Address correspondence to Dr. Karl L. Magleby, Department of Physiology and Biophysics, University of Miami School of Medicine, P.O. Box 016340, Miami, FL 33101-6430. E-mail: kmagleby{at}miami.edu
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ABSTRACT |
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Key Words: Ca2+-activated K+ channel RCK domain DHS-I estrogen Slo3
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INTRODUCTION |
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In addition to activation by Ca2+i and depolarization, BK channels can be modulated by at least four different types of accessory ß subunits (McManus et al., 1995; Dworetzky et al., 1996
; Tanaka et al., 1997
; Chang et al., 1997
; Wallner et al., 1999
; Jiang et al., 1999
; Meera et al., 2000
; Ramanathan et al., 2000
; Weiger et al., 2000
; Xia et al., 2000
; Pluger et al., 2000
; Brenner et al., 2000a
,b
; Petkov et al., 2001
). When coexpressed with
subunits, the ß1 subunit has two different effects. It greatly increases the apparent Ca2+ sensitivity (McManus et al., 1995
; Meera et al., 1996
; Nimigean and Magleby, 1999
; Cox and Aldrich, 2000
; Nimigean and Magleby, 2000
; Ramanathan et al., 2000
) and decreases the voltage sensitivity (Cox and Aldrich, 2000
; Nimigean and Magleby, 2000
). The decrease in voltage sensitivity is apparent as a shallower dependence of macroscopic conductance or Po on voltage. The increase in the apparent Ca2+ sensitivity is indicated in two ways: 510-fold less Ca2+i is required with the ß1 subunit for 50% activation of the channel at a fixed membrane potential, or conversely, 60100 mV less depolarization is required for 50% activation of the channel at a fixed Ca2+i. As might be expected from such pronounced effects on the Ca2+ sensitivity of BK channels, the ß1 subunits are required for proper function in the tissues where they are expressed. For example, knocking out the ß1 subunit in smooth muscle of mice leads to chronic hypertension (Pluger et al., 2000
; Brenner et al., 2000b
) and also decreased frequency of contraction in urinary muscle (Petkov et al., 2001
).
This study further examines the mechanism by which the ß1 subunit modulates the gating of the BK channel by exploring which structure features of the subunit are involved in the dual action of the ß1 subunit, of increasing the apparent Ca2+ sensitivity and decreasing the voltage sensitivity. Attention is directed toward the S9-S10 tail region that includes the Ca2+ bowl.
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MATERIALS AND METHODS |
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Using the wild-type construct of the subunit of the BK channel initially cloned by Pallanck and Ganetzky (1994)
, as modified (McManus et al., 1995
) and provided by Merck Research Laboratories, we made two different mutations to disrupt the Ca2+ bowl by either replacing one aspartic acid with asparagine (D965N) changing FDLQDDDDDPD to FDLQNDDDDPD, or by deleting two consecutive aspartic acid residues in the Ca2+ bowl: deletion of both D965 and D966 (FLDQDDDDDPD to FLDQ__DDDPD). The indicated residue numbers are those in the EMBL/GenBank/DDBJ under accession no. MMU09383 (Pallanck and Ganetzky, 1994
) before modification by Merck. The deletions were made by using the Stratagene QuickChange site-directed mutagenesis kit and checked by sequencing. The human ß1 subunit (NM_004137) and bovine ß1 subunit (L26101) were kindly provided by Merck Research Laboratories. Unless otherwise indicated, the experiments were performed with the human ß1 subunit.
The experiments examining the effect of the bovine ß1 subunit on channels formed from mouse subunits with deletion of D965 and D966 in the Ca2+ bowl were performed using human embryonic kidney (HEK) 293 cells. As described in Nimigean and Magleby (1999)
, these cells were transiently transfected with the above-mentioned constructs together with Plasmid pGreen Lantern-1 (GIBCO BRL) that encodes for a green fluorescent protein as a marker for gene expression.
Electrophysiology and Solutions
Both single-channel and macropatch currents were recorded with the patch clamp technique (Hamill et al., 1981) from patches of membrane excised from Xenopus oocytes using an Axopatch 200B amplifier. All experiments were done using inside out patches, except when outside out patches were used for application of 17 ß-estradiol to the extracellular surface. For single-channel recording, patches containing a single BK channel were identified by extended recordings at high levels of Ca2+ and/or depolarized potentials expected to readily activate the channels. In the absence of injection of cRNA, endogenous BK channels (Krause et al., 1996
) were observed so infrequently (less than once in 50 patches) that it is unlikely that any of the single-channel recordings were from endogenous BK channels. In any case, all the results reported were consistently observed, suggesting that they were from expressed channels. For macropatch recording, the expressed channels would far outnumber any endogenous channels. Injection of cRNA for the core or the tail alone does not give rise to measurable currents (Wei et al., 1994
; Meera et al., 1997
).
The pipette solution contained (mM) KCl 158, TES 5, and usually 10 µM GdCl3 to block the endogenous stretchactivated channels (Yang and Sachs, 1989). The bath solution contained (mM): KCl 158, TES 5, EGTA 1, HEDTA 1, and sufficient added Ca2+ to bring the free Ca2+ levels to those indicated (Nimigean and Magleby, 1999
). (For Fig. 7 only, the KCl was 150 mM.) All solutions were adjusted to pH 7.0. Solutions with no added Ca2+ had a calculated free Ca2+ of <10-8 M. Such solutions will be referred to as 0 Ca2+ solutions because Ca2+i at these concentrations has essentially no effect on the gating of the channel (Meera et al., 1996
; Nimigean and Magleby, 2000
). Indicated voltages refer to the intracellular potential. Experiments were performed at room temperature (1822°C).
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Data Analysis
Single-channel data were typically filtered at 510 kHz and sampled at 200 kHz directly to disk using pClamp7 or pClamp8. Analysis of the digitized records was then performed using custom programs, as described previously (McManus et al., 1987; McManus and Magleby, 1988
; Nimigean and Magleby, 1999
). Burst analysis was performed using a critical gap calculated from the closed dwell-time distributions to separate bursts, as detailed previously (Magleby and Pallotta, 1983
; Nimigean and Magleby, 1999
). Macropatch currents were analyzed with Clampfit in pClamp8. Conductance-voltage (G-V) curves were normally constructed from tail currents, but in a few experiments when Ca2+ was not present, peak currents were used.
Each G-V curve was fitted with a Boltzmann function (Eq. 1), normalized to the peak current,
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RESULTS |
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To examine the contributions of the core and tail to the modulation of the channel by the accessory ß1 subunit, we examined three different chimeric constructs. In the first, the tail of Slo3 was joined to the core of Slo1 with the linker between S8 and S9 intact (Slo1 core/Slo3 tail joined channel). In the second, the Slo1 core was coexpressed with the Slo1 tail (Slo1 core/Slo1 tail channel), and in the third the Slo1 core was coexpressed with the Slo3 tail (Slo1 core/Slo3 tail channel). For the channels expressed in two parts, the unconserved linker between S8 and S9 was removed (Wei et al., 1994; Schreiber et al., 1999
). Previous experiments have shown that Slo1 core/Slo1 tail channels are functionally expressed and have properties similar to full-length Slo1 channels (Meera et al., 1997
; Schreiber and Salkoff, 1997
; Schreiber et al., 1999
; Moss and Magleby, 2001
), and that the Slo1 core/Slo3 tail channels are functionally expressed and have greatly reduced Ca2+ sensitivity (Schreiber et al., 1999
; Moss and Magleby, 2001
).
The ß1 Subunit Increases the Apparent Ca2+ Sensitivity of the Slo1 Core/Slo1 Tail Channel While Reducing its Voltage Sensitivity
The ß1 subunit increases the apparent Ca2+ sensitivity of the BK channel by increasing the open probability Po at a fixed Ca2+i (McManus et al., 1995; Meera et al., 1996
; Cox and Aldrich, 2000
; Ramanathan et al., 2000
), mainly through an increase in the burst duration (Nimigean and Magleby, 1999
, 2000
). To explore whether this characteristic action of the ß1 subunit was retained when the
subunit of the BK channel was expressed as two separate parts, we coexpressed the Slo1 core and Slo1 tail with and without the ß1 subunit. Fig. 2, A and B, which presents representative single-channel records obtained at three different Ca2+i, show that the presence of the ß1 subunit greatly increased Po and the burst duration at all three Ca2+i. For example, with 2.5 µM Ca2+i, the Po increased 10-fold from 0.053 ± 0.012 in the absence of the ß1 subunit to 0.54 ± 0.08 with the ß1 subunit, and the burst duration increased 12-fold, from 4.3 ± 1.0 to 53.5 ± 14.2 ms (30 mV, n = 5).
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Consistent with the observations of Cox and Aldrich (2000) for full-length Slo1, the ß1 subunit also decreased the voltage sensitivity of the two part Slo1 core/Slo1 tail channel, from 20.2 ± 1.0 mV/e-fold change to 26.3 ± 2.1 mV/e-fold change (P < 0.05; data from 0, 1.1, 5.7, 20, and 100 µM Ca2+i). This decreased voltage sensitivity was evident as a decrease in the slope of the G/V curves. Thus, expressing Slo1 in two parts did not change the characteristic modulatory effects of the ß1 subunit, of increasing the apparent Ca2+ sensitivity (expressed as increases in burst duration, Po and the leftward shift in V0.5), and of decreasing the voltage sensitivity. Consequently, the
40 amino acids that comprise the linker between RCK1 and RCK2, that are missing in the Slo1 core/Slo1 tail channel, are not required for the ß1 subunit to induce its characteristic effects.
In Fig. 2, E and F, it can be seen that the shift in V0.5 induced by the ß1 subunit for the two part Slo1 core/Slo1 tail channel becomes progressively less as the Ca2+i was reduced, with little shift at 0 Ca2+i, as has been reported previously for full-length Slo1 (Meera et al., 1996; Cox and Aldrich, 2000
; Ramanathan et al., 2000
). This observation might suggest that the effect of the ß1 subunit on increasing Po requires Ca2+i, but this is not the case. Nimigean and Magleby (2001)
found that the ß1 subunit still increased burst duration and Po an order of magnitude in 0 Ca2+i at 30 mV, and the ß1 subunit increases currents from macro patch recordings in 0 Ca2+i for voltages <175 mV (Fig. 2 E; Cox and Aldrich, 2000
). The reduced ability of the ß1 subunit to shift V0.5 at low Ca2+i arises in large part from the secondary effect of the ß1 subunit to decrease the voltage sensitivity of the channel. Large depolarizations are required to half activate the BK channel with 0 Ca2+i. At such large depolarizations, the decreased voltage dependence of the channel due to the ß1 subunit becomes prominent, and consequently, the decreased activation due to the decreased voltage dependence cancels out the primary effects of the ß1 subunit to increase burst duration and Po. These interacting effects can be seen in Fig. 4 of Nimigean and Magleby (2000)
for 0 Ca2+i, where the ß1 subunit increases burst duration and Po about an order of magnitude at 30 mV, and these ß1 subunit induced increases become progressively less as the channel is depolarized.
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Replacing the Slo1 tail with the Slo3 tail converted the channel from a highly Ca2+-sensitive channel to one with little Ca2+ sensitivity, as reported previously (Schreiber et al., 1999; Moss and Magleby, 2001
). This is shown in the representative records in Fig. 3, where changing Ca2+ from 0 to 100 µM had little effect on either Po or the gating (Fig. 3 A). In contrast to the marked effects of the ß1 subunit on the gating of the Slo1 core/Slo1 tail channel (Fig. 2, A and B), the ß1 subunit had little effect on the gating of the Slo1 core/Slo3 tail channel (Fig. 3, A and B). The results are summarized in Fig. 4. The ß1 subunit greatly increased Po, mean burst duration, and mean open time, and decreased mean closed time for channels with the Slo1 tail, while having little effect on these parameters for channels with the Slo3 tails. In some experiments, channels with Slo3 tails could enter a mode of activity with longer bursts than those in Fig. 3, A and B, somewhat similar to the bursting activity seen with channels with Slo1 tails, as in Fig. 2, A and B. Data from this mode were excluded from the analysis.
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The results in Figs. 2 and 3 show that the ß1 subunit decreased the voltage sensitivity, independently of whether the tail of the channel was the Slo1 tail (Fig. 2 E) or the Slo3 tail (Fig. 3 E). With Slo1 tails, the increase in burst duration and Po induced by the ß1 subunit dominated the decrease in voltage sensitivity and V0.5 was shifted to the left. With Slo3 tails, Po was not increased by the ß1 subunit so the decreased voltage dependence was apparent, shifting V0.5 to the right. The results in Figs. 2 and 3 show, then, that the effect of the ß1 subunit on decreasing the voltage sensitivity was independent of whether the channels had Slo1 tails or Slo3 tails, but that the Slo1 rather than the Slo3 tail was required for the ß1 subunit to induce its characteristic effects of increasing Po through increases in mean burst duration.
As observed by Moss and Magleby (2001), we also observed that Slo1 core/Slo3 tail channels had less voltage sensitivity than Slo1 core/Slo1 tail channels (20.2 ± 1.0 mV/e-fold change in Po versus 22.6 ± 0.4 mV/e-fold change in Po), although this difference was not significant in this current study. The values for the voltage sensitivity observed in this current study using macro patches, which average data from many channels, were less than in the study of Moss and Magleby (2001)
using analysis of data from one channel at a time. An explanation for this is that the natural variation in the Po among BK channels (McManus and Magleby, 1991
) flattens the I/V curves for data averaged from multiple channels (Matthews, 1998
; Ruiz et al., 1999
).
The ß1 Subunit Is Functionally Associated with Both Two Part and Full-length Slo1 Core/Slo3 Tail Channels
It is clear from the Figs. 3 and 4 that the ß1 subunit does not increase burst duration and Po when the Slo1 tail is replaced by the Slo3 tail. This lack of effect may arise because: (a) ß1 subunits are not appropriately expressed in the presence of the Slo3 tails, (b) ß1 subunits are expressed but do not functionally associate with the Slo1 core/Slo3 tail channel, or (c) ß1 subunits are expressed and associate, but the proper machinery in the Slo3 tail is missing for the ß1 subunit to carry out its modulating effects.
The observation in Fig. 3 E that the ß1 subunit decreases the voltage sensitivity of Slo1 core/Slo3 tail channels, shifting V0.5 1020 mV positive (Fig. 3 F), suggests that the ß1 subunit is expressed and that it does associate in some manner with the Slo1 core/Slo3 tail channels. Nevertheless, the type of association for a decrease in voltage sensitivity may be different than for the increase in burst duration and Po. Consequently, as a further test of functional association, we took advantage of the BK channel opening agonists DHS-I and estradiol as probes. Previous studies have shown that DHS-I (McManus et al., 1995; Giangiacomo et al., 1998
; Brenner et al., 2000b
) and estradiol (Valverde et al., 1999
; Dick and Sanders, 2001
) activate BK channels only in the presence of the ß1 subunit. If DHS-I and estradiol also activate Slo1 core/Slo3 tail channels only in the presence of the ß1 subunit, then this would suggest that the ß1 subunit is also functionally associated with Slo1 core/Slo3 tail channels.
Representative results for testing this hypothesis are shown in Fig. 5. In the absence of the ß1 subunit, DHS-I had no effect on the gating of the Slo1 core/Slo3 tail channel (Fig. 5 A), while in the presence of the ß1 subunit, DHS-I dramatically increased Po by increasing the burst duration (Fig. 5 B). The effects of DHS-I were readily reversible. Similar results were observed in four additional experiments of this type, where DHSI had no effect on Slo1 core/Slo3 tail channels in the absence of the ß1 subunit, while increasing burst duration from 1.7 ± 0.1 ms to 6.2 ± 0.8 ms and Po from 0.024 ± 0.006 to 0.13 ± 0.004 in the presence of the ß1 subunit (30 mV). The DHS-Iinduced increase in burst duration and Po for Slo1 core/Slo3 tail channels is similar to what we observed for the Slo1 core/Slo1 tail channels (unpublished data) and to what has been reported previously for the Slo1 (full length) channel (McManus et al., 1995; Giangiacomo et al., 1998
). Our results with DHS-I suggest that the ß1 subunit is functionally associated with the Slo1 core/Slo3 tail channel. Otherwise, DHS-I should have no effect.
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To test further whether the ß1 subunit functionally associates with the Slo1 core/Slo3 tail channel, we examined whether estrogen (17 ß-estradiol) would activate the channel in the presence of the ß1 subunit. Estrogen had no effect on Slo1 core/Slo3 tail channels in the absence of the ß1 subunit (Fig. 5 C), but reversibly activated the Slo1 core/Slo3 tail channels in the presence of the ß1 subunit (Fig. 5 D). Similar results were observed in three additional experiments of this type. Estrogen had no effect on Slo1 core/Slo3 tail channels in the absence of the ß1 subunit, while greatly increasing channel activity and Po fivefold, from 0.0015 ± 0.0006 to 0.007 ± 0.0004, in the presence of the ß1 subunit (-30 mV). These effects of estrogen on Slo1 core/Slo3 tail channels are similar to those reported previously for Slo1 channels (Valverde et al., 1999). Consistent with the single-channel results, recordings from macro patches showed that both DHS-I and 17 ß-estradiol introduced (leftward) shifts in V0.5 of -30.4 ± 3.2 mV and -33.3 ± 3.7 mV, respectively, in the presence of the ß1 subunit, but not in its absence (unpublished data).
The observations that the two channel opening agents, DHS-I and estrogen, that require the presence of the ß1 subunit to activate BK channels, also activate Slo1 core/Slo3 tail channels in the presence of the ß1 subunit, but not in its absence, suggest that the ß1 subunit is functionally associated with Slo1 core/Slo3 tail channels. Hence, the inability of the ß1 subunit to increase burst duration of Slo1 core/Slo3 tail channels, even though the ß1 subunit is functionally associated with the channel, suggests that either the Slo1 tail (or some part of the Slo1 tail) is required for the ß1 subunit to have its characteristic effects, or that the Slo3 tail blocks the effects of the ß1 subunit acting at a site separate from the tail.
Slo1 core/Slo3 tail channels can exhibit wanderlust kinetics, as observed for dSlo (Silberberg et al., 1996), if insufficient Slo3 tail cRNA is injected (unpublished data). Consequently, we examined the effects of the ß1 subunit, DHS-I, and 17 ß-estradiol on joined Slo1 core/Slo3 tail channels. We found that the joined Slo1 core/Slo3 tail channel typically had more stable kinetics than two part channels. Using these more stable joined Slo1 core/Slo3 tail channels we have observed similar results with the ß1 subunit, DHS-I, and 17 ß-estradiol to those observed for the two part Slo1 core/Slo3 tail channels (unpublished data). Hence, the S8-S9 linker between the two RCK domains, which was present in the joined Slo1 core/Slo3 tail channel and not in the two part Slo1 core/Slo3 tail channel, is not required for the effects of the ß1 subunit, DHS-I, or estrogen on the Slo1 core/Slo3 tail channel, but may influence the stability of the gating and Po, perhaps by assuring that there is one Slo3 tail for each Slo1 core.
A Functional Ca2+ Bowl Is Not Required for the ß1 Subunit to Have its Characteristic Effects on the BK Channel
As shown in a previous section, the ß1 subunit no longer increased burst duration and Po after the Slo1 tail was replaced by the Slo3 tail. A major difference between the Slo1 tail and Slo3 tail is that the Slo3 tail does not contain a Ca2+ bowl, thought to be a high affinity Ca2+ binding site (Wei et al., 1994; Schreiber and Salkoff, 1997
; Schreiber et al., 1999
; Bian et al., 2001
; Braun and Sy, 2001
; Bao et al., 2002
). Hence, perhaps the inability of the ß1 subunit to increase burst duration and Po in the presence of the Slo3 tail is due solely to the disruption of the Ca2+ bowl. To test this possibility, the effects of the ß1 subunit on Slo1 channels with disrupted Ca2+ bowls were examined. Based on the work of Schreiber and Salkoff (1997)
, a point mutation (D965N, see MATERIALS AND METHODS) was made in the Ca2+ bowl of Slo1 (full length) to essentially remove the high affinity Ca2+ sensitivity of the channel. Both single-channel and macroscopic currents were then recorded from the channel in the presence and absence of the ß1 subunit to see if an intact Ca2+ bowl is required for the ß1 subunit to have its characteristic effects of increasing burst duration and Po.
Representative results are presented in Fig. 6 A, which shows single-channel records from Slo1, Slo1 with a mutated Ca2+ bowl (D965N), and Slo1 with the mutated Ca2+ bowl in the presence of the ß1 subunit. As expected (Schreiber and Salkoff, 1997), the Ca2+ bowl mutation dramatically decreased the Ca2+ sensitivity. With 20 µM Ca2+i, the Po for the wild-type channel was typically > 0.7 (top trace), compared with only 0.04 for the mutated channel (middle trace). In spite of the greatly reduced Ca2+ sensitivity of Slo1 with a mutated Ca2+ bowl, the ß1 subunit still had its characteristic effects of increasing burst duration and Po (Fig. 6 A, bottom trace). Similar results were observed in six additional experiments of this type. With 20 µM Ca2+i, the ß1 subunit increased the burst duration of D965N 21-fold, from 3.2 ± 0.8 to 68.5 ± 17.5 ms and Po 16-fold, from 0.016 ± 0.002 to 0.25 ± 0.03. Results are summarized in Fig. 4.
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It should be noted that the values of the ß1 subunitinduced leftward shifts were less for the mutated channel than for the wild-type channel at the same level of Ca2+i. However, if one adjusts the data for the differences in Po, then the shifts with and without the ß1 subunit were the same for the two types of channel. For example, D965N with 20 µM Ca2+i had a Po of 0.016, which was similar to the Po of the wild type channel with 12 µM Ca2+i. In both these cases the ß1 subunitinduced leftward shift in V0.5 was 35 mV. It will be recalled from an earlier section that the ß1 subunitinduced leftward shift becomes less when the Po is less, because the high positive voltages required to half-activate the channel for low initial Po emphasize the decreased voltage sensitivity induced by the ß1 subunit, which cancels out the facilitatory effects of the ß1 subunit. Similar effects of the ß1 subunit on channels with and without a functional Ca2+ bowl suggests that the Ca2+ bowl is not required for the ß1 subunit to decrease either the voltage sensitivity or increase the apparent Ca2+ sensitivity.
As a further test of whether the ß1 subunit still had it characteristic effects after disrupting the Ca2+ bowl, we also examined a deletion mutation to the Ca2+ bowl (deletion of D965 and D966), which had the greatest effect of reducing the Ca2+ sensitivity of all the mutations to the Ca2+ bowl examined by Schreiber and Salkoff (1997). Representative single-channel records from Slo1, the deletion mutation of Slo1, and the deletion mutation of Slo1 plus the ß1 subunit are shown in Fig. 7 for 8 µM Ca2+i (50 mV). As expected, the deletion mutation greatly decreased Po, and coexpression of the mutant with the ß1 subunit then increased Po by increasing the burst duration (Fig. 7 A). Results from four mutated channels without the ß1 subunit and three mutated channels in the presence of the ß1 subunit are shown in Fig. 7 B. The ß1 subunit increased burst duration
10-fold, independent of Ca2+i for changes in Ca2+i >3 orders of magnitude, including 0 Ca2+i. The retention of the characteristic effects of the ß1 subunit on channels with disrupted Ca2+ bowls for both the oocyte expression system with the human ß1 subunit (Fig. 6) and in the HEK cell expression system with the bovine ß1 subunit (Fig. 7), indicates that the ß1 subunit still induces its characteristic effects when the Ca2+ sensitivity is greatly reduced due to mutations in the Ca2+ bowl, and that these observations are independent of the expression system and the species of the ß1 subunit.
From Figs. 6 D and 7 B it can be seen that, although the Ca2+ bowl mutations greatly reduce the Ca2+ sensitivity of Slo1 channels, the channels still retain Ca2+ sensitivity. This is consistent with previous studies that suggest that mutations to the Ca2+ bowl do not remove all the Ca2+ binding sites (Schreiber and Salkoff, 1997; Schreiber et al., 1999
; Bian et al., 2001
; Braun and Sy, 2001
; Shi and Cui, 2001
; Zhang et al., 2001
; Bao et al., 2002
; Xia et al., 2002
).
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DISCUSSION |
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Consistent with previous observations, we found in the absence of the ß1 subunit, that replacing the Slo1 tail with the Slo3 tail (Slo1 core/Slo3 tail channel) removed most of the Ca2+ sensitivity for Ca2+ < 100 µM (Fig. 3 and Schreiber et al., 1999; Moss and Magleby, 2001
), and mutating the Ca2+ bowl greatly decreased the Ca2+ sensitivity, such that levels of Ca2+i that gave Pos > 0.7 before the mutations gave Pos < 0.05 after mutation (Figs. 6 A and 7 A, and Schreiber and Salkoff, 1997
; Schreiber et al., 1999
; Bian et al., 2001
; Braun and Sy, 2001
; Bao et al., 2002
; Xia et al., 2002
).
We found that the ß1 subunit still had its characteristic effects of increasing the apparent Ca2+ sensitivity (through increases in burst duration and Po) and decreasing the voltage sensitivity when the Slo1 channels were expressed in two parts, from separate S0-S8 cores and S9-S10 tails (Slo1 core/Slo1 tail channel), rather than from full-length subunits (Fig. 2). This indicates that the missing 40-amino acid S8-S9 linker is not required for the ß1 subunit to exert either of its two different effects.
When the Slo3 tail replaced the Slo1 tail, the ß1 subunit no longer increased burst duration and Po, but still decreased the voltage sensitivity. The lack of effect of the ß1 subunit on burst duration and Po for channels with Slo3 tails was not due to a lack of functional association between the ß1 subunit and the chimeric channel, as the specific channel openers DHS-I and estrogen that only activate BK channels in the presence of the ß1 subunit, also only activated the channels with Slo3 tails in the presence of the ß1 subunit. The observation that the ß1 subunit decreased the voltage sensitivity of the channels with Slo3 tails provides additional support that the ß1 subunit was functionally associated with the channels with Slo3 tails. Since the ß1 subunit functionally associates with channels with either Slo1 tails or Slo3 tails, then some difference of the Slo3 tail from the Slo1 tail must prevent the ß1 subunit from increasing burst duration and Po for channels with Slo3 tails. In contrast, the ß1 subunit decreased voltage sensitivity for channels with either Slo1 tails or Slo3 tails. These differential effects of the ß1 subunit on channels with different tails suggests that the ß1 subunitinduced increase in burst duration and Po (apparent Ca2+ sensitivity) is mediated through the tail domain of the channel, while the ß1 subunit induced decrease in voltage sensitivity may be mediated through the core domain.
One difference between Slo1 tails and Slo3 tails is the virtual absence of the Ca2+ bowl in Slo3 tails. However, we found that disrupting the Ca2+ bowl in the tails of Slo1 channels by mutation did not prevent the ß1 subunit from increasing the apparent Ca2+ sensitivity of Slo1 channels. This finding, together with the previous observations, suggests that one or more structural features of the Slo1 tail may be required for the ß1 subunit to exert its characteristic effects of increasing apparent Ca2+ sensitivity through increases in burst duration and Po, but the Ca2+ bowl is not one of them.
Could sites other than the Ca2+ bowl contribute to the apparent increased Ca2+ sensitivity observed with the ß1 subunit? In addition to the Ca2+ bowl, BK channels may have one or more additional high affinity Ca2+ sites (Braun and Sy, 2001; Bao et al., 2002
; Xia et al., 2002
), as well as a low affinity site activated by mM Ca2+i or Mg2+i (Shi and Cui, 2001
; Zhang et al., 2001
). The low affinity site has been shown to be functional for channels with either Slo1 tails or Slo3 tails, consistent with its location on the core of the channel (Shi and Cui, 2001
; Xia et al., 2002
). Thus, our observation that the ß1 subunit had little effect on burst duration and Po for channels with Slo3 tails when compared with the pronounced effects on channels with Slo1 tails, suggests that a functional low affinity site is not a major factor contributing to the actions of the ß1 subunit on these parameters. If it were, then the ß1 subunit should have increased burst duration and Po for channels with Slo3 tails. It also seems unlikely that the ß1 subunit is acting through major changes in the affinities of any additional high affinity sites, as previous studies have shown that the critical level of Ca2+i that first starts to increase channel activity is little changed by the ß1 subunit (Cox and Aldrich, 2000
; Nimigean and Magleby, 2000
).
Together, all of these observations suggest that neither the Ca2+ bowl nor the low affinity site nor other high affinity sites are required for the ß1 subunit to exert its major action on increasing apparent Ca2+ sensitivity through increases in burst duration and Po. These conclusions are consistent with the observations that the ß1 subunit still exerts its major effect on burst duration and Po in the absence of Ca2+i (Nimigean and Magleby, 2000). These conclusions do not exclude that the ß1 subunit may act on the allosteric machinery downstream from Ca2+ binding sites. Even though the major actions of the ß1 subunit appear to be through Ca2+-independent mechanisms, the ß1 subunit does have some Ca2+ dependent effects on the gaps between bursts (Nimigean and Magleby, 2000
) and the apparent Ca2+ affinity (Cox and Aldrich, 2000
), which will not be considered here.
If the major actions of the ß1 subunit on increasing Po and burst duration are not through action on Ca2+ binding sites, as discussed above, then the absence of the Ca2+ bowl or other high affinity sites on the Slo3 tail is not the explanation for why the ß1 subunit does not increase burst duration and Po when the Slo1 tail is replaced with the Slo3 tail. Possible explanations are that there are specific structural features (other than Ca2+ binding sites) on the Slo1 tail that are not present on the Slo3 tail, that are required for the action of the ß1 subunit, or that the Slo3 tail, either directly or allosterically, prevents the action of the ß1 subunit. It is unlikely that the Slo3 tail blocks the action of the ß1 subunit, as two channel opening agents (DHS-I and estrogen) that activate Slo1 channels only in the presence of the ß1 subunit, also activated channels with Slo3 tails only in the presence of the ß1 subunit (Fig. 5), indicating that ß1 subunit is not blocking the gating of the channel when the Slo3 tail is present. If anything, the channel is typically more active with the Slo3 tail (Schreiber et al., 1999; Moss and Magleby, 2001
). Perhaps the ß1 subunit and the Slo3 tail are increasing Po by acting through a common mechanism, such that once the mechanism is employed by the Slo3 tail, it becomes saturated so that the ß1 subunit can contribute no further effect. This possibility seems unlikely, however, since, for comparisons at the same Po, the Slo3 tail typically decreases mean burst duration, mean open time, and the mean duration of gaps between bursts (Moss and Magleby, 2001
), whereas the ß1 subunit has the opposite effect on these parameters (Nimigean and Magleby, 1999
).
Studies by Jiang et al. (2002a)(b
) on the structure of a bacterial Ca2+gated potassium channel, MethK, suggest that the Ca2+-dependent gating of MethK is controlled by eight RCK domains (regulators of the conductance of K+). Four of these arise from a COOH terminus domain attached to each of the four
subunits (RCK1) and four are assembled separately from solution as soluble proteins (RCK2). The eight RCK domains assemble to produce four fixed and four flexible interfaces to form a gating ring that hangs beneath the channel on the intracellular side. Jiang et al. (2002a)
propose that the binding of two Ca2+ at each of the four flexible interfaces changes the structure of the gating ring so that each RCK1 domain pulls on a flexible linker attached to the intracellular end of each inner helix (S6 equivalent of Slo1), opening the channel.
By analogy to MethK, RCK1 in BK channels would include S7 and S8, and RCK2 would include at least S9. Whether the Ca2+ bowl and S10 should be functionally considered as part of RCK2 or as an additional attachment, since they are contained in a serine proteinase-like domain (Moczydlowski et al., 1992; Moss et al., 1996a
,b
), is unclear (see Fig. 1). With four
subunits, each with a sequential RCK1 and RCK2 domains, BK channels would also have eight RCK domains like MethK. The Ca2+ coordinating sites in MethK, D184, E210, and E212, appear to be replaced with L, Q, and L, respectively, in RCK1 of BK channels (see alignment in Jiang et al., 2002a
), suggesting that the Ca2+ sites in BK channels are located in different places than in MethK. Nevertheless, the idea that a Ca2+-induced movement at flexible interfaces between RCK domains leads to gating of the BK channel can serve as starting point for discussion of mechanism.
Our observation that the unconserved linker between S8 and S9 in BK channels was not required for the ß1 subunit to have its characteristic effects would suggest that this linker has little function except to attach RCK2 to RCK1 (see Fig. 1). This conclusion is consistent with the observation (see above) that the linker is missing altogether in MethK channels, where the RCK2 equivalent domain is a separate protein. Based on the model of Jiang et al. (2002a), replacing the Slo1 tail with the Slo3 tail in our experiments would replace the four native RCK2 domains of Slo1 with four foreign RCK2 domains from Slo3, a BK like channel with low Ca2+ sensitivity. This substitution appears to decrease the energy barriers for the open-closed transition, as channels with Slo3 tails have increased activity and Po in 0 Ca2+i together with decreased mean open and closed times at the same Po (Schreiber et al., 1999
; Moss and Magleby, 2001
).
The simplest interpretation of the actions of the ß1 subunit to increase the apparent Ca2+ sensitivity in light of the model of Jiang et al. (2002a), is that the ß1 subunit alters some energy barriers for the movement of the gating ring to favor reentry into the open conformation and to decrease the rate constants for closing in order to increase both the numbers of openings per burst and the mean open times. The proper RCK2 domain (Slo1 tail rather than Slo3 tail) is required for the ß1 subunit to induce these changes in apparent Ca2+ sensitivity, while having little effect on the ß1 subunitinduced decrease in voltage sensitivity, which may arise from other areas of the channel.
Current allosteric models for the activation of BK channels suggest that voltage and Ca2+ sensors act relatively independently of one another to modulate the open-closing transitions (Horrigan et al., 1999; Horrigan and Aldrich, 1999
; Cox and Aldrich, 2000
; Cui and Aldrich, 2000
; Zhang et al., 2001
; Niu and Magleby, 2002
). Independent allosteric activators could provide a means for the ß1 subunit to exert its differential effects on channel activity, of increasing Po through increases in burst duration and decreasing Po through decreases in the voltage sensitivity. Contact of the ß1 subunit with at least two regions of the channel, each associated with a different allosteric activator could provide these differential effects.
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FOOTNOTES |
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Dr. Moss's present address is the Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
* Abbreviations used in this paper: DHS-I, dehydrosoyasaponin I; RCK, regulator of the conductance of potassium.
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ACKNOWLEDGMENTS |
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Submitted: 5 August 2002
Revised: 2 October 2002
Accepted: 4 October 2002
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REFERENCES |
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