Correspondence to: Crina M. Nimigean, Department of Physiology and Biophysics, R430, P.O. Box 016430, Miami, FL 33101-6430. Fax:305-243-6898 E-mail:cnimigea{at}chroma.med.miami.edu.
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Abstract |
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Coexpression of the ß1 subunit with the subunit (mSlo) of BK channels increases the apparent Ca2+ sensitivity of the channel. This study investigates whether the mechanism underlying the increased Ca2+ sensitivity requires Ca2+, by comparing the gating in 0 Ca2+i of BK channels composed of
subunits to those composed of
+ß1 subunits. The ß1 subunit increased burst duration ~20-fold and the duration of gaps between bursts ~3-fold, giving an ~10-fold increase in open probability (Po) in 0 Ca2+i. The effect of the ß1 subunit on increasing burst duration was little changed over a wide range of Po achieved by varying either Ca2+i or depolarization. The effect of the ß1 subunit on increasing the durations of the gaps between bursts in 0 Ca2+i was preserved over a range of voltage, but was switched off as Ca2+i was increased into the activation range. The Ca2+-independent, ß1 subunit-induced increase in burst duration accounted for 80% of the leftward shift in the Po vs. Ca2+i curve that reflects the increased Ca2+ sensitivity induced by the ß1 subunit. The Ca2+-dependent effect of the ß1 subunit on the gaps between bursts accounted for the remaining 20% of the leftward shift. Our observation that the major effects of the ß1 subunit are independent of Ca2+i suggests that the ß1 subunit mainly alters the energy barriers of Ca2+-independent transitions. The changes in gating induced by the ß1 subunit differ from those induced by depolarization, as increasing Po by depolarization or by the ß1 subunit gave different gating kinetics. The complex gating kinetics for both
and
+ß1 channels in 0 Ca2+i arise from transitions among two to three open and three to five closed states and are inconsistent with Monod-Wyman-Changeux type models, which predict gating among only one open and one closed state in 0 Ca2+i.
Key Words: maxi-K channel, KCa channel, single-channel, mSlo, Monod-Wyman-Changeux
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INTRODUCTION |
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Large conductance Ca2+-activated K+ channels (BK channels or maxi-K channels)1 are found in a wide variety of tissues, where they regulate excitability through a negative feedback mechanism ( subunits alone (
channels) or of
subunits together with various ß subunits (
+ß channels). The larger pore-forming
subunits, which are encoded by the gene at the slo locus, were first cloned from Drosophila (slowpoke phenotype), and bear homology to the superfamily of voltage-gated K+ channels, including a pore-forming region between the S5 and S6 transmembrane segments, and an S4 voltage-sensing domain (
Several distinct auxiliary ß subunits for the BK channel have been cloned: ß1, ß2, and ß3 ( subunits in several ways. The ß1 subunit increases the apparent Ca2+ sensitivity by decreasing the Ca2+i required for half activation of the channel (
In a recent study, we showed that the ß1 subunit increases the apparent Ca2+ sensitivity of BK channels by stabilizing the channel in the bursting states (
We now investigate this possibility by examining the effects of the ß1 subunit on the gating of unliganded BK channels, by studying the gating of and
+ß1 channels in the virtual absence of Ca2+i. Such experiments are possible since BK channels can gate in effective 0 Ca2+i (
Previous studies ( subunits, thus allowing lower levels of Ca2+i to activate the BK channel by shifting the Po vs. Ca2+i curve to the left. We examined the mechanism of this switch and found that the observation that the ß1 subunit increases mean burst duration ~20-fold, independent of Ca2+i, is sufficient to account for 80% of the increase in Ca2+ sensitivity indicated by the leftward shift in the Po vs. Ca2+i curve. The remaining 20% of the leftward shift arises because the ß1 subunit no longer increases (and may decrease slightly) the durations of gaps between bursts in the presence of Ca2+i. Thus, the functional switch has both Ca2+-independent and -dependent components, with the Ca2+-independent component accounting for the majority of the increase in Ca2+ sensitivity. While the effect of the ß1 subunit on increasing burst duration is always present, independent of Ca2+i, it is only in the presence of Ca2+i, when the Po becomes significant, that this ß1 subunit-induced increase in burst duration has a physiological effect. For example, increasing Po 20-fold, from 0.0002 to 0.004 in very low Ca2+i, would have little effect on current, whereas increasing Po 20-fold, from 0.02 to 0.40 in higher Ca2+i, could have a dramatic physiological effect.
The complex bursting kinetics in 0 Ca2+i for both and
+ß1 channels was found to arise from transitions among a minimum of two to three open states and three to five closed states. Gating among such a large number of unliganded states is inconsistent with gating mechanisms based on Monod-Wyman-Changeux type models for ligand-activated tetrameric allosteric proteins (
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METHODS |
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Heterologous Expression of BK Channels in Human Embryonic Kidney 293 Cells
Human embryonic kidney (HEK) 293 cells were transiently transfected with expression vectors (pcDNA3) encoding the subunit (mSlo from mouse; Genbank accession number MMU09383) and ß1 subunit (bovine ß; Genbank accession number
L26101) of the BK channels kindly provided by Merck Research Laboratories, and also with an expression vector encoding the green fluorescent protein (GFP, Plasmid pGreen Lantern-1; GIBCO BRL). Cells were transfected transiently using the Lipofectamine Reagent (Life Technologies) according to the protocol provided by GIBCO BRL. The GFP was used to monitor successfully transfected cells. HEK cells are optimal for transfection and expression after they have been in culture for ~34 wk. The cells are cultured using standard tissue culture media: DMEM with 5% fetal bovine serum (Life Technologies) and 1% penicillin-streptomycin solution (Sigma-Aldrich) and passaged at ~100% confluency using PBS with 5 mM EDTA to loosen cells from the bottom of the dish. For transfection, cells at 3040% confluency in 30-mm Falcon dishes used later for recording were first washed with antibiotic and serum-free DMEM, and then incubated with a mixture of the plasmids (total of 1 µg DNA per dish), Lipofectamine Reagent (optimal results at 7 µl) and Opti-MEM I reduced serum medium (Life Technologies). The mixture was left on cells for 11.5 h, after which it was replaced with standard tissue culture media. The culture media was again replaced after 24 h to remove debris and dead cells. The cells were patch-clamped 23 d after transfection when the culture medium was replaced with standard extracellular saline solution that contained (mM) 2.04 CaCl2, 2.68 KCl, 1.48 MgCl2, 0.05 MgSO4, 125 NaCl, 0.83 NaH2PO4, 20 NaHCO3, and 2 HEPES, pH 7.4.
In the coexpression experiments, a fourfold molar excess of plasmid encoding the ß1 subunit was used to drive coassembly with the subunits (
and ß1 subunits and the GFP increased the probability that if the GFP was expressed, the included subunits would also be expressed.
Solutions
The intracellular solution contained 175 mM KCl, 5 mM TES pH buffer, and 10 mM EGTA and 10 mM HEDTA to buffer the Ca2+ (see below). The extracellular solution contained either 150 or 175 mM KCl and 5 mM TES and had no added Ca2+ or Ca2+ buffers. Both the intracellular and extracellular solutions were adjusted to pH 7.0. The amount of Ca2+ added to the intracellular solution to obtain approximate free Ca2+ concentrations of 0.001100 µM was calculated using stability constants for EGTA (
Single-Channel Recording and Analysis
Currents flowing through single (or in some cases multiple) BK channels in patches of surface membrane excised from HEK 293 cells transfected with clones for either the or the
and ß1 subunits were recorded using the patch-clamp technique (
Single-channel current records were low-pass filtered with a four-pole Bessel filter to give a final effective filtering (-3 dB) of typically 10 kHz (range 4.510 kHz), and were sampled by computer at a rate of 125250 kHz. The methods used to select the level of filtering to exclude false events that could arise from noise, measure interval durations with half-amplitude threshold analysis, and use stability plots to test for stability and identify modes have been described previously, including the precautions taken to prevent artifacts in the analysis (
Data from multichannel patches were only analyzed for very low Ca2+i, where the activity was so low that simultaneous openings of two or more channels were seldom if ever observed. For the multichannel patches, the open probability was calculated by dividing the total open time by the total record length, and then by the number of channels in the patch. The mean durations of the gaps between bursts for the multichannel patches were estimated by determining these parameters as if the data were from a single channel, and then multiplying the estimates by the numbers of channels in the patch. The mean closed times for the multichannel patches were determined in the following way: the sum of all durations of the gaps between the bursts during the total recording time, multiplied by the number of channels in the patch, was added to the sum of all the durations of the closed intervals within bursts, and then the value was divided by the total number of closed intervals in the record. There was no need to correct estimates of the mean open time and mean number of openings per burst, since at such low Po, only one channel was open at any given time.
The methods used to log-bin the intervals into dwell-time distributions, fit the distributions with sums of exponential components using maximum likelihood fitting techniques (intervals less than two dead times were excluded from the fitting), and determine the number of significant exponential components with the likelihood ratio test have been described previously (
The method of defining a critical gap (closed interval) to identify bursts is detailed in
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RESULTS |
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The ß1 Subunit Increases both Po and Burst Duration in the Virtual Absence of Ca2+i
To investigate whether the ß1 subunit requires Ca2+i for its action, we used single-channel analysis to examine the gating of and
+ß1 channels in the effective absence of Ca2+i (<1 nM), which will be referred to as 0 Ca2+i. (It will be shown in a later section that effective 0 Ca2+i was achieved.) Fig 1 A shows single-channel currents recorded in 0 Ca2+i at +30 mV from an
channel and also from an
+ß1 channel. The occasional openings and bursts of openings are separated by the long closed intervals of many seconds that form the gaps between bursts. The long gaps between bursts in 0 Ca2+i give very low open probability. The average Pos for the entire records from which each excerpt was obtained were 0.00056 for the
channel and 0.0039 for the
+ß1 channel, for a sevenfold increase in Po. The mean Po for 15
channels and 21
+ß1 channels at 0 Ca2+i is plotted in Fig 2 A (left-most points), where the presence of the ß1 subunit increased Po ~10-fold on average, from ~0.0002 to 0.002.
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In conjunction with the large increase in Po, the ß1 subunit stabilized the bursting states, increasing mean burst duration in 0 Ca2+i. For the channels in Fig 1, mean burst duration increased from 1.1 to 28.8 ms, for a 26-fold increase. The dramatic increase in mean burst duration can be seen in Fig 1 B, where selected bursts are presented on a faster time base. The ß1 subunit consistently increased burst duration in 0 Ca2+i. Mean burst duration for 15 channels and 21
+ß1 channels at 0 Ca2+i is plotted in Fig 2 C (left-most points). The presence of the ß1 subunit increased mean burst duration 21-fold in 0 Ca2+i, from 0.59 to 12.4 ms. Thus, the ß1 subunit exerts its characteristic effects of increasing Po by retaining the gating in the bursting states in 0 Ca2+i, just as it does in the presence of Ca2+i. For comparison, the effects of the ß1 subunit on gating in the presence of Ca2+i (1.8 µM) are shown in Fig 1 C, where the ß1 subunit also increased burst duration and Po, as described previously (
The ß1 Subunit Alters the Gating Parameters from 0 to Higher Ca2+i
To examine further the effects of the ß1 subunit on the gating at 0 Ca2+i and to compare these effects with those at higher Ca2+i, we measured an array of kinetic parameters (Po, mean burst duration, mean duration of gaps between bursts, mean open time, mean closed time, and mean number of openings per burst) for and
+ß1 channels and plotted them against Ca2+i in Fig 2. Over the entire range of Ca2+i, from 0 to higher levels, the ß1 subunit increased mean burst duration ~20-fold (Fig 2 C). This increase in the mean burst duration arose from both an increase in mean open time (Fig 2 E) and in the mean number of openings per burst (Fig 2 G). The ß1 subunit also increased the mean durations of the gaps between bursts approximately threefold in 0 Ca2+i, while having little effect on the durations of the gaps at higher Ca2+i (Fig 2 D). The mean closed time was little affected by the ß1 subunit in 0 Ca2+i (Fig 2 F) because the ß1 subunit-induced increase in the duration of the gaps between bursts (Fig 2 D) was compensated for by a decrease in the fraction of closed intervals that were gaps between bursts (Fig 2 H).
Evidence for the Effective Absence of Ca2+
In Fig 2 it can be seen that all the measured kinetic parameters (Po, mean burst duration, mean duration of gaps between bursts, mean open time, mean closed time, and mean number of openings per burst) remained relatively unchanged as Ca2+i was increased more than two orders of magnitude (from 0.00018 to 0.018 µM). If Ca2+i were to bind to the channel and affect activity over this wide range of Ca2+i, then the kinetic parameters that define gating should change. Since little change was observed, these observations suggest that the channel remained functionally unliganded for Ca2+i < 0.1 µM. Hence, even though trace Ca2+i was likely present, functional 0 Ca2+i was achieved.
The ß1 Subunit Shifts the Po vs. Ca2+i Curve to the Left
The characteristic leftward shift in the Po vs. Ca2+i curve induced by the ß1 subunit (
ß1 Subunit Exerts Its Effects Over a Range of Voltages
The data in Fig 2 were obtained at a single voltage of +30 mV. To examine whether the effects of the ß1 subunit in 0 Ca2+i are dependent on voltage, we collected data in 0 Ca2+i over a range of voltages. Examples of single-channel currents obtained at +60 and +90 mV for and
+ß1 channels are shown in Fig 3. The ß1 subunit increased the durations of the bursts as well as the gaps between bursts at both voltages. The increase in burst duration with the ß1 subunit in 0 Ca2+i is clearly shown in Fig 3 C for both voltages, where representative bursts are presented on a faster time base.
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The mean effects of the ß1 subunit on the gating kinetics over a range of voltages in 0 Ca2+i are shown in Fig 4 for eight patches with channels and nine patches with
+ß1 channels. For both
and
+ß1 channels, depolarization increased Po through increases in mean burst duration, mean open time, and the mean number of openings per burst, and decreases in the mean closed time and in the mean duration of the gaps between bursts (Fig 4). The same characteristic effects of the ß1 subunit that were observed in 0 Ca2+i in Fig 2 were then superimposed on these effects of depolarization. Over the examined range of voltage (+30 to +100 mV), the ß1 subunit increased mean burst duration ~20-fold (Fig 4 B) and the mean duration of the gaps between bursts two- to threefold (Fig 4 C). The increase in mean burst duration was due to increases in both mean open time (approximately eightfold, Fig 4 D), and the mean number of openings per burst (approximately threefold, Fig 4 F). The ß1 subunit had little effect on mean closed time even though the mean closed time drastically decreased with depolarization (Fig 4 E).
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Evident in Fig 4 is that the estimates of the parameters that describe single-channel kinetics for and
+ß1 channels (mean open time, mean closed time, mean burst duration, mean duration of gaps between bursts, and mean number of openings per burst) are generally parallel with each other on a logarithmic scale, indicating that the ratios of the kinetic parameters for the
+ß1 to the
channels remain relatively constant over the examined range of +30 to +100 mV. As a first approximation, a constant ratio suggests that the ß1 subunit may act like a gain control, independent of voltage, such that the kinetic parameters measured in the presence of the ß1 subunit are simply the result of multiplication between the kinetic parameters in the absence of the ß1 subunit and a constant factor, which depends on the parameter measured.
Interestingly, the magnitude of the fractional increase in Po with the ß1 subunit decreased with depolarization (Fig 4 A). Some decrease with depolarization might be expected since depolarization increases Po and Po saturates near 0.96 for both and
+ß1 channels. However, the decreased effect of the ß1 subunit on Po with depolarization was apparent at low Pos as well. Projection of imaginary lines through the data in Fig 4 A suggests that at very depolarized voltages, the ß1 subunit may no longer have an effect of increasing Po and may even decrease it. A similar trend, however slight, is also apparent in Fig 4B and Fig D, for mean burst duration and mean open time, suggesting that the ß1 subunit may have reduced effects on these parameters at greatly depolarized voltages.
The ß1 Subunit Does Not Act Like an Increase in Membrane Potential
Previous results ( and
+ß1 channels had markedly different gating kinetics at the same Po, achieved by changing Ca2+i. We now apply the same type of analysis to investigate whether the ß1 subunit acts like an increase in voltage.
If voltage and the ß1 subunit worked through the same mechanism, and
+ß1 channels should display identical gating kinetics at the same Po, achieved by changing voltage. This was not the case. Increasing Po with the ß1 subunit in 0 Ca2+i was associated with greatly increased burst duration and a smaller increase in the duration of the gaps between bursts. In contrast, increasing Po with depolarization was associated with small increases in burst duration and large decreases in the duration of gaps between bursts.
Although these differential effects on kinetics are apparent from the examination of Fig 1 Fig 2 Fig 3 Fig 4, they are more easily seen in Fig 5, where the Po of an channel was increased with depolarization to match the Po of an
+ß1 channel. The dramatic differences in single-channel kinetics at similar Pos for
and
+ß1 channels are readily apparent in the current traces inset in Fig 5. These effects of voltage and the ß1 subunit on kinetics are quantified in Fig 5 by the open dwell-time distributions (left) and the closed dwell-time distributions (right) for both the
and the
+ß1 channels. At similar Pos, both the mean open times and the mean durations of the gaps between bursts were about an order of magnitude less for the
channel than for the
+ß1 channel (vertical lines), while the relative number of closed intervals that were gaps between bursts was greater for the
channel than for the
+ß1 channel. These marked differences in the kinetics of
and
+ß1 channels at the same Po (achieved by changing voltage) suggest that depolarization and the ß1 subunit act through different mechanisms.
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The ß1 Subunit Acts as a Gain Control on Bursting Kinetics, Independent of whether the Channel Is Activated by Voltage or Ca2+i
To explore whether the ß1 subunit has the same effect on the bursting kinetics, independent of whether Po is changed by voltage or Ca2+i, mean burst duration and the mean duration of gaps between bursts were plotted against Po for both and
+ß1 channels in Fig 6. The circles plot data obtained over a range of Ca2+i (0 to 18 µM) at +30 mV, and the squares plot data obtained over a range of voltages (+30 to +100 mV) in 0 Ca2+i. Filled symbols plot data from
+ß1 channels and open symbols plot data from
channels. At any given Po, both the mean burst duration and the mean duration of gaps between bursts were 10-fold longer in the
+ß1 channels as compared with the
channels, independent of whether the Po was achieved by changing Ca2+i or voltage. This 10-fold effect of the ß1 subunit on the bursting parameters was independent of Po, as indicated by the parallel shifts over four orders of magnitude of change in Po.
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The results in Fig 6 suggest that the ß1 subunit acts mainly as a gain control on the bursting parameters, independent of Po or whether the channel is activated by Ca2+i or by voltage. This is the case since a parallel shift on a logarithmic coordinate, as in Fig 2, Fig 4, and Fig 6, is consistent with a multiplicative (gain) effect. Hence, as a first approximation, the gain effect of the ß1 subunit appears to be independent of Ca2+i (Fig 2), voltage (Fig 4), and Po, over the examined range of conditions.
The constant shift in the mean durations of gaps between bursts in Fig 6 B in the presence of the ß1 subunit may appear paradoxical, since it was observed in Fig 2 D that the ß1 subunit increased the durations of the gaps between bursts approximately threefold in 0 Ca2+i, but had little effect on the durations of the gaps once Ca2+i was increased. The difference between Fig 6 and Fig 2 is that the data in Fig 6 are plotted against Po rather than Ca2+i. At a fixed Ca2+i, the ß1 subunit greatly increased burst duration, leading to an increase in Po for +ß1 channels. The same Po could then be achieved in
channels by increasing their activity through depolarization or increased Ca2+i. This increased activity is associated with large decreases in the durations of the gaps between bursts and smaller increases in burst duration (Fig 2 and Fig 4). Hence, at the same Po,
channels must have much smaller gaps between bursts than
+ß1 channels, as observed (Fig 6 B), to compensate for the much longer duration bursts of
+ß1 channels (Fig 6 A).
Ca2+i Switches Off the ß1 Subunit-induced Increase in the Duration of Gaps between Bursts
As indicated previously, for 0 Ca2+i, the ß1 subunit increased the durations of gaps between bursts approximately threefold (Fig 2 D). For Ca2+i > ~0.2 µM, the ß1 subunit no longer lengthened the durations of the gaps between bursts (perhaps even decreased them slightly), consistent with
80% of the ß1 Subunit-induced Increase in Ca2+ Sensitivity Is Independent of Ca2+i
To determine to what extent the ß1 subunit-induced shift in Ca2+ sensitivity is Ca2+ independent, we examined how much of the ß1 subunit-induced shift in Ca2+ sensitivity could be accounted for by assuming that the sole effect of the ß1 subunit was to multiply burst duration a constant amount, independent of Ca2+i. We first developed an empirical model to generate the Po vs. Ca2+i data for channels, and then calculated the predicted Po vs. Ca2+i curve for
+ß1 channels by assuming that the only effect of the ß1 subunit was to multiply burst duration a constant amount, independent of Ca2+i.
Open probability is defined by Equation 1:
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(1) |
Since the durations of the closed intervals within bursts are brief compared with both the durations of the open intervals and the durations of the gaps between bursts, Po can be approximated by:
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(2) |
where burst represents the mean burst duration and gap represents the mean duration of gaps between bursts.
The continuous lines in Fig 2C and Fig D, are empirical descriptions of mean burst duration and the mean duration of gaps between bursts, respectively, as functions of Ca2+i for channels (see figure legends). These empirical descriptions for burst and gap were then used with Equation 2 to calculate the Po vs. Ca2+i curve for the
channels, plotted as continuous lines in Fig 2A and Fig B. It can be seen that this method of predicting Po gave a reasonable description of the Po vs. Ca2+i data for the
channels for both semilogarithmic and double logarithmic plots.
To determine to what extent the increased Ca2+ sensitivity induced by the ß1 subunit could be predicted by assuming that the sole effect of the ß1 subunit was to increase burst duration a constant (multiplicative) amount, independent of Ca2+i, the Po vs. Ca2+i curve for +ß1 channels was calculated exactly as it was for the
channels, except that mean burst duration (burst) in Equation 2 was multiplied by a constant factor 22. The results of the calculation (Fig 2, AC, dashed lines) show a leftward shift in the Po vs. Ca2+i curve that accounts for 80% of the increase in Ca2+ sensitivity induced by the ß1 subunit. This simple multiplicative effect also described the effect of the ß1 subunit on burst duration (Fig 2 C, dashed line). Thus, the assumption that the sole effect of the ß1 subunit was to increase mean burst duration a constant multiplicative amount, independent of Ca2+i, could describe the ß1 subunit-induced increase in burst duration and 80% of the leftward shift in the Po vs. Ca2+i curve induced by the ß1 subunit. It follows, then, that 80% of the ß1 subunit-induced increase in Ca2+ sensitivity (the apparent Ca2+ switch) can be accounted for by a Ca2+-independent mechanism.
The remaining 20% of the ß1 subunit-induced increase in Ca2+ sensitivity did appear to be Ca2+ dependent. When the Ca2+-dependent effect of the ß1 subunit on the gaps between bursts was taken into account by describing gap in Equation 2 with the dashed line in Fig 2 D (rather than by the continuous line), 100% of the ß1 subunit-induced leftward shift in the Ca2+ sensitivity could be accounted for, as shown by the dotted line in Fig 2A and Fig B. Furthermore, when the Ca2+-dependent component was included, the Po in 0 Ca2+i was also correctly predicted (Fig 2 A, dotted line). (Results essentially indistinguishable from those presented in this section were obtained when the calculations included the effects of the durations of the intervals within bursts.)
We cannot exclude that there may be Ca2+-dependent effects of the ß1 subunit on mean open time and on the mean number of openings per burst (Fig 2E and Fig G). Unfortunately, estimates of these two parameters, unlike burst duration, are highly dependent on the flickers (brief closed intervals within bursts). Since flickers may arise from closed states beyond the activation pathway (
Unliganded BK Channels Gate in a Minimum of Two to Three Open and Three to Five Closed States
To obtain further insight into the gating mechanism of unliganded and
+ß1 channels, an estimate of the number of kinetic states entered during gating in 0 Ca2+i for each channel were obtained by fitting dwell-time distributions of open- and closed-interval durations with sums of exponential components. The number of significant exponential components gives a measure of the minimal number of kinetic states entered during gating (
and
+ß1 channels and the closed distributions were described by five significant closed components for
channels and four significant closed components for
+ß1 channels. Estimates from eight patches containing
channels and 12 patches containing
+ß1 channels are presented in Fig 7 for data obtained over a range of voltages in 0 Ca2+i.
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Both and
+ß1 channels typically gated in a minimum of two to three open states and three to five closed states in 0 Ca2+i. (Similar results were found when analysis was restricted to the 27 data sets from the seven patches containing a single channel.) These estimates can be compared with a minimum of typically three to four open and five to six closed states for
and
+ß1 at higher levels of activity in the presence Ca2+i (
In 0 Ca2+i, the estimates of the mean number of detected open states for channels (2.2 ± 0.4, mean ± SD) was not significantly different (P > 0.25, Mann-Whitney test) from the mean number of detected open states for the
+ß1 channels (2.4 ± 0.6). The estimate of the mean number of detected closed states for
channels (3.4 ± 1.0) was significantly less (P < 0.05) than the number of detected closed states entered for
+ß1 channels (4.1 ± 0.8). An increased number of detected closed states for
+ß1 channels in 0 Ca2+i could reflect a difference in the actual numbers of states typically entered during gating in 0 Ca2+i, or an increased ability to detect closed states for
+ß1 channels because the intervals are spread over a greater range of times than for
channels.
Our findings of gating among multiple open and closed states in 0 Ca2+i are consistent with a study using single-channel recording just published by subunit. The fewer closed states detected in their study may reflect that fewer closed states were typically entered at the more depolarized voltages used in their study or that the detection of closed states was more difficult because of the compressed dwell time distributions at the higher Pos in their study.
Rejection of the Monod-Wyman-Changeux Model for Gating in 0 Ca
Models considered for the gating of BK channels have often been based on the Monod-Wyman-Changeux (MWC) model for allosteric proteins (
The opening-closing transitions in the MWC model are concerted, with all four subunits undergoing simultaneous conformational changes. From Scheme 1, it can be seen that the gating will be confined to the two unliganded states in the absence of Ca2+i, giving only one open and one closed state. The observations in Fig 7, that both and
+ß1 channels typically gate in a minimum of two to three open and three to five closed states in 0 Ca2+i are clearly at odds with Scheme 1, and require that the MWC model be rejected as a mechanism for the gating of these channels.
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DISCUSSION |
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The accessory ß1 subunit of BK channels greatly increases their Ca2+ sensitivity by reducing the Ca2+i required for half activation 510-fold, giving a characteristic leftward shift in the Po vs. Ca2+i plots ( and ß1 subunits is not yet established. One possibility is that the functional coupling between
and ß1 subunits requires Ca2+i (
If the dominant action of the ß1 subunit does not require Ca2+-dependent processes, then the paradoxical possibility arises that the mechanism underlying the ß1 subunit-induced increase in Ca2+ sensitivity also does not require Ca2+i. To explore this possibility, we examined to what extent an assumption of Ca2+-independent action could account for the leftward shift that gave rise to the increased Ca2+ sensitivity. We found that 80% of the leftward shift could be accounted for by assuming that the only effect of the ß1 subunit was to increase burst duration ~20-fold, independent of Ca2+i (Fig 2A and Fig C, dashed lines). Thus, a Ca2+-independent mechanism was sufficient to account for 80% of the increased Ca2+ sensitivity induced by the ß1 subunit.
The remaining 20% of the shift in Ca2+ sensitivity reflects a Ca2+-dependent mechanism. The ß1 subunit increased the durations of the gaps between bursts approximately threefold in the absence of Ca2+i, and this increase disappeared (and the gap durations became slightly briefer based on the fitted lines) as Ca2+i was raised sufficiently to just increase channel activity (Fig 2 D). When this Ca2+-dependent effect of the ß1 subunit on gaps between bursts was taken into account, the remaining 20% of the leftward shift in Ca2+ sensitivity could be accounted for (Fig 2A and Fig B, dotted lines).
Whatever the mechanism for the Ca2+ dependence of the ß1 subunit on the durations of gaps between bursts at the transition between 0 and low Ca2+i, it seems unlikely to reflect a ß1 subunit-induced increase in Ca2+i binding rates, as the ß1 subunit then had little effect on the durations of the gaps between bursts for further increases in Ca2+i that decreased the durations of gaps between bursts two orders of magnitude (Fig 2 D). This relative lack of effect of the ß1 subunit on the gaps between bursts in the presence of Ca2+i has been described previously (
Our finding that the ß1 subunit was always functionally coupled to the subunit, independent of Ca2+i (Fig 1 Fig 2 Fig 3 Fig 4), differs from that of
+ ß1 complex into a functionally coupled state. This difference in conclusions could arise from a number of factors. First, our experiments used single-channel recording, which allowed high resolution analysis at very low Pos, while their experiments used macro currents, where activity at low Po would be more difficult to study. Using single-channel recording, we observed a 10-fold increase in Po in 0 Ca2+i in the presence of the ß1 subunit (from 0.0002 to 0.002 at + 30 mV), while they reported no change in Po under similar 0 Ca2+i. Second, we directly measured the effects of the ß1 subunit on the gating in 0 Ca2+i, while they estimated the effects from the projected voltages required for half activation (V0.5) in 0 Ca2+i. Third, our experiments used bovine ß1 and mouse
subunits, while theirs used human ß1 and
subunits. The difference in primary structure of the ß1 subunits (84% homology) and
subunits (96% homology) in the two studies might lead to different mechanisms of modulation by the ß1 subunit.
The contributions of these three factors to the differences in conclusions are not clear, but the most likely explanation is that the ß1 subunit has pronounced effects on Po in 0 Ca2+i at moderate depolarized potentials, as we observed, while having little effect on Po at large depolarizations, as used by
We did find that the ß1 subunit had a Ca2+-dependent component, but this component accounted for only 20% of the increased Ca2+ sensitivity, and arose mainly from a Ca2+-dependent switching off of the ß1 subunit-induced lengthening of the gaps between bursts (Fig 2 D). Consistent with our observations of functional coupling in 0 Ca2+i,
If the ß1 subunit is always coupled, then it should be possible to predict the effects of the ß1 subunit on the kinetic parameters in the absence of Ca2+i by projecting data obtained in the presence of Ca2+i to the abscissa at 0 Ca2+i. Such projections are difficult on the loglog plots used in this paper to emphasize the kinetics at low Ca2+i, because a value of 0 Ca2+i is never reached on a log axis. However, such projections can be made from the semilogarithmic plots presented in our previous study. For example, the predicted value of burst duration for +ß1 and
channels obtained by projecting a linear regression line to 0 Ca2+i from the data obtained from 15 to 1.8 µM Ca2+i was 16 and 0.7 ms, respectively (
The direct observations in Fig 1 and Fig 2, and the projected observations discussed above, indicate that the ß1 subunit exerts its characteristic effects of increasing Po and mean burst duration through an increase in the mean open time and the number of openings per burst in Ca2+i so low that the channel is essentially unliganded. Consequently, since the ß1 subunit imposes its characteristic effects on channel gating in the absence of Ca2+i, it follows that the ß1 subunit is coupled to the channel in the absence of Ca2+i, and can generate its signature effects without changing any Ca2+-binding rates. Furthermore, the observation on the double logarithmic plots in Fig 2 A that both the and
+ß1 channels appear to have similar critical Ca2+i for initiating the Ca2+-dependent activation (between 0.18 and 0.9 µM Ca2+i) indicates that the ß1 subunit may have relatively little effect on the initial Ca2+-binding rates, for if it had a pronounced effect, the Ca2+-induced increase in Po (and underlying changes in the other gating parameters) should occur at appreciably lower Ca2+i for
+ß1 channels than for
channels.
Consistent with a lack of increase in Ca2+-dependent rate constants, observations in our previous study indicate that the ß1 subunit does not act by mimicking the effects of increased Ca2+i ( and
+ß1 channels (
The observations in this present study indicate that the major effect of the ß1 subunit is to produce approximately parallel shifts (on logarithmic coordinates) in the magnitudes of the examined bursting parameters, when compared with channels (Fig 2 and Fig 4). Such parallel shifts on logarithmic coordinates are consistent with a multiplicative (gain) effect of the ß1 subunit on the examined parameters, and this gain effect was observed over three orders of magnitude of Po, independent of whether the channel was activated by Ca2+i or by voltage (Fig 6). How might such a gain effect on the bursting parameters occur?
The gating of BK channels is described by a model comprised of five parallel subschemes, each with five open and five closed states, in which the subschemes differ from one another by having either 0, 1, 2, 3, or 4 Ca2+i bound to the states in each subscheme ( channels in 0 Ca2+i by
In 0 Ca2+i, none of the states involved in gating would have bound Ca2+. Binding Ca2+ would then increase Po by altering the rate constants to decrease the durations of gaps and to increase the durations of bursts, and this would be the case for both and
+ß1 channels (Fig 2).
Since the ß1 subunit had little effect on the minimal number of kinetic states entered during gating in 0 Ca2+i (Fig 7) or in the presence of Ca2+i (
Whereas the ß1 subunit increases burst duration ~20-fold, independent of Ca2+i, its smaller effect of increasing the durations of the gaps between bursts approximately threefold was only observed in 0 Ca2+i (Fig 2 D). A threefold increase in the durations of the gaps between bursts in 0 Ca2+i, but not in higher Ca2+i, would arise if the ß1 subunit selectively slowed k(n), where n = 0, threefold, while having little effect on k(n) where n = 14. If this were the case, Ca2+i would switch off the lengthening effect of the ß1 subunit on gap duration. Alternatively, the addition of Ca2+i might remove the lengthening effect of the ß1 subunit by driving the gating away from the altered transition pathways involved in the lengthening, or by selectively changing these pathways. Under conditions of 0 Ca2+i, the ~20-fold increase in burst duration overrides the smaller threefold increase in gap duration, giving rise to the observed 10-fold increase in Po with the ß1 subunit in 0 Ca2+i. In the presence of Ca2+i, the ß1 subunit no longer lengthens the duration of the gaps between bursts (and may shorten them slightly), so the increase in burst duration can give rise to an even greater increase in Po, which becomes limited as Po saturates near its maximum of 0.96.
As indicated above, the ß1 subunit only slows k(n) in the absence of Ca2+i when n = 0. The presence of Ca2+i switches off the inhibitory effect of the ß1 subunit in 0 Ca2+i of increasing gap duration. This switching occurs over a range of Ca2+i between 0.2 and 2 µM (Fig 2 D). This Ca2+-dependent removal of the inhibition accounted for ~20% of the shift in the apparent Ca2+ sensitivity, while the Ca2+-independent increase in burst duration accounted for the other 80% of the shift in apparent Ca2+ sensitivity (Fig 2A and Fig B).
Conclusion
Ca2+i is not required for the coupling of the ß1 subunit to the BK channel. In the absence of Ca2+i, the ß1 subunit increases mean burst duration ~20-fold and also increases the duration of the gaps between bursts approximately threefold. The increase in burst duration facilitates channel activity and the increase in gap duration inhibits channel activity, for an increase in Po of ~10 fold in 0 Ca2+i. The ß1 subunit-induced ~20-fold increase in mean burst duration is Ca2+ independent, is retained over wide ranges of Ca2+i and voltage, and accounts for 80% of the increased Ca2+ sensitivity associated with the ß1 subunit. The ß1 subunit-induced approximately threefold increase in the duration of gaps between bursts is switched off (inhibited) by the addition of Ca2+i. This removal of the ß1 subunit-induced inhibition accounts for the remaining 20% of the increased Ca2+ sensitivity associated with the ß1 subunit. Thus, the major effect of the ß1 subunit on increasing Ca2+ sensitivity occurs through changes in Ca2+-independent rate constants.
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Footnotes |
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Portions of this work were previously published in abstract form (Nimigean, C.M., and K.L. Magleby. 2000. Biophys. J. 78:91A; and Nimigean, C.M., B.L. Moss, and K.L. Magleby. 1999. Soc. Neurosci. Abstr. 25:985).
1 Abbreviations used in this paper: BK channel, large conductance Ca2+-activated K+ channel; GFP, green fluorescent protein; HEK cells, human embryonic kidney cells; MWC, Monod-Wyman-Changeux.
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Acknowledgements |
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We thank Merck Research Laboratories for providing the mslo (initially cloned by
This work was supported by a fellowship from the American Heart Association, Florida Affiliate to C.M. Nimigean, and grants from the National Institutes of Health (AR32805) and the Muscular Dystrophy Association to K.L. Magleby.
Submitted: 7 February 2000
Revised: 12 April 2000
Accepted: 13 April 2000
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References |
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