was then
divided by the mean ??GCa of WT mSlo1 under the same condi-
tions of [Ca2?]i and [Mg2?]i. In Fig. 10 C, ?GV values were mea-
sured and calculated at V ? V1/2.
Ca2? sensitivity in BKCa channel activation is sometimes evalu-
ated by fitting the response of Po to [Ca2?]i to Hill equation at a
fixed voltage to obtain K1/2 and Hill coefficient. Ca2? sensitivity
determined in such a manner is pertinent only to the specific
voltage at which the data is analyzed. It does not reflect the over-
all Ca2? sensitivity of a channel because for the same channel
Ca2? sensitivity defined by the fit of Hill equation is different at
different voltages (Cui et al., 1997; Bian et al., 2001; Zhang et al.,
2001). When the voltage range of activation is shifted by a muta-
tion, at a fixed voltage the Ca2? sensitivity estimated by Hill equa-
tion would naturally change even if the overall Ca2? sensitivity of
the channel is actually not changed. The error bars in all figures
show standard error of means from three to nine patches of data.
Monod-Wyman-Changeux (MWC) Model
In Fig. 11, MWC model fits used the following equation:
Kc ? dissociation constant of [Ca2?] in the closed state; Ko ? dis-
sociation constant of [Ca2?] in the open state; c ? Ko/Kc is a
measure of Ca2? sensitivity of activation; L(0) ? [C]/[O] at 0
[Ca2?]i; n ? 4 or 8. MWC model code was written and executed
in MATLAB 6.5 (The Mathworks Inc.).
The first step to computationally simulate the mSlo1 and dSlo1
AC region was to create structural models for each protein. Us-
ing the MthK structure as a starting point (PDB code 1ID1), we
created the required mutations and small insertions using PLOP
(Jacobson et al., 2004). Using the molecular dynamics package
( ) e
Gromacs (Lindahl et al., 2001) we first minimized both structures
using the OPLS/AA force field and explicit SPC solvent. The sys-
tems were then heated in 50 K steps to 300 K, equilibrated at 300 K
for 10 ns, and followed by production runs of 20 ns. In all cases we
used 2 fs time steps with bond constraints, Particle Mesh Ewald,
and coordinates were saved every 1 ps. To compare the dynamics
of these two domains, we used Principal Component Analysis. The
covariance matrix calculated from each production run was diago-
nalized and the eigenvectors corresponding to the largest eigen-
value (i.e., the most significant mode) were used. The motion
along the principal eigenvectors of each protein showed marked
dynamical differences between mSlo1 and dSlo1. To further quan-
tify this dynamical difference, we computed the root mean square
fluctuations of the ? carbons for each protein. Since the proteins
are of identical length and have the same secondary structure, this
comparison highlights the effect of sidechain substitutions on the
motion of the protein backbone.
R E S U L T S
dSlo1 Exhibits Greater Ca2? Sensitivity than mSlo1
BK channel homologues mSlo1 and dSlo1 exhibit high
sequence homology and amino acid identity (Adelman
et al., 1992, Butler et al., 1993). However, there are
significant differences in their channel activation and
macroscopic current properties in response to changes
in voltage and [Ca2?]i as shown in Fig. 1. In the absence
of [Ca2?]i (see MATERIALS AND METHODS), signifi-
cant currents could be evoked in mSlo1 at positive po-
tentials (the voltage at half maximum activation, V1/2
?180 mV), whereas, virtually no current was evoked in
dSlo1 for the same condition, with the result that volt-
age dependence of channel activation was far-right
shifted on the voltage axis and could not be determined
(Fig. 1 B). Similarly, when [Ca2?]i was increased to 5.7
?M, the same voltage protocol elicited larger mSlo1
currents than dSlo1 (Fig. 1 A, top). However, at 89 ?M
[Ca2?]i, comparable amounts of mSlo1 and dSlo1 cur-
rents were observed (Fig. 1 A, bottom), such that at this
[Ca2?]i, the voltage dependence of open probability
was the same for both mSlo1 and dSlo1 (Fig. 1 B).
Thus, the same amount of increase in [Ca2?]i (0–89 ?M
or 5.7–89 ?M) results in a more pronounced increase
of dSlo1 activation than that of mSlo1 for the same volt-
age protocols (Fig. 1, A and B). The voltage range of
channel activation for dSlo1 exhibits a larger leftward
shift (?V1/2 ?160 mV) than mSlo1 (?V1/2 ?68 mV) for
the same change of [Ca2?]i from 5.7 to 89 ?M (Fig. 1
B), imparting higher Ca2? sensitivity to dSlo1 as com-
pared with mSlo1 (see MATERIALS AND METHODS
for more details). Higher Ca2? sensitivity of dSlo1 as
compared with mSlo1 is also seen in the free energy of
channel activation contributed by Ca2? binding when
[Ca2?]i increases. The increase in [Ca2?]i from 5.7 to 89
?M contributes ?20 kcal/mole to dSlo1 activation as
compared with ?9 kcal/mole to mSlo1 activation (Fig.
1 C). More importantly, the ??GCa contributed to dSlo1
230The AC Region Regulates BKCa Gating
activation by an increase in [Ca2?]i from 5.7 to 89 ?M is
about the same as the ??GCa contributed to mSlo1 acti-
vation by an increase in [Ca2?]i from 0 to 89 ?M (Cui
and Aldrich, 2000) (Fig. 1 C). The above results demon-
strate that (1) the activation properties of dSlo1 and
mSlo1 channels in the absence of Ca2? are vastly differ-
ent, and (2) the Ca2? sensitivity of dSlo1 is higher com-
pared with mSlo1.
NH2-terminal Region in the RCK1 Domain Modulates Ca2?
Sensitivity of Activation
A comparison of the sequences of mSlo1 and dSlo1
shows differences at many locations throughout the en-
tire peptide (Adelman et al., 1992; Butler et al., 1993).
To determine the structural basis of the higher Ca2?
sensitivity of dSlo1, we constructed chimeric channels
of mSlo1 and dSlo1, named Chim1 to Chim7, by re-
placing parts of the sequence of the dSlo1 protein with
corresponding sequences of mSlo1. The aim was to
identify the structural domain in the mSlo1 protein
that would reduce Ca2? sensitivity of the background
dSlo1 channel. To compare Ca2? sensitivity of the chi-
meric channels with that of dSlo1 and mSlo1, it is desir-
able that Ca2? sensitivity for each channel is measured
over the complete range of [Ca2?]i, from zero to satu-
rating levels. However, it was not possible to measure
Ca2? sensitivity over the same Ca2? range for all the chi-
meric channels because, like dSlo1 (Fig. 1 B), the volt-
age range of activation of some chimeric channels is
too positive to be measured at low [Ca2?]i. This is illus-
trated in Fig. 2 for two of the chimeric channels, Chim2
and Chim6 (for description and sequence informa-
tion of chimeric channels, refer to MATERIALS AND
METHODS). We observed that the channel activation
properties of Chim2 are similar to that of dSlo1 as seen
in Fig. 1. At 0 [Ca2?]i the channel could not be acti-
vated even at voltages more positive than 250 mV. In
this case, the lowest [Ca2?]i at which we could measure
a portion of the G-V relation of Chim2 was 2 ?M (Fig. 2
A). Therefore, the Ca2? sensitivity was measured be-
tween a [Ca2?]i change from 2 ?M to a saturating level
for this channel. Fig. 2 A shows that for an identical
change in [Ca2?]i from 2 to 100 ?M, the G-V relation of
mSlo1 shifts much less on the voltage axis than that of
Chim2. On the other hand, Chim6 exhibits channel
properties more similar to mSlo1, such that for the
same voltage protocols, the currents evoked are compa-
rable to mSlo1 at all [Ca2?]i (Fig. 2 B, current traces).
Although the G-V relation of Chim6 at all [Ca2?]i is
right shifted as compared with mSlo1 (Fig. 2 B, bot-
tom), a portion of the G-V relation at 0 [Ca2?]i can be
measured. Therefore, the Ca2? sensitivity was measured
ity than mSlo1. (A) Current traces of mSlo1 and dSlo1.
Test potentials were ?80 to ?200 mV and ?150 to ?200
mV for 5.7 ?M and 89 ?M [Ca2?]i, respectively, holding
and repolarization potentials were ?50 mV. (B) Steady-
state G-V relations of mSlo1 and dSlo1. Smooth curves
are fits of the Boltzmann equation (see MATERIALS
AND METHODS) with parameters for mSlo1, in 0
[Ca2?]i: V1/2 ? 179 mV, z ? 1.2, in 5.7 ?M [Ca2?]i,
V1/2 ? 64.6 mV, z ? 1.4, in 89 ?M [Ca2?]i, V1/2 ? ?2.8
mV, z ? 1.2; for dSlo1, in 5.7 ?M [Ca2?]i, V1/2 ? 157 mV,
z ? 1.3, in 89 ?M [Ca2?]i, V1/2 ? ?2.9 mV, z ? 1.1. (C)
Free energy provided by Ca2? binding for channel acti-
vation when [Ca2?]i changes from 0 or 5.7 ?M to 89
?M, as indicated in parentheses under the abscissa. m,
mSlo1; d, dSlo1.
dSlo1 activation exhibits higher Ca2? sensitiv-
Krishnamoorthy et al. 231
between a full range of [Ca2?]i change from 0 to a satu-
rating level for this channel. Similar to Chim2, the Ca2?
sensitivity of Chim6 is compared with that of the WT
mSlo1 measured at the same [Ca2?]i range (Fig. 2 B).
To compare the Ca2? sensitivity of chimeric channels
with that of mSlo1 within the maximum [Ca2?]i range,
we needed to find the saturating [Ca2?]i for channel
activation. It has been shown previously that the high
affinity Ca2? binding sites for mSlo1 activation is nearly
saturated at [Ca2?]i ? 80 ?M (Cox et al., 1997; Cui et
al., 1997). Fig. 3 shows that Ca2?-dependent activation
of dSlo1 also saturates at similar [Ca2?]i. Fig. 3 (A and
of mSlo1 and dSlo1 retain
features of their native WT
channels. (A, top) Current
traces of Chim2 and mSlo1 at
[Ca2?]i of 2, 10, and 100 ?M.
Test potentials were ?80 to
?200 mV, holding and re-
polarization potentials were
?50 mV. (Bottom) Steady-
state G-V relations of Chim2
and mSlo1. Smooth curves
are fits of the Boltzmann
equation with parameters for
Chim2, in 2 ?M [Ca2?]i:
V1/2 ? 240.1 mV, z ? 0.95, in
10 ?M [Ca2?]i: V1/2 ? 76.1
mV, z ? 1.5, in 100 ?M
[Ca2?]i: V1/2 ? 43.5 mV, z ?
1.5; for mSlo1, in 2 ?M
[Ca2?]i: V1/2 ? 84.8 mV, z ?
1.5, in 10 ?M [Ca2?]i: V1/2 ?
31.4 mV, z ? 1.5, in 100 ?M
[Ca2?]i: V1/2 ? 14.4 mV, z ?
1.4. (B, top) Current traces of
Chim6 and mSlo1 at [Ca2?]i
of 0, 10, and 500 ?M. Test po-
tentials were ?80 to ?200 mV, holding and repolarization potentials were ?50 mV. (Bottom) Steady-state G-V relations of Chim6 and
mSlo1. Smooth curves are fits of the Boltzmann equation with parameters for Chim6, in 0 [Ca2?]i: V1/2 ? 206.7 mV, z ? 1.3, in 10 ?M
[Ca2?]i: V1/2 ? 101.1 mV, z ? 1.2, in 500 ?M [Ca2?]i: V1/2 ? 52.5 mV, z ? 1.2; for mSlo1, in 0 [Ca2?]i: V1/2 ? 189.7 mV, z ? 1.1, in 10 ?M
[Ca2?]i: V1/2 ? 33.8 mV, z ? 1.7, in 500 ?M [Ca2?]i: V1/2 ? ?13.2 mV, z ? 1.8.
[Ca2?]i. (A and B) Current traces of dSlo1 for indicated
[Ca2?]i elicited by voltages from ?150 to 200 mV. (C)
Steady-state G-V curves of dSlo1. Solid curves are fits of the
Boltzmann equation. V1/2 obtained from the fits of the G-V
relations at each [Ca2?]i is indicated. Dotted lines indicate
MWC model simulations of the G-V relation at [Ca2?]i
89, 100, 200, and 300 ?M using the same parameters as
indicated in Fig. 10 C.
Activation of dSlo1 saturates around 89 ?M
232The AC Region Regulates BKCa Gating
B) shows that the current traces of WT dSlo1 recorded
at 89 and 300 ?M are very similar. The steady-state G-V
relations of WT dSlo1 at [Ca2?]i 5.7, 11.2, 28.5, 89, 100,
200, and 300 ?M are shown in Fig. 3 C. Doubling
[Ca2?]i from 89 to 200 ?M caused little shift in the G-V
relation while a doubling of [Ca2?]i from 5.7 to 11.2
?M caused a G-V shift of ?50 mV, suggesting that at 89
?M [Ca2?]i, the activation is close to being saturated.
We also fitted the data at [Ca2?]i of 89, 100, 200, and
300 ?M with the MWC model using the same parame-
ters indicated in Fig. 10 (Fig. 3 C), which also suggest
that at 89 ?M [Ca2?]i, the activation is close to being
saturated. These results indicate that the activation of
both mSlo1 and dSlo1 channels saturates at [Ca2?]i ?
89 ?M and the activation differs little within the [Ca2?]i
range between 89 and 300 ?M. Therefore, we have
used [Ca2?]i between 89 and 500 ?M as the saturating
[Ca2?]i in the experiments involving measurement of
Based on the observations similar to those described
above, we measured the Ca2? sensitivity (??GCa) be-
tween the maximum [Ca2?]i intervals possible for each
chimeric channel, and then compared it to the ??GCa
measured for WT mSlo1 between the same [Ca2?]i in-
tervals. Fig. 4 A plots the ??GCa values of dSlo1 and
chimeric channels normalized against that of mSlo1
measured between the same [Ca2?]i intervals, respec-
tively. Shown at the left are cartoons illustrating the
mSlo1 domain substituting dSlo1 counterpart in each
chimera and on the right are the [Ca2?]i intervals at
which ??GCa was measured (Fig. 4 A). In chimeric
channels Chim1–Chim7, replacement of dSlo1 started
with the tail of mSlo1 in Chim1 and progressively cov-
ered all the important regions that have been shown in
previous studies to affect the Ca2? sensitivity of BKCa
channels. Replacing the tail of dSlo1 with its mSlo1
counterpart (Chim1), which included Ca2? bowl, the
putative Ca2? binding site (Schreiber and Salkoff,
1997; Schreiber et al., 1999; Bian et al., 2001; Bao et
al., 2004), did not have much effect on Ca2? sensitivity;
nor did Chim2 and Chim3, where the COOH termi-
nus of the RCK1 domain was included in the replace-
ment. However, Chim4, in which the replacement in-
cluded the NH2-terminal part of the RCK1 domain,
brought the Ca2? sensitivity closer to that of WT
mSlo1. Further addition of mSlo1 sequence did not
significantly alter the Ca2? sensitivity further, as seen in
Chim5, Chim6, and Chim7. In addition to the lowest
and highest possible [Ca2?]i for each chimeric chan-
nel, we also measured channel activation at one or
more intermediate [Ca2?]i and compared the V0.5–
[Ca2?]i plots with that of WT mSlo1 and dSlo1 as
important for Ca2? sensitivity. (A) Ca2? sensitivity of
activation in chimeric channels of mSlo1 and dSlo1.
The vertical axis shows schematic representation of
chimera constructs with dSlo1 portions shaded gray
and mSlo1 black. Rectangles are transmembrane
segments or RCK1 domains (Jiang et al., 2002a),
ovals are the Ca2? bowl (Schreiber et al., 1999). Free
energy increase in response to increase in [Ca2?]i
(shown at the right) for each chimera and WT
channel was normalized against that for mSlo1. (B)
Plot of V1/2 versus [Ca2?]i for mSlo1 (thin black line),
dSlo1 (thick black line), and the chimeric channels
as defined in A. V1/2 values are obtained by fitting the
G-V relations of the channels at various [Ca2?]i with
the Boltzmann equation.
The AC region in the RCK1 domain is
Krishnamoorthy et al.233
shown in Fig. 4 B. The maximal slope of the V0.5–
[Ca2?]i for dSlo1 (thick black line) is significantly
larger than for mSlo1 (thin black line), indicating
higher Ca2? sensitivity. As in Fig. 4 A for Ca2? sensitiv-
ity, Chim1, Chim2, and Chim3 exhibit a maximal slope
of V0.5–[Ca2?]i similar to dSlo1 (thick lines), while
Chim4 to Chim7 have a maximal V0.5–[Ca2?]i slope
similar to mSlo1 (thin lines) (Fig. 4 B).
Thus, a difference of a 43 amino acid stretch in the
RCK1 domain (?B-?C, Fig. 6 A), between Chim3 and
Chim4, switched the Ca2? sensitivity of the channel
from being dSlo1-like to being mSlo1-like (Fig. 4). To
examine if this stretch is sufficient to switch Ca2? sensi-
tivity of the BKCa channel, we replaced just the NH2-ter-
minal part of the RCK1 domain (?A-?C, henceforth re-
ferred to as AC, Fig. 6 A) in dSlo1 by its mSlo1 counter-
part (d[mAC]). Fig. 4 A shows that the AC stretch from
mSlo1 alone is able to reduce Ca2? sensitivity of dSlo1.
Conversely, when the same region in mSlo1 was re-
placed by the dSlo1 counterpart (m[dAC]), the Ca2?
sensitivity increased (Fig. 4 A). To confirm whether the
AC region is able to switch the Ca2? sensitivity between
mSlo1 and dSlo1 at all [Ca2?]i and not just at the
extremities, we measured and plotted the voltage for
half-maximal activation, V1/2 as well as the equivalent
charge, z, versus [Ca2?]i for a range of [Ca2?]i from 0 to
89 ?M (Fig. 5, A and B). Both plots confirm the obser-
vation from Fig. 4 that switching the AC region be-
tween mSlo1 and dSlo1 was sufficient to switch the phe-
notype of Ca2? sensitivity between the channels for all
[Ca2?]i between 0 and saturating levels.
and dSlo1. Plot of (A) V1/2 and (B) z versus [Ca2?]i for mSlo1,
dSlo1, m[dAC], and d[mAC]. V1/2 and z are obtained by fitting
the G-V relations of the channels at various [Ca2?]i with the
The AC region switches Ca2? sensitivity between mSlo1
binding site in the AC region. (A) Sequence alignment of the AC
region of the RCK1 domain (Jiang et al., 2002a) from mSlo1
(Butler et al., 1993), dSlo1 (Adelman et al., 1992), and the archeon
MthK (Jiang et al., 2002a). Numbers indicate the position of the
rightmost residues in the primary sequence of respective proteins.
Secondary structures ?A-C and ?A-C are indicated by underlines.
Boxed amino acids labeled as motifs 1, 2, and 3 are regions
showing significant sequence differences between mSlo1 and
dSlo1. Motif 1 is important for Ca2?-dependent activation (Shi et
al., 2002; Xia et al., 2002). Effects of switching motif 1 between
mSlo1 and dSlo1 are shown in B and C. (B, left) Steady-state G-V
relations of the mutant channel m[d1] (motif 1 from dSlo1 in the
mSlo1 background). Solid lines are fits of the Boltzmann equation
with the following parameters: at 0 [Ca2?]i, V1/2 ? 185 mV, z ? 1.2;
at 89 ?M [Ca2?]i, V1/2 ? 15.9 mV, and z ? 1.2. Dotted lines are
G-V relations of mSlo1. (B, right) Steady-state G-V relations of the
mutant channel d[m1] (motif 1 from mSlo1 in the dSlo1 back-
ground). Solid lines are fits of the Boltzmann equation with the
following parameters: at 89 ?M [Ca2?]i, V1/2 ? ?52.5 mV, z ?
0.83; at 5.7 ?M [Ca2?]i, V1/2 ? 95.1 mV and z ? 0.89; at 0 [Ca2?]i,
the G-V relation was too right shifted for z and V1/2 values to be
determined. Dotted lines are G-V relations of dSlo1. (C) Free
energy of activation provided by Ca2? binding in mSlo1, d[m1],
m[d1], and dSlo1 when [Ca2?]i increased from 0 to 89 ?M (for
m[d1]) and from 5.7 to 89 ?M (for d[m1] and dSlo1), normalized
against corresponding free energy values for mSlo1.
Ca2? sensitivity change is not related to the Ca2?
234The AC Region Regulates BKCa Gating
We observe that the extent of increase in Ca2? sensi-
tivity in m[dAC] is not equal to the extent of decrease
in Ca2? sensitivity in d[mAC] (Fig. 4 A). We postulate
that this is because the modulation of Ca2? sensitivity
involves many parts of the channel protein and replac-
ing just one of the components in mSlo1 by dSlo1 (in
this case, the AC region) is not enough to result in a
complete gain of function, i.e., an increase in Ca2? sen-
sitivity. A loss of function is however more easily ob-
tained by changing one of the components and this is
seen by the significant decrease in Ca2? sensitivity of
d[mAC] compared with WT dSlo1. Hence, results of
Figs. 4 and 5 indicate that the AC region of the RCK1
domain is important in determining the Ca2? sensitivity
of channel activation in mSlo1 and dSlo1.
Metal Binding Sites in the AC Region Are Not Responsible
for the Difference in Ca2? Sensitivity
Previous studies demonstrated that Ca2? activates BKCa
channels by an allosteric mechanism, i.e., Ca2? binds to
sites distant from the activation gate and opens the
channel by changing the conformation of channel pro-
tein (McManus and Magleby, 1991; Cox et al., 1997;
Jiang et al., 2002a,b). Thus, an alteration of either the
Ca2? binding sites or the structure linking binding sites
to the activation gate can change Ca2? sensitivity of
BKCa gating. At present, the location of the Ca2? bind-
ing sites for BKCa activation has not been completely
elucidated. Previous results have led to the proposal
that perhaps more than one site per subunit contribute
to channel gating and are located in the cytosolic re-
gions (Xia et al., 2004), possibly in the Ca2? bowl
(Schreiber and Salkoff, 1997) and the RCK domains
(Bao et al., 2002; Xia et al., 2002), or in the core of the
channel that includes transmembrane segments and
connecting loops (Braun and Sy, 2001; Piskorowski and
Aldrich, 2002). Sequence differences between mSlo1
and dSlo1 at these putative Ca2? binding sites are not
likely to be responsible for the differences in Ca2? sen-
sitivity because switching sequences between the two
channels in these locations failed to alter Ca2? sensitiv-
ity (Chim 1, Chim 5, and Chim 7 in Fig. 4).
Closer inspection of the AC regions of mSlo1 and
dSlo1 highlighted three groups of amino acids, Motif1,
Motif2, and Motif3, which show significant differences
in amino acid identity (Fig. 6 A). Of these, Motif1 con-
tains a putative Ca2? binding site (Xia et al., 2002),
which is conserved between mSlo1 and dSlo1 (D367 in
mSlo1 or D381 in dSlo1), but differs in amino acids
flanking this conserved site (Fig. 6 A). It is reasonable
to suppose that differences in one or all of these motifs
are responsible for reversing the Ca2? sensitivity pheno-
type between mSlo1 and dSlo1. To test this hypothesis,
we made chimeric mutant channels where the three
motifs were switched between mSlo1 and dSlo1 either
individually or in combination. Figs. 6 and 7 summarize
the observations of the effect of motif switching on
the phenotype of Ca2? sensitivity. Neither d[m1] nor
m[d1] showed any change in Ca2? sensitivity when
compared with their respective native phenotype (Fig.
6, B and C). The free energy of channel activation pro-
vided by Ca2? binding, ??GCa, is dependent on the
background but not motif1 of the channel such that
??GCa of m[d1] is similar to that of WT mSlo1, whereas
mSlo1 and dSlo1 either singly or in combination does not
switch Ca2? sensitivity of BKCa gating. (A) Steady-state G-V relations
of m[d-Motif2] and m[d-Motif3]. Dotted lines are G-V relations
of mSlo1. Parameters of the Boltzmann fits (solid lines): for
m[d-Motif2], at 0 [Ca2?]i, V1/2 ? 195.7 mV, z ? 1.1; at 100 ?M
[Ca2?]i, V1/2 ? 8.2 mV, z ? 1.2; for m[d-Motif3], at 0 [Ca2?]i,
V1/2 ? 213.2 mV, z ? 1.1; at 100 ?M [Ca2?]i, V1/2 ? 9.2 mV, z ?
1.2. (B) Steady-state G-V relations of m[d-Motif12] and m[d-
Motif13]. Dotted lines are G-V relations of mSlo1. Parameters
of the Boltzmann fits (solid lines): for m[d-Motif12], at 0 [Ca2?]i,
V1/2 ? 186.5 mV, z ? 1.2; at 100 ?M [Ca2?]i, V1/2 ? 22.2 mV, z ?
1.1; for m[d-Motif13], at 0 [Ca2?]i, V1/2 ? 211.9 mV, z ? 1.4; at 100
?M [Ca2?]i, V1/2 ? 38.4 mV, z ? 1.3. (C) Free energy of activa-
tion provided by Ca2? binding for the chimeric channels. Free en-
ergy change for each channel was in response to [Ca2?]i change
shown in parentheses for mSlo1 plotted alongside each group.
Switching sequence differences in AC region between
Krishnamoorthy et al.235
??GCa of d[m1] is similar to WT dSlo1 (Fig. 6 C). At
0 [Ca2?]i, the voltage-dependent activation of m[d1]
has similar characteristics as WT mSlo1, while that of
d[m1] is similar to WT dSlo1 (Fig. 6 B). Switching
Motif2 or Motif3 of mSlo1 to dSlo1 did not affect Ca2?
sensitivity of mSlo1 (Fig. 7 A). Additionally, the mutant
channels in which motif pairs were switched also failed
to switch the Ca2? sensitivity phenotype (Fig. 7 B). In
each case, the ??GCa value matched that of its native
mSlo1 channel (Fig. 7 C). In all cases, the mutations
shifted the positions of the G-V on the voltage axis from
that of the WT channels at all [Ca2?]i (Fig. 6 B and Fig.
7, A and B). However, these shifts are relatively small
and did not significantly affect the free energy of chan-
nel activation provided by Ca2? binding. The results of
Figs. 6 and 7 indicate that the reversal of phenotype
seen as a result of a switch of the AC regions between
mSlo1 and dSlo1 is not the result of changes to the
Ca2? binding site or individual amino acid differences
between mSlo1 and dSlo1 in the AC region. Rather, the
AC region as a whole is responsible for the differences
in Ca2? sensitivity between mSlo1 and dSlo1 channels.
Besides the differences in the boxed motifs (Fig. 6
A), the AC region of mSlo1 and dSlo1 contains a con-
served Mg2? site (Shi et al., 2002; Xia et al., 2002). In-
tracellular Mg2? activates mSlo1 and dSlo1 similarly by
binding to this low-affinity metal binding site (Kd ?
mM), which is nonspecific to divalent cations (Shi et
al., 2002; Xia et al., 2002) (Fig. 8 A). We considered the
possibility that the affinity of this metal binding site for
Ca2? might increase in dSlo1 such that the excess Ca2?
sensitivity observed in dSlo1 could be the effect of Ca2?
binding to this conserved metal site, even when Ca2? is
present only in micromolar concentrations. To test this
hypothesis, we made a mutation in the Mg2? binding
site, E413R in dSlo1. E413R not only effectively abol-
ishes Mg2? sensitivity in dSlo1 (Fig. 8, B and C) but also
leaves the Ca2? sensitivity of the channel unchanged
from WT dSlo1 (Fig. 8 D). This result demonstrates
that the increased Ca2? sensitivity in dSlo1 seen in Fig.
1 is not due to Ca2? binding to the low affinity metal
Conformational Differences in AC Region May Cause the
Difference in Ca2? Sensitivity between mSlo1 and dSlo1
The results in Figs. 4–7 suggest that, instead of affecting
Ca2? binding, AC region may act as a structure linking
binding sites to the activation gate and modulate Ca2?
sensitivity of BKCa channels. Consistent with this mecha-
nism, we found that only when the sequence compris-
ing the AC region as a whole was exchanged between
mSlo1 and dSlo1, was the phenotype of Ca2? sensitivity
also exchanged (Figs. 4–7). This result suggests that a
combination of all the sequence differences in AC re-
gion may contribute and lead to a different conforma-
tion of the AC region between mSlo1 and dSlo1, which
then results in altered conformational transitions dur-
ing channel activation and hence Ca2? sensitivity. Pres-
ently, there is no structural information available to al-
low a direct comparison between the conformations of
mSlo1 and dSlo1. To examine if the conformation of
the AC region in mSlo1 is different than in dSlo1 we
performed molecular dynamics simulations on the AC
region of dSlo1 and mSlo1 (Fig. 9), using homology
models based on the crystal structure of MthK (Jiang et
al., 2002a) (see MATERIALS AND METHODS). We ob-
served that dSlo1 AC appears more compact than the
mSlo1 AC, and that the range of motion in the first
affinity metal binding site. (A) Steady-state G-V relations of mSlo1
and dSlo1 at 89 ?M [Ca2?]i and indicated [Mg2?]i. Boltzmann fits
(smooth curves) gave following parameters: mSlo1, for 0 [Mg2?]i,
V1/2 ? ?2.8 mV, z ? 1.2, for 10 mM [Mg2?]i, V1/2 ? ?73.8 mV, z ?
1.1; dSlo1, for 0 [Mg2?]i, V1/2 ? ?2.9 mV, z ? 1.1, for 10 mM
[Mg2?]i, V1/2 ? ?69.2 mV, z ? 0.94. (B) Steady-state G-V relations
of the E413R mutant dSlo1 channel. Boltzmann fits (solid curves)
gave the following: E413R, for 5.7 ?M [Ca2?]i, V1/2 ? 163 mV, z ?
1.1, for 89 ?M [Ca2?]i, 0 [Mg2?]i, V1/2 ? ?24.6 mV, z ? 0.95, for
89 ?M [Ca2?]i, 10 mM [Mg2?]i, V1/2 ? ?46.7 mV, z ? 0.87. Dotted
lines are G-V relations for dSlo1 at 5.7 ?M [Ca2?]i and 89 ?M
[Ca2?]i, 0 [Mg2?]i. (C) Free energy provided by Mg2? binding for
channel activation in mSlo1, dSlo1, and E413R dSlo1 when
[Mg2?]i changes from 0 to 10 mM, measured at [Ca2?]i of 89 ?M.
(D) Free energy provided by Ca2? binding for channel activation
in WT and E413R dSlo1 when [Ca2?]i changes from 5.7 to 89 ?M.
High Ca2? sensitivity in dSlo1 is not related to the low
236The AC Region Regulates BKCa Gating
principal eigenmode is significantly different for the
two proteins (Fig. 9, top). Further, the two structures
show considerable differences in the fluctuations of
their backbone throughout the AC region (Fig. 9, bot-
tom). These simulations were performed on isolated
AC regions, indicating that the AC regions from dSlo1
and mSlo1 have intrinsic differences in their conforma-
tion and dynamics even without considering effects
of possible interactions between AC region and other
parts of the channel.
AC Region Modulates Channel Activation Depending on
Ca2? Binding and States of Gating
If AC region modulates Ca2?-dependent gating by alter-
ing the conformational changes induced by Ca2? bind-
ing, it may affect channel gating differently depending
on whether or not the Ca2? binding sites are occupied.
This is what we observed (Fig. 10) when we studied the
activation of mSlo1, dSlo1, m[dAC], and d[mAC] at 0
and 89 ?M [Ca2?]i, where the Ca2? binding sites are ei-
ther empty or nearly saturated (Fig. 3) (Cox et al.,
1997; Cui et al., 1997). In Fig. 10 A, it is immediately ap-
parent that AC region affects the voltage-dependent
channel activation at 0 [Ca2?]i. When the channel con-
tains AC region of dSlo1 (WT dSlo1 and m[dAC]),
little current could be measured even at ?240 mV,
mSlo1 and dSlo1. Top panels show the extrema (green and orange)
of the motion along the principal eigenvector for the AC region
from mSlo1 (left) and dSlo1 (right) (see MATERIALS AND
METHODS). Part of helix B highlighted in purple shows the
region of largest dynamic difference between the structures of
mSlo1 and dSlo1. Bottom panel shows the RMS fluctuations for
the C?’s of the above structure. The purple box denotes the same
region (?B) highlighted above. Amino acid in the sequence at the
bottom corresponds to the C? whose dynamics are plotted above.
Molecular graphics were produced using visual molecular dynam-
ics (VMD) (Humphrey et al., 1996).
Molecular dynamics simulations of the AC region of
depends on Ca2? occupancy. (A) Current traces of mSlo1, dSlo1,
d[mAC], and m[dAC] at 0 [Ca2?]i. Test voltages were from ?80 to
200 mV with a holding and repolarization potential of ?50 mV.
(B) Steady-state G-V relations of above channels at 0 (open sym-
bols) and 89 ?M (filled symbols) [Ca2?]i. Smooth curves are fits to
the Boltzmann equation. At 89 ?M [Ca2?]i, for mSlo1: V1/2 ? ?2.8
mV, z ? 1.2; for d[mAC]: V1/2 ? 7.7 mV, z ? 1.4; for dSlo1: V1/2 ?
?2.9 mV, z ? 1.1; for m[dAC]: V1/2 ? 41.6 mV, z ? 1.2. At 0
[Ca2?]i, for mSlo1: V1/2 ? 179 mV, z ? 1.2; for d[mAC]: V1/2 ? 181
mV, z ? 1.3. The voltage range of dSlo1 and m[dAC] activation at
0 [Ca2?]i is too positive to record any current. (C) Box plot of
?GV ? zV1/2 for the above channels at 0 and 89 ?M [Ca2?]i.
The percentile values shown are 10, 25, 50, 75, and 90 for each
channel. ?GV for m[dAC] and dSlo1 at 0 [Ca2?]i are too large to
Modulation of BKCa activation by the AC region
Krishnamoorthy et al.237
whereas the same voltage protocol elicited substantial
currents in the channels containing AC region of
mSlo1 (WT mSlo1 and d[mAC]). Subsequently, the
G-V relation at 0 [Ca2?]i for d[mAC] is similar in slope
and voltage range to that of WT mSlo1, whereas, in
m[dAC], as in WT dSlo1, the G-V curve was so far right
shifted that the shape could not be determined (Fig. 10
B). On the other hand, voltage dependence of activa-
tion for all four channels at nearly saturating (89 ?M)
[Ca2?]i are similar (Fig. 10 B). Fig. 10 C shows that at
nearly saturating [Ca2?]i, when Ca2? binding sites are
occupied, free energy of activation provided by voltage
(?GV) is about the same for all four channels, regard-
less of the origin of AC region. At 0 [Ca2?]i, however,
?GV for WT mSlo1 and d[mAC] is similar while that for
WT dSlo1 and m[dAC] is too large to be measured.
Thus, changing AC region in the channel causes a
large difference in voltage-dependent energy required
to open the channel when Ca2? binding sites are empty,
but has little effect on channel gating when Ca2? bind-
ing sites are occupied.
To further investigate the effects of AC region on
Ca2? sensitivity we obtained G-V relations of mSlo1,
dSlo1, m[dAC], and d[mAC] channels at various
[Ca2?]i between 0 and 89 ?M and fit them (Fig. 11)
with the 10-state MWC model (Cox et al., 1997). Al-
though the MWC model does not precisely describe the
voltage and Ca2?-dependent gating of BKCa channels
(Horrigan and Aldrich, 2002), it has been successfully
used to describe major characteristics of BKCa gating
(Cox et al., 1997; Magleby, 2003) and alterations by mu-
tations and coexpression with ? subunits (Cox and Al-
drich, 2000; Zhang et al., 2001; Shi and Cui, 2001; Bao
et al., 2002; Xia et al., 2002; Magleby, 2003). In the
model, the conformation of the Ca2? binding site(s)
changes from the closed state to the open state, result-
ing in a different dissociation constant, Kc and Ko, for
Ca2? binding to the closed and open states, respectively.
Ca2? binds to the open channel with higher affinity,
hence it shifts the closed–open equilibrium toward the
open conformation by factor “c” (c ? Ko/Kc). Since
more than one high-affinity Ca2? binding site in each
Slo1 subunit could contribute to activation, Fig. 11 A
shows the fits obtained for the G-V relations using the
MWC model of four Ca2? binding sites (n ? 4), one per
subunit, and Fig. 11 B shows the fits obtained for eight
Ca2? binding sites (n ? 8), two per subunit with similar
Kd’s (Bao et al., 2002; Xia et al., 2002).
In both cases, n ? 4 and n ? 8, dSlo1 has smaller val-
ues for the c factor than mSlo1, signifying that dSlo1 is
more sensitive to the effects of Ca2? binding than
mSlo1 (Fig. 11 C). The value of the c factor depends on
the origin of AC region (Fig. 11 C). Switching AC re-
gion of mSlo1 to that of dSlo1 (m[dAC]) increased
Ca2? sensitivity. Conversely, replacing AC region of
dSlo1 by its mSlo1 counterpart (d[mAC]) decreased
Ca2? sensitivity. These results suggest that higher Ca2?
sensitivity in dSlo1 is due to the greater ability of its AC
region to change Ca2? binding affinity during channel
gating in comparison with that of mSlo1.
It is striking that the value of Ko, the dissociation con-
stant for Ca2? binding to the open state, for all four
affects Ca2? binding to closed
channels. (A and B) G-V rela-
tions of mSlo1,
d[mAC], and m[dAC] chan-
nels at [Ca2?]i of 0, 1.7, 2.3,
5.7, 11.2, 28.5, and 89 ?M.
Each dataset was fit (smooth
curves) by the MWC model
(Eq. 4), n ? 4 (A) or 8 (B).
(C) Parameters of fits ob-
tained from A and B.
The AC region
238The AC Region Regulates BKCa Gating
channels is similar, regardless of the origin of AC re-
gion (Fig. 11 C). However, the dissociation constant of
Ca2? binding to the closed state, Kc, of dSlo1 is higher
than that of mSlo1, implying that Ca2? binds with
greater affinity to mSlo1 than to dSlo1 in the closed
state. Such a difference in Kc can be largely accounted
for by the switch of AC region (m[dAC] and d[mAC];
Fig. 11 C). These results suggest that the conformation
of AC region influences the conformation of Ca2?
binding sites that is apparent only when the channel is
closed, but with little effect when the channel is open.
D I S C U S S I O N
AC Region Is a Crucial Link in the Allosteric Machinery that
Couples Ca2? Binding to Channel Opening
We found that dSlo1 has higher Ca2? sensitivity than
mSlo1 such that for an identical increase in [Ca2?]i the
energy of activation provided by Ca2? binding, ??GCa,
in dSlo1 was about twice that of mSlo1 (Fig. 1). This dif-
ferential sensitivity was not due to a different Ca2? bind-
ing site in these channels because of two observations.
(1) Sequence differences in all putative metal binding
sites did not alter Ca2? sensitivity (Figs. 4, 5, 6, and 8).
(2) The value of Ko, the dissociation constant of Ca2?
binding in the open conformation, is the same (?1
?M) for both mSlo1 and dSlo1 (Fig. 11). Rather, we
find that the differences in conformation and dynamics
of the NH2-terminal (AC) region of the RCK1 domain is
responsible for the differential Ca2? sensitivity (Figs. 4,
5, and 9) such that when switched between mSlo1 and
dSlo1, the voltage- and Ca2?-dependent gating proper-
ties of these channels are largely reversed (Figs. 4, 5, 10,
and 11). Consistent with this mechanism, modulation
by AC region is Ca2? dependent as well as activation
state dependent (Figs. 10 and 11); the identity of AC re-
gion matters only when the Ca2? binding sites are empty
(Fig. 10) and in the closed states (Fig. 11). These effects
of AC region result in dSlo1 having a larger free energy
change provided by Ca2? binding during channel open-
ing (Figs. 1 and 10), i.e., dSlo1 has higher Ca2? sensitiv-
ity of activation. These results demonstrate that AC re-
gion is important in the allosteric coupling between
Ca2? binding and channel opening and may determine
the phenotype of Ca2?-dependent activation.
The role of AC region in allosteric coupling between
Ca2? binding and channel opening is also consistent
with its position in the structure of gating ring. The
crystal structure of MthK shows that RCK domains in
the gating ring interact with one another only at the
fixed and flexible interfaces, both of which are formed
by amino acids outside of AC region (Jiang et al.,
2002a). The NH2 terminus of AC region is directly con-
nected to the S6 activation gate (Liu et al., 1997; Web-
ster et al., 2004) through a short peptide linker while
the COOH terminus is upstream to ?-D and ?-E that
form the fixed interface with another adjacent RCK do-
main (Jiang et al., 2002a). A recent study demonstrated
that the linker between S6 and the RCK1 domain in
BKCa channels is important in controlling channel gat-
ing, suggesting that BKCa channels may have a similar
mechanism for Ca2?-dependent gating as MthK such
that a conformational change in the gating ring in-
duced by Ca2? binding opens the channel by pulling
the activation gate (Niu et al., 2004). The structural ar-
rangement suggests that both the linker and AC region
may be pulled during the conformational change in
the gating ring, while the fixed interface acts as a piv-
otal point. Consistent with the importance of the AC re-
gion in Ca-dependent activation found in this study, a
mutation in the AC region has been shown to alter
Ca2? sensitivity of the channel and is linked to epilepsy
and paroxysmal dyskinesia (Du et al., 2005).
Possible Mechanisms of AC Region Function in Ca2?-
dependent Gating of BKCa
Niu et al. (2004) showed that the voltage dependence
of BKCa channel open probability (Po–V relation) in the
absence of Ca2? exhibits a linear shift in voltage range
with respect to the length of the linker between the in-
ner helix and the gating ring. Based on this observa-
tion, they proposed that the channel is gated by a
spring-like mechanism and the change of linker length
changes the force of the spring. The structural identity
of the spring, however, was not clear. We find that the
changes in voltage dependence and Ca2? sensitivity
caused by a change in the AC region from mSlo1 to
dSlo1 are similar to the reported functional changes
caused by an increased linker length (Niu et al., 2004),
i.e., a shift in the voltage dependence to more positive
voltage ranges in the absence of Ca2? (Fig. 10) and an
increased Ca2? sensitivity (Figs. 1, 4, 5, 10, and 11). Fur-
thermore, in both studies, the voltage dependence of
channel activation is affected much more prominently
at 0 [Ca2?]i than at saturating [Ca2?]i (Niu et al., 2004)
(Fig. 10). Based on these similarities, it is reasonable to
suppose that the AC region functions as a likely spring
component in controlling voltage dependence and
Ca2? sensitivity of BKCa gating. Thus, changing the
linker length alters the compression of the spring and
hence the force on channel gating (Niu et al., 2004),
while exchanging the AC region between dSlo1 and
mSlo1 may alter either the compression or the stiffness
of the spring.
It is not known at present how AC region couples
Ca2? binding to channel opening. Based on the results
from Figs. 10 and 11, we propose a likely mechanism of
such coupling. When the channel is not bound to Ca2?,
AC regions from mSlo1 and dSlo1 cause a large differ-
Krishnamoorthy et al. 239
ence in the free energy of the channel gating (Fig. 10),
indicating that channel function is very sensitive to the
conformational and dynamical differences between AC
regions. However, it is striking that such an energetic
change derived from the structural variation largely dis-
appeared when the channel was saturated with Ca2?
(Fig. 10). This result could only be explained by two
possibilities. (1) Ca2? binding changes channel confor-
mation in a manner that AC region no longer contrib-
utes to the free energy of channel activation. Hence the
structural variations in AC region no longer matter. (2)
AC region contributes energetically to channel gating
at all [Ca2?]i’s. However, although the structural varia-
tions are critical to channel gating at low [Ca2?]i, they
matter little to channel gating at high [Ca2?]i. The
latter explanation seems unlikely given that the re-
sponse to low [Ca2?]i is sensitive to sequence variation
throughout the AC region (Figs. 4–7, 9, and 11); and
it is difficult to imagine that the sum contribution
of these multiple differences would coincidentally be
equal in saturating [Ca2?]i.
Therefore, these results left us with the more likely
conclusion that AC region may not contribute energet-
ically to channel gating at saturating [Ca2?]i. This con-
clusion is equivalent to a mechanism wherein AC re-
gion inhibits the channel in the absence of Ca2?, while
Ca2? binding relieves the inhibition and allows the
channel to gate by less voltage-dependent free en-
ergy. This mechanism is consistent with the findings in
the cAMP modulation of HCN channel gating, where
the COOH-terminal cyclic nucleotide-binding domain
(CNBD) inhibits channel activation and the binding of
cAMP relieves this inhibition (Wainger et al., 2001).
Similar mechanism for BKCa channel activation by the
Ca2? bowl has been previously proposed (Schreiber et
al., 1999). In addition, we have also observed that the
difference in the AC region structure alters channel
conformation at closed state but not open state (Fig.
11), which is consistent with the mechanism that the
AC region inhibits channel from opening by stabilizing
the closed state.
A model on the mechanism of how AC region modu-
lates BKCa gating can be used to summarize the above
discussions (Fig. 12). In this model, AC region stabi-
lizes the closed conformation when the Ca2? binding
sites are empty. When the Ca2? binding sites are satu-
rated or the channel is in the open state, AC region no
longer affects BKCa gating (Fig. 12), which, in terms of
a spring model, is as if a spring were slackened. How-
ever, the model in Fig. 12 does not require that AC re-
gion behaves like a spring. Regardless of whether or
not AC region per se changes conformation during
channel gating, the model will be similar as long as the
conformational changes of the channel induced by
Ca2? binding result in AC region no longer affecting
the activation gate. It is worth pointing out that this
model requires a conformational change in the closed
state upon Ca2? binding, which differs from the MWC-
based models that assume a conformational change
only occurring during the closed–open transition.
An interesting outcome of the parameter fits to the
MWC model is the 20-fold difference in the values of
L(0) between mSlo1 and dSlo1 (Fig. 11 C). In the
MWC model used here, L(0) is a lumped parameter
representing the steady-state equilibrium of the volt-
age-dependent closed–open transition that occurs in
the absence of Ca2? binding. Thus, L(0) may be taken
as a measure of the voltage-dependent activation en-
ergy that combines the energy of voltage sensor activa-
tion as well as channel opening. While the L(0) value
for mSlo1 is consistent with those published before
(Cox et al., 1997; Cui and Aldrich, 2000; Shi and Cui,
2001), no such reference exists for dSlo1. However, we
note the higher value of L(0) as being consistent with
the experimental observation that in the absence of
Ca2? binding, the voltage dependence of activation of
the channel is extremely right shifted (Fig. 1 B and Fig.
10). Indications of such differences have been also ob-
served in previous studies (Adelman et al., 1992; Wei et
al., 1994; Silberberg et al., 1996; Moss et al., 1999). This
could be interpreted as a suggestion that the allosteric
the absence of bound Ca2? and in the closed conformation, the
AC region adopts a conformation that inhibits channel opening.
Ca2? binding (?Ca2?) or channel opening by depolarization
(?V) removes this inhibition, rendering the channel gate more
favorable to the open conformation. In the open conformation or
when the Ca2? binding sites are occupied, the AC region has little
effect on channel gating.
Spring effect of the AC region in channel gating. In
240 The AC Region Regulates BKCa Gating
mechanisms involved in the voltage-dependent gating
of dSlo1 may be quite different from that of mSlo1. Ex-
ploring this aspect of dSlo1 activation in more detail
warrants a different set of experiments and probably a
different set of kinetic schemes, which is beyond the
scope of the present study. The change in voltage de-
pendence, however, should not affect our fitting results
on the Ca2? sensitivity using the MWC model because
it has been shown that Ca2? and voltage activate the
channel through separate activation pathways, and the
change of one pathway by mutations or ?-subunit asso-
ciation does not affect the other significantly (Cox and
Aldrich, 2000; Cui and Aldrich, 2000; Shi and Cui,
2001; Horrigan and Aldrich, 2002; Niu and Magleby,
2002; Hu et al., 2003; Orio and Latorre, 2005).
Recent structural studies demonstrate that the ionic
pore and the activation gate of various ion channels
adopt a similar structure (Doyle et al., 1998; Jiang et al.,
2002b, 2003; Kuo et al., 2003), while they are associated
with different gating modules that sense various stimuli
such as voltage and intracellular ligands to control the
closed–open transitions of the channel. BKCa is acti-
vated by both voltage and intracellular divalent cations.
It is likely that a general and conserved principle of
BKCa gating in coupling the stimuli to channel gate is
adopted by both the intracellular gating ring and the
transmembrane voltage sensor. If AC region modulates
BKCa channel gating by inhibiting channel from open-
ing while Ca2? binding relieves this inhibition as sug-
gested in the above model (Fig. 11), the voltage-depen-
dent mechanism may also activate the channel by re-
lieving an inhibition, similar to a mechanism suggested
for voltage-gated Shaker K? channels (Armstrong, 2003).
Obviously, further investigation is required to test such
The mSlo1 clone was kindly provided to us by Larry Salkoff
(Washington University) and the dSlo1 clone by John Adelman
(Vollum Institute, Portland, OR). Data for Mg2?-dependent acti-
vation of WT dSlo1 was obtained by Lei Hu and Huanghe Yang.
We thank Urvi Shah, Frank Horrigan, and Richard Aldrich
for comments on the manuscript and Gary Yellen for helpful
This work was supported by grants from National Institutes
of Health (R01-HL70393), American Heart Association (Estab-
lished Investigator Award), and The Whitaker Foundation (to J.
Cui). J. Cui is Associate Professor of Biomedical Engineering on
the Spencer T. Olin Endowment.
Lawrence G. Palmer served as editor.
Submitted: 4 May 2005
Accepted: 15 July 2005
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