Address correspondence to Daniel H. Cox, 750 Washington St., NEMC Hospitals, Box 7868, Boston, MA 02111. Fax: 617-636-0576; E-mail: dan.cox{at}tufts.edu
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ABSTRACT |
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Key Words: mSlo Slo potassium channel Ca2+ bowl Ca2+ binding
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INTRODUCTION |
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BKCa channels (Fig. 1)
are structurally similar to K+ channels gated only by voltage (KV). Four identical subunits, known by their gene name slo, form a fully functional channel (Shen et al., 1994), and the amino-terminal third of the Slo protein contains several membrane-spanning regions, including a pore domain and a S4 helix that likely forms the channel's voltage sensors (Adelman et al., 1992
; Butler et al., 1993
; Diaz et al., 1998
; Cui and Aldrich, 2000
). Unlike KV channels, however, the Slo subunit contains a large intracellular domain that confers Ca2+ sensitivity on a voltage-gated structure (Wei et al., 1994
; Schreiber et al., 1999
; Moss and Magleby, 2001
). The COOH-terminal part of this intracellular domain is termed the channel's "tail," while the portion of Slo that precedes the tail is termed the "core" (Fig. 1) (Wei et al., 1994
). Neither core nor tail contain canonical Ca2+-binding motifs, and so the origin of the BKCa channel's Ca2+ sensitivity remains unresolved.
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Arguing against an exclusive or even dominant role for the Ca2+ bowl in Ca2+ sensing, however, are the following observations: when the same aspartates were mutated in the full-length channel, the channel remained Ca2+ sensitive, its G-V relation still shifting well over 100 mV in response to changes in [Ca2+] (Bian et al., 2001); and this has been the case for all Ca2+ bowl mutations so far reported (Schreiber and Salkoff, 1997
; Bian et al., 2001
; Braun and Sy, 2001
). No mutation in the Ca2+ bowl has been shown to substantially alter the concentration range over which the channel responds to Ca2+, and mutations outside the Ca2+ bowl have also been shown to cause rightward G-V shifts at constant [Ca2+] (Diaz et al., 1998
; Cui and Aldrich, 2000
; Braun and Sy, 2001
). Thus, if there is a Ca2+-binding site in the Ca2+ bowl, then there are likely to be others outside this region as well (Schreiber and Salkoff, 1997
; Schreiber et al., 1999
; Bian et al., 2001
).
To investigate further the role of the Ca2+ bowl in the Ca2+-sensing mechanism of the BKCa channel, we made a series of mutations in the mSlo Ca2+ bowl and analyzed their effects on the energetics of Ca2+-dependent gating. Our data argue that Ca2+ bowl mutations can eliminate the ability of a high-affinity Ca2+-binding site to promote channel opening, and therefore suggest that a Ca2+-binding site is present in this region. In addition, however, we have identified another site, at the end of the channel's seventh hydrophobic domain, that when mutated, also impairs Ca2+ sensing. And when the two sites are mutated together, all high-affinity Ca2+ response is lost. Thus, we propose that the BKCa channel contains two types of high-affinity Ca2+-binding sites, and in the experiments detailed below we estimate their binding characteristics.
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MATERIALS AND METHODS |
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Mutagenesis
All mutations were made with the Quick Change Sitedirected mutagenesis kit (Stratagene), and mutations were identified by sequencing around the point of the mutation. Further, the coding regions of the following channels were sequenced in their entirety (wild-type mSlo [mbr5], 896903, M513I,
896903+M513I,
899903+M513I).
Electrophysiology
Electrophysiological recordings were performed essentially as described previously (Cox and Aldrich, 2000). All recordings were done in the inside-out patch-clamp configuration (Hamill et al., 1981
). Patch pipettes were made of borosilicate glass (VWR micropipettes), and had resistances of typically 12 Mohms in our recording solutions. Their tips were coated with wax (Sticky Wax) and fire polished before use. Data were acquired using an Axopatch 200B patch clamp amplifier (Axon Instruments, Inc.) or a list EPC-7 and a Macintosh-based computer system using "Pulse" acquisition software (HEKA Electronik) and the ITC-16 hardware interface (Instrutech). Records were digitized at 20-µs intervals and low pass filtered at 10 KHz. All experiments were performed at room temperature, 2224°C. Before current records were analyzed and displayed, capacity and leak currents were subtracted using a P/5 leak subtraction protocol with a holding potential of -120 mV and voltage steps opposite in polarity to those in the experimental protocol.
Solutions
In general, recording solutions were composed of the following (in mM). Pipette solution: 80 KMeSO3, 60 N-methyl-glucamine-MeSO3, 20 HEPES, 2 KCl, 2 MgCl2, pH = 7.20. Internal solution: 80 KMeSO3, 60 N-methyl-glucamine-MeSO3, 20 HEPES, 2 KCl, 1 HEDTA or 1 EGTA, and CaCl2 sufficient to give the appropriate free Ca2+ concentration; pH, 7.20. EGTA (Sigma-Aldrich) was used as the Ca2+ buffer for solutions containing <0.8 µM free [Ca2+]. HEDTA (Sigma-Aldrich) was used as the Ca2+ buffer for solutions containing between 0.8 and 10 µM free [Ca2+], and no Ca2+ chelator was used in solutions containing 100 µM free [Ca2+]. 50 µM (+)-18-crown-6-tetracarboxylic acid (18C6TA) was added to all internal solutions to prevent Ba2+ block at high voltages.
The appropriate amount of total Ca2+ (100 mM CaCl2 standard solution; Orion Research, Inc.) to add to the base internal solution containing 1 mM HEDTA or 1 mM EGTA to yield the desired free Ca2+ concentration was calculated using the program Max Chelator (Bers et al., 1994), which was downloaded from www.stanford.edu/~cpatton/maxc.html, and the proton and Ca2+-binding constants of Bers (supplied with the program) for pH = 7.20, T = 23°C, and ionic strength = 0.15. The ability of 18C6TA to chelate Ca2+, as well as K+ and Ba2+, was also considered in these calculations using the following dissociation constants: Ca2+ 10-8 M (Dietrich, 1985
), K+ 3.3 x 10-6 M (Dietrich, 1985
), Ba2+ 1.6 x 10-10 M (Diaz et al., 1996
). Free [Ca2+] was measured with a Ca-sensitive electrode (Orion Research, Inc.) and the measured value was reported. Free [Ca2+] measurements were precise to within
8%. Standard Ca2+ solutions for calibration of the Ca-sensitive electrode were purchased from World Precision Instruments (10-810-2 M). Endogenous [Ca2+] in our internal solution before addition of Ca2+ chelator was estimated from the deviation from linearity of the Ca-sensitive electrode's response at 10 µM added [Ca2+], and was 1620 µM. Endogenous [Ca2+] was then compensated for when making Ca2+-buffered solutions.
During our experiments, the solution bathing the cytoplasmic face of the patch was exchanged using a sewer pipe flow system (DAD 12) purchased from Adams and List Assoc. Ltd.
In the experiments of Fig. 5 , Mg2+ was often added to the internal solution. When this was the case, the following solutions were used. For the solution containing nominally 0 [Ca2+] and 10 mM [Mg2+], our base internal solution described above was used with the addition of 5 mM rather than 1 mM EGTA, and 12.1 mM MgCl2 (Sigma-Aldrich 4.9 M stock). For the solution containing 100 µM [Ca2+] and 10 mM [Mg2+] our base internal solution was used without added chelator, and 90 µM CaCl2 and 10 mM MgCl2 were added. For solutions containing 100 mM [Mg2+] our base internal was also used, except N-methyl-glucamine was not included, and then either 15 mM EGTA and 112 mM MgCl2 were added to create a solution containing nominally 0 [Ca2+] and 100 mM free [Mg2+], or 0 EGTA, 100 mM MgCl2, and 90 µM CaCl2 were added to create a solution containing 100 µM free [Ca2+] and 100 mM free [Mg2+].
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RESULTS |
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Because G-V compression can be attributed to an alteration in the voltage-sensing aspect of gating (Cox et al., 1997; Cox and Aldrich, 2000
; Cui and Aldrich, 2000
), it is reasonable to question whether the Ca2+-binding properties of the mSlo channel have been altered at all by the 899903A mutation. However, the mutation's relatively small effects on G-V steepness and position at very low [Ca2+] and its large effect on G-V position at high [Ca2+] make a compelling case for a true change in the channel's Ca2+-binding properties. This can be evaluated quantitatively by interpreting these data in terms of the voltage-dependent Monod-Wyman-Changeaux model (VD-MWC), which supposes a central voltage-dependent closed-to-open conformational change that is allosterically regulated by Ca2+ binding (Cox et al., 1997
; Cox and Aldrich, 2000
) (see diagram in Fig. 6
B). According to this model, the position of the channel's G-V relation as indexed by its half-activation voltage (V1/2) is given by the following function of [Ca2+]
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![]() | (1) |
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If this idea is correct, then mutations in the Ca2+ bowl more severe than 899903A would not be expected to display a more severe phenotype, as under this hypothesis Ca2+ binding at the Ca2+ bowl is completely lost with 899903A. To test this prediction, we mutated to alanine four additional residues in the heart of the Ca2+ bowl (895903), three aspartates and one glutamine. Remarkably, despite the loss of 7 of 10 Ca2+ bowl acidic side chains, the 895903A channel's equilibrium gating behavior is still appreciably Ca2+ sensitive, and very similar to that of the less severe 899903A mutant (Fig. 2 E and Table I). Other than a small leftward (rather than rightward) G-V shift at 3 nM [Ca2+], neutralizing an additional three acidic and therefore potentially Ca2+-coordinating side chains had no further effect on G-V position as a function of [Ca2+] (Fig. 2 D and Table I). Thus, the smaller 899903A mutation appears to be sufficient to completely eliminate Ca2+ binding at the Ca2+ bowl.
To examine, however, whether deleting rather than altering Ca2+-bowl residues might create a more severe phenotype, we also made the following deletion mutants: 895903,
896903,
898903, and
899903 deletions of 9, 8, 6, and 5 residues, respectively. Unfortunately, the
895903 mutant did not produce stable currents. The others, however, did express well and displayed phenotypes that are similar to that of the alanine-substitution mutants described above: little effect at 3 nM and a fairly large reduction in the power of 100 µM [Ca2+] to shift the mSlo G-V relation leftward (Fig. 3)
. In fact, the phenotype of the smaller deletion (
899903) is virtually identical to that of 895903A, and that of the larger deletions differs primarily at 100 µM [Ca2+], where the G-V curves for
896903 and
898903 are 74 and 79 mV right-shifted relative to wild-type, rather than 56 mV as is observed for 895903A (Fig. 3 C and Table I). Despite this difference, the similar phenotypes of the five Ca2+-bowl mutants described above suggest the disruption of a common function, and we might tentatively conclude that the alanine-substitution mutants (899903A, 895903A) and the smaller deletion (
899903) cause a near complete loss of function at the Ca2+ bowl, whereas the larger deletions (
896903,
898903) eliminate Ca2+-bowl function.
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Mg2+ Eliminates the Double Mutant's Ca2+ Sensitivity
BKCa channel gating can be modulated by intracellular Mg2+ (Golowasch et al., 1986) and millimolar concentrations of Mg2+ have recently been shown to activate the mSlo channel via a site in its core domain that also has a low affinity for Ca2+ (Shi and Cui, 2001
; Zhang et al., 2001
). As the double mutants described above show only a low-affinity Ca2+ response, we tested whether the remaining Ca2+ sensitivity of the M513I+
896903 mutant is due to Ca2+ binding at this low-affinity, Mg2+-sensitive site. We examined the shift of the double mutant's G-V relation in response to 100 µM [Ca2+] in the absence and presence of 10 and 100 mM [Mg2+]. If Ca2+ binding at the Mg2+-sensitive site is responsible for the double mutant's low-affinity Ca2+ response, then 100 mM [Mg2+] would be expected to occupy this site and eliminate the effect of Ca2+, whereas 10 mM [Mg2+] would be expected to inhibit but not eliminate the effect of Ca2+ (Shi and Cui, 2001
; Zhang et al., 2001
).
As shown in Fig. 5 A, these predictions were born out in our data. In the presence of 10 mM [Mg2+] the 896903+M513I channel's G-V shift, when exposed to 100 µM [Ca2+], is reduced from 45 ± 3.6 mV to 24 ± 3.4 mV, and in the presence of 100 mM [Mg2+] it is completely eliminated (
V1/2 = -2 ± 6.8 mV, see Fig. 5 A, bottom). Furthermore, when the same experiment is performed with the wild-type channel (Fig. 5 B), 100 mM [Mg2+] does not completely eliminate the effect of 100 µM [Ca2+], but rather reduces
V1/2 by an amount similar to its effect on the double mutant (Fig. 5 B bottom). These results argue that Mg2+ only effectively blocks the channel's low-affinity Ca2+ binding sites (as has been reported, Shi and Cui, 2001
; Zhang et al., 2001
), and that the weak Ca2+ response of the double mutant is due to low-affinity Ca2+ binding at Mg2+-sensitive sites. Furthermore, the high-affinity response of the wild-type channel must therefore arise from Ca2+ binding at sites whose functional effects are completely eliminated by the double mutant. This is remarkable, because it suggests the presence of three types of Ca2+-binding sites on the mSlo channel, one of low affinity whose effects can be blocked by high concentrations of Mg2+, and two of higher affinity, one whose effects are eliminated by mutations in the Ca2+ bowl, and another whose effects are eliminated by mutation at M513.
Estimating the Ca2+-binding Characteristics of the Channel's High-Affinity Sites
The mutations described above must cause complete loss of function at their respective sites; otherwise, the double mutant would respond to Ca2+ even after its low-affinity sites are occupied by Mg2+. Thus, they provide an opportunity to estimate the binding characteristics of the sites they disrupt. To do this, however, requires extracting from the wild-type and mutant G-V data the degree to which Ca2+ binding is affecting G(0)o-c, the free energy difference between open and closed in the absence of an applied voltage. That is, we would like to plot
G(0)o-c rather than V1/2 as a function of [Ca2+], so as to examine a physically meaningful Ca2+-dependent quantity that is independent of the mechanism by which the channel responds to voltage, and yet readily interpretable in terms of the binding properties of the channel's Ca2+-binding sites.
A strategy which has been used to do this in the past is to fit each G-V relation with a Boltzmann function and from the parameters of the fit estimate G(0)o-c as
G(0)o-c = zFV1/2 (Cox and Aldrich, 2000
; Cui and Aldrich, 2000
; Jiang et al., 2001
; Shi and Cui, 2001
), and in fact this was the strategy we used above in examining the 899903A mutant. In effect, it assumes a two-state model for the channel's voltage-sensing mechanism, and then on the basis of this model estimates the effect of Ca2+ binding on the free energy difference that the applied voltage must overcome to open the channel. As discussed above, for the voltage-dependent MWC model (Fig. 6 B) this strategy is strictly correct (Cox et al., 1997
; Cox and Aldrich, 2000
; Cui and Aldrich, 2000
).
Results of such an analysis for the wild-type channel and the 896903 and M513I mutants are plotted in Fig. 6 F, together with standard V1/2 vs. [Ca2+] plots (Fig. 6 D). New data have been added for each channel at 71, 130, and 360 nM [Ca2+] so as to get a better sense of the shapes of the resulting
G(0)o-c vs. [Ca2+] relations. Upon examining these relations, we see that the zFV1/2 values, or equivalently the
G(0)o-c values, of the three channels are very similar at 3 nM [Ca2+], indicating that the intrinsic free energy difference between open and closed is unaltered by the mutations. Also, 100 µM [Ca2+] causes a smaller change in the
G(0)o-c values of the two mutants than it does that of the wild-type channel, reflecting a weaker influence of Ca2+ binding on the channel's closed-to-open conformational change. More interesting, however, are the shapes of the three
G(0)o-c vs. [Ca2+] curves. Over the [Ca2+] range 3 nM to 0.8 µM, where V1/2 for all three channels is changing appreciably (see Fig. 6 D),
G(0)o-c for all three channels remains nearly constant (Fig. 6 F). This is unexpected, because, according to the current view, it is only through altering
G(0)o-c that Ca2+ binding alters V1/2. That is, Ca2+ binding is thought to shift the mSlo G-V relation, not because it alters the channel's voltage-sensing mechanism, but because it decreases the energy barrier this mechanism must work against. Thus, V1/2 is not expected to change without a concomitant change in
G(0)o-c, and the incongruity we observe between Fig. 6, D and F, suggests that either this view is not correct, or the method we have used to estimate
G(0)o-c is inaccurate, at least at [Ca2+] less than
1 µM.
To test the latter possibility, we used a model of BKCa channel gating developed by Horrigan et al. (1999) that is more sophisticated in its voltage-sensing mechanism than the VD-MWC model. It supposes four voltage sensors, one in each subunit, that move independently to promote channel opening. Voltage-sensor movement is not required for channel opening, however, but rather promotes opening in a manner similar to Ca2+ binding in an MWC system. It also supposes a weak voltage dependence associated with its central closed-to-open conformational change, and not including Ca2+ binding, it consists of the 10 states illustrated in Fig. 7
A. Adding then four identical Ca2+-binding sites that also allosterically promote opening leads to the 50-state model depicted in Fig. 7 B (Cox and Aldrich, 2000
; Rothberg and Magleby, 2000
). Although apparently complex, this model contains only seven free parameters, and yet it has been shown to account for mSlo macroscopic currents over wide ranges of [Ca2+] and voltage both with and without the BKCa ß1 subunit (Cox and Aldrich, 2000
) and, as well, gating currents at nominally 0 [Ca2+] (Horrigan and Aldrich, 1999
; Horrigan et al., 1999
). Furthermore, a scheme of the same form was developed simultaneously to account for mSlo's single-channel gating behavior (Rothberg and Magleby, 2000
). Thus, the model illustrated in Fig. 7 B, although overly simple in its Ca2+-sensing mechanism, represents the latest view of the relationship between Ca2+ binding and voltage sensing in BKCa-channel activation.
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Interestingly, although the 50-state model assumes no direct effect of Ca2+ binding on voltage sensing, the Boltzmann method of estimating G(0)o-c severely underestimates the model's true
G(0)o-c at [Ca2+] below
1 µM, where its G-V curves become both increasingly shallower and poorly fitted by a Boltzmann function, characteristics also observed in the data (see Figs. 2 4). Furthermore, the underestimation occurs in such a way that the estimated
G(0)o-c vs. [Ca2+] relation is almost constant between 0 and 1 µM [Ca2+], when the true relation is changing by
23%. Thus, the unexpected, flat characteristic we observed in our analysis of the data in Fig. 6 is also observed here, and it thus seems likely that in Fig. 6 F we are indeed underestimating
G(0)o-c at low [Ca2+] when using zFV1/2 as an estimator.
To attempt to better estimate G(0)o-c, we took advantage of the understanding of the voltage-dependent mechanism of mSlo gating developed by Horrigan et al. (1999)
. Instead of using a Boltzmann function, we fit each G-V curve with the following function, which describes the G-V relation of the scheme diagramed in Fig. 7 A (Horrigan and Aldrich, 1999
; Horrigan et al., 1999
).
![]() | (2) |
Here, JO(0) and JC(0) represent the equilibrium constants for voltage-sensor movement in each subunit at 0 mV when the channel is open or closed, respectively, z represents the gating charge associated with each voltage sensor, q represents the gating charge associated with the model's central conformational change, and ACa is a Ca2+-dependent factor that is logarithmically related to G(0)o-c. That is, assuming only that Ca2+ binding promotes opening, and the voltage-dependent gating mechanism described by the scheme in Fig. 7 A, for any [Ca2+], we may write:
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Thus, we fit the wild-type and mutant G-V curves with Eq. 2, fixing the voltage-dependent gating parameters to values we have used previously to describe mSlo gating (Cox and Aldrich, 2000), and from the ACa parameter and Eq. 3 calculated
G(0)o-c. This method of estimating
G(0)o-c is precisely accurate for the 50-state model, and precisely accurate as well for any model with a voltage-sensing mechanism described by the scheme in Fig. 7 A, regardless of the number and type of Ca2+-binding sites it contains, as long as those sites do not directly interact with the channel's voltage sensors.
Estimates of G(0)o-c as a function of [Ca2+] for the wild-type channel and three of the mutants described above are plotted in Fig. 8
A. Supporting the idea that we have improved our estimates of
G(0)o-c at low [Ca2+], the
G(0)o-c vs. [Ca2+] relations for all four channels are no longer flat in the 3 nM to 0.8 µM [Ca2+] range, but now better follow changes in V1/2. In Fig. 8 B we have shifted each curve in A vertically so as to have a value of zero at 3 nM [Ca2+], and thereby created plots of
G(0)o-c, the change in
G(0)o-c as a function of [Ca2+], a measure of the influence of [Ca2+] on the energetics of channel opening. And in Fig. 8 C we have plotted difference curves obtained by subtracting each mutant's curve in Fig. 8 B from the wild-type curve. These curves measure the Ca2+ influence lost by each mutation and thus are reflective of the properties of the binding site each mutation disrupts.
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![]() | (4) |
This relation has two free parameters, KC and KO, and listed in Table II
are estimates of these parameters for the sites disrupted by each mutation. Examining the results from the 896903 and M513I mutations, we see that both types of high-affinity Ca2+-binding site appear to function similarly, each having an affinity for Ca2+ of 3.53.8 µM when the channel is closed and 0.800.94 µM when the channel is open, and each maximally altering
G(0)o-c by approximately -3.4 kJ/mol as Ca2+ binds, for a total of approximately -13.5 kJ/mol for each type of site. Also listed under site 1 in Table II, however, are affinity-constant estimates derived from the WT
899903 difference curve in Fig. 8 C, because, as discussed below, it is not clear which mutant,
896903 or
899903, represents elimination of just Ca2+ bowl function. The values derived from the WT-
899903 difference curve suggest a somewhat higher Ca2+ affinity for site 1, both in the closed and open conformation (KC = 1.8 µM, KO = 0.6 µM), with a maximum
GCa(0)o-c per site of -2.4 kJ/mol for a total of -9.5 kJ/mol.
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DISCUSSION |
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Are There Two High-Affinity Sites?
It is natural to question whether mutations that create similar phenotypes alter the same or different processes. One could imagine that although separated by 370 amino acids in the channel's primary sequence, the Ca2+ bowl and M513 are oriented such that mutations at both sites alter the same process by which Ca2+ binding promotes opening. The observation that the combined effect of mutations at both sites is considerably stronger than mutations at either site alone, however, argues against this hypothesis. In fact, the double mutants, 896903+M513I and
899903+M513I, do not respond appreciably to [Ca2+] below
10 µM, whereas the single mutants,
896903,
899903, and M513I, each start to respond to [Ca2+] (as does the wild-type channel) at
0.1 µM. Thus, mutations at the two sites are qualitatively additive, and likely alter separate binding sites. Indeed, we find that the energetic effects of
899903 and M513I, as shown in Fig. 9 B, are very close to quantitatively additive. The difference between the measured
Go-c value (3 nM to 100 µM) of the double mutant M513I+
899903 and the value predicted by strict additivity is 2.25 kJ/mol, a value that is <10% of the
Go-c (3 nM to 100 µM) of the wild-type channel, and less than is typically taken to indicate nonindependence in studies of interactions between mutations (1.5 kT or 3.69 kJ/mol; Schreiber and Fersht, 1995
; Naranjo and Miller, 1996
; Ranganathan et al., 1996
). Thus, it would appear that the
899903 and the M513I mutations are acting independently and on separate Ca2+-binding sites.
We cannot rigorously exclude the possibility, however, that the 899903 and M513I mutations affect a single binding site in such a way that they happen to behave additively. In fact, the combined effects of the
896903 and M513I mutations are less than additive by 5.47 kJ/mol, or 20% of the of the wild-type channel's
Go-C value (3 nM to 100 µM). Here, one mutation partially obscures the effects of the other, suggesting a common process is altered. This result could also be explained, however, by supposing that the binding of Ca2+ at one site influences binding at the other, or more simply that in addition to disrupting the Ca2+ bowl, the
896903 mutation has a partial effect at the second site, a conclusion that also requires the presence of two types of high-affinity sites. Thus, we think two distinct high-affinity Ca2+-binding sites are likely.
Supporting this conclusion, Bian et al. (2001) have found that the Ca2+-bowl mutation 897901N reduces the dSlo channel's Hill coefficient for Ca2+ activation by a factor of
2 at many voltages, a result consistent with the idea that the Ca2+-bowl mutation disrupts one type of high-affinity Ca2+-binding site while leaving another intact. This same mutation also reduces Ca2+ binding to a fusion protein consisting of the last 280 amino acids of dSlo by approximately half. Furthermore, Schreiber and Salkoff (1997)
have argued for a second type of mSlo Ca2+-binding site based on the observation that mutations at the Ca2+ bowl alter the effects of Ca2+, but leave the activating effects of Cd2+ unaltered. And in another series of experiments they divided the mSlo tail domain into four regions (AD) and found that both region B, which contains the Ca2+ bowl, and region C which does not, can each confer partial Ca2+ sensitivity upon an insensitive mSlocoremSlo3tail chimeric channel. Thus, the two high-affinity site interpretation we have given our results is not at odds with previous work.
Do the Mutations Create Complete Loss of Function?
Does each mutation completely eliminate the effect of Ca2+ binding at the site it disrupts? The answer to this question appears to be yes, at least for the 896903 and the M513I mutations, as when both mutations are made together all high-affinity Ca2+ response is lost, leaving only a low-affinity response blocked by high concentrations of Mg2+. Indeed, Shi and Cui (2001)
and Zhang et al. (2001)
have extensively examined the ability of Mg2+ to activate the mSlo channel. Both groups concluded that Mg2+ activates the channel via a site separate from the channel's high-affinity Ca2+-binding sites. This site was estimated to have an affinity for Ca2+ of 0.61 mM when the channel is open and 23 mM when the channel is closed, and an affinity for Mg2+ of 26 mM when the channel is open and 1020 mM when the channel is closed (Zhang et al., 2001
). For a channel with four such sites, a simple VD-MWC type model predicts that this low-affinity site would start to influence the position of the mSlo G-V relation at just above 10 µM [Ca2+], just as we observed with our double mutants (simulations not shown). And it would be
90% occupied by 100 mM [Mg2+] and therefore almost fully activated. Thus, our double mutant
896903+M513I acts exactly as one would predict if it creates a channel whose high-affinity Ca2+-binding sites have lost their function, but whose low-affinity Mg2+-sensitive, Ca2+-binding sites are still intact (Figs. 4 and 5). These results therefore lend further credence to the notion that the BKCa channel has low-affinity, Mg2+-sensitive Ca2+-binding sites that are separate from its high-affinity sites, and they pave the way for the study of these low-affinity sites in isolation.
No Other Binding Sites
The results of our double mutant experiments also argue that the mSlo channel contains no other high-affinity Ca2+-binding sites besides those disrupted by mutations at the Ca2+ bowl and M513 and no low-affinity sites that are not Mg2+sensitive. If either of these suppositions were not correct, then the 896903+M513I channel would be expected to respond to increases in [Ca2+] in the presence high concentrations of Mg2+ via these as yet unaccounted for sites. Generally, no such response is observed. It should be mentioned, however, that although we saw no significant change in the G-V position of the
896903+M513I channel as [Ca2+] was increased from <3 nM to 100 µM in the presence of 100 mM [Mg2+], we did see a small but consistent change in the shape of the channel's G-V relation. The relation became more shallow by 27% (Fig. 5) as [Ca2+] was raised. Whether this is due to weaker binding of Mg2+ at low voltages, or some real remaining Ca2+ effect will require further investigation.
Estimates of Ca2+-binding Characteristics
Identifying mutations that cause a complete loss of function at mSlo's high-affinity Ca2+-binding sites affords a unique opportunity to estimate the binding characteristics of these sites. Results of our analysis suggest that the site disrupted by M513I, which we refer as site 2, accounts for 50% of the -26.7 kJ/mol that 100 µM [Ca2+] imparts to the channel closed-to-open conformational change, whereas the Ca2+ bowl site (site 1) imparts 3550%, depending on which mutation (
89990 or
896903) is analyzed. Thus, type 1 and 2 sites appear to contribute roughly equally to the high-affinity effects of Ca2+. Assuming one binding site of each type per subunit (four per channel), and taking our estimates from the
896903 mutant, we estimate KO for both sites to be 0.80.9 µM and KC to be 3.53.8 µM. Thus, both sites appear to have very similar binding characteristics. If, however, the channel has an unequal number of sites of each type, as might arise if one type of site is formed at the interface between subunits while the other is not, this may not be the case. But our estimates of the energetic contributions of each type of site (-9.5 to -13.6 kJ/mol for site 1 and -13.4 kJ/mol for site 2) would remain valid.
How Do the Mutations Create a Loss of Function?
How do the mutations we have described exert their effects? Must they act directly at the binding site? Indeed, one might suppose that mutations at either position affect Ca2+ sensitivity, not because they affect Ca2+ binding, but because they affect the ability of Ca2+ binding to influence opening. Suppose, as suggested by Schreiber et al. (1999), that there is a Ca2+-binding domain that swings as if on a hinge after Ca2+ binds, and in so doing promotes opening. One might then suppose that a given mutation breaks the hinge without affecting Ca2+ binding. Thermodynamic constraints dictate, however, that in order for Ca2+ binding to promote opening, the affinity of the binding site must increase as the channel opens, and conversely, if a binding site's affinity increases as the channel opens, binding must promote opening. Thus, for a mutation to eliminate the effectiveness of a binding site, it must either prevent binding altogether, or prevent the change in affinity that occurs at the binding site as the channel opens. Thus, binding and the transduction of binding to opening are inextricably linked, and the mutations we have described must therefore affect Ca2+ binding.
One might still ask, however, whether the mutations we have studied disrupt residues that actually make contact with Ca2+? Our data do not rule out the possibility that some protein endogenous to the oocyte associates with mSlo to form its Ca2+ sensors, as is the case for the small-conductance Ca2+-activated K+ channel, which uses constitutively associated calmodulin as its Ca2+ sensor (Xia et al., 1998; Wissmann et al., 2002
). It is conceivable, then, that mutations at the Ca2+ bowl or M513 disrupt such an association. For the Ca2+ bowl mutations, however, there are good reasons to think this is not the case. First, these mutations remove acidic side chains that could provide the negative charge density normally associated with Ca2+ coordination. Second, two laboratories have found that Ca2+ will bind to recombinant Slo fusion proteins overexpressed and purified from bacteria, and will do so in gel overlay assays where auxiliary proteins would not likely remain associated (Bian et al., 2001
; Braun and Sy, 2001
). And third, as mentioned above, the Ca2+-bowl mutation 897901N inhibits this binding by approximately half (Bian et al., 2001
). Thus, it seems likely that the Ca2+ bowl does in fact form a Ca2+-binding site, although this is not directly demonstrated by our data.
The situation for the M513I mutation is less clear. Methionines are not commonly Ca2+ coordinators, so it may be that M513I interferes with the linkage between binding and opening (by linkage we mean that which causes the binding site to increase its affinity as the channel opens). This could be the movement of a key atom closer to the Ca2+ ion in its site, as is the case with cGMP when the cyclic nucleotidegated channel opens (Varnum et al., 1995). Or perhaps the M513I mutation inhibits an intersubunit association of intracellular domains that may occur after Ca2+ binds. Such an association has been shown to occur after Ca2+ binds to the SK channel (Schumacher et al., 2001
), and M513 lies at the very end of mSlo's seventh hydrophobic domain (S7), a region that might reasonably bury its hydrophobic side chains by dimerization, perhaps with its counterpart on a neighboring subunit, when prodded by Ca2+ binding.
Pursuing this idea further, M513 also lies at the very end of mSlo's RCK domain, which spans from just after S6 to the end of S7 (Jiang et al., 2001). In the recent crystal structure of the E. coli K+ channel's RCK domain (Jiang et al., 2001
), the
G helix, where M513 would be predicted to lie, participates in a dimer interface. Thus, although the homology in this region between the E. coli K+ channel and mSlo is low, and the E. coli channel does not contain a methionine at the position corresponding to mSlo's 513, by structural analogy we might still speculate that M513 is important for the formation of dimers between mSlo's intracellular domains.
Alternatively, it could also be that M513I disrupts a Ca2+-binding site formed by the RCK domain itself, since much of this domain has a structure commonly associated with ligand-binding proteins known as a Rossman fold (Branden and Tooze, 1991) and Rossman folds have been shown to form binding sites for a variety of small molecules, including ions (Lee et al., 1995
). So, although M513 is not in mSlo's predicted Rossman fold, its close proximity suggests its mutation may destabilize a Ca2+-binding site formed therein (Jiang et al., 2001
).
It is interesting to note that the mSlo3 channel, a Ca2+-insensitive mSlo homologue, contains neither a Ca2+ bowl nor a methionine at position 513 (Schreiber et al., 1998) and thus its lack of Ca2+ sensitivity is easily explained in terms of our results. Less easy to explain, however, is the observation that a channel containing the mSlo core and the mSlo3 tail, and thus one that has lost its Ca2+ bowl but retains M513, is in large-part Ca2+ insensitive (Schreiber et al., 1999
; Moss and Magleby, 2001
). This result could mean that there are elements of the mSlo tail besides those we have mutated that are required for the proper functioning of a binding site in the core of the channel, or conversely, that M513 interacts with a Ca2+-binding site in the mSlo tail, besides the Ca2+ bowl, that is not present in the mSlo3 tail. Clearly, confirmation of any of these hypotheses must await new physiological and structural data.
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FOOTNOTES |
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ACKNOWLEDGMENTS |
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This work was supported by grant R01HL64831 from the National Institutes of Health and by a grant from The Jessie B. Cox Charitable Trust and The Medical Foundation.
Submitted: 14 May 2002
Revised: 11 June 2002
Accepted: 11 June 2002
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REFERENCES |
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