Correspondence to: Karl L. Magleby, Department of Physiology and Biophysics, University of Miami School of Medicine, P.O. Box 016430, Miami, FL 33101-6430. Fax:(305) 243-6898 E-mail:kmagleby{at}miami.edu.
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Abstract |
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The COOH-terminal S9S10 tail domain of large conductance Ca2+-activated K+ (BK) channels is a major determinant of Ca2+ sensitivity (Schreiber, M., A. Wei, A. Yuan, J. Gaut, M. Saito, and L. Salkoff. 1999. Nat. Neurosci. 2:416421). To investigate whether the tail domain also modulates Ca2+-independent properties of BK channels, we explored the functional differences between the BK channel mSlo1 and another member of the Slo family, mSlo3 (Schreiber, M., A. Yuan, and L. Salkoff. 1998. J. Biol. Chem. 273:35093516). Compared with mSlo1 channels, mSlo3 channels showed little Ca2+ sensitivity, and the mean open time, burst duration, gaps between bursts, and single-channel conductance of mSlo3 channels were only 32, 22, 41, and 37% of that for mSlo1 channels, respectively. To examine which channel properties arise from the tail domain, we coexpressed the core of mSlo1 with either the tail domain of mSlo1 or the tail domain of mSlo3 channels, and studied the single-channel currents. Replacing the mSlo1 tail with the mSlo3 tail resulted in the following: increased open probability in the absence of Ca2+; reduced the Ca2+ sensitivity greatly by allowing only partial activation by Ca2+ and by reducing the Hill coefficient for Ca2+ activation; decreased the voltage dependence 28%; decreased the mean open time two- to threefold; decreased the mean burst duration three- to ninefold; decreased the single-channel conductance
14%; decreased the Kd for block by TEAi
30%; did not change the minimal numbers of three to four open and five to seven closed states entered during gating; and did not change the major features of the dependency between adjacent interval durations. These observations support a modular construction of the BK channel in which the tail domain modulates the gating kinetics and conductance properties of the voltage-dependent core domain, in addition to determining most of the high affinity Ca2+ sensitivity.
Key Words: Ca2+-activated K+ channel, maxi K+ channel, TEA, MWC model, gating kinetics
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INTRODUCTION |
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Large-conductance Ca2+-activated potassium channels (BK channels)* are activated by both intracellular Ca2+ and membrane depolarization. Consequently, BK channels can provide a direct link between Ca2+-dependent cellular processes and membrane excitability (for reviews see
The cloning of BK channels has paved the way toward approaching an understanding of the molecular mechanism of activation. The cDNAs encoding the pore-forming subunit were first obtained from Drosophila (dSlo;
subunit can be divided into two domains: (1) a core domain and (2) a COOH-terminal tail domain (
The purpose of our present study is to further define the functional role of the COOH-terminal tail domain. Its role as the primary Ca2+ sensor requires that there be an interaction between the core and tail domains so that Ca2+ binding can regulate channel gating (
Previous experiments have shown that the separate core (S0S8) and tail (S9S10) domains of BK channels expressed by themselves do not form functional channels (
Consistent with the findings of
Portions of this work were previously published in abstract form (
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MATERIALS AND METHODS |
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Expression of Cloned Channels in Xenopus Oocytes
Constructs encoding wild-type mSlo1 and mSlo3 channels, as well as the separate mSlo1 core domain, and the separate mSlo1 and mSlo3 tail domains were a gift from Dr. Lawrence Salkoff and Dr. Matthew Schreiber (Washington University School of Medicine, St. Louis, MO). Details on the constructs can be found in 1 µg/µl. The mSlo1 core cRNA was then mixed 1:1 (vol/vol) with either the mSlo1 tail or mSlo3 tail cRNA. Because the cRNA encoding the core domain is about two to three times longer than the cRNA encoding either tail domain, the tail domain cRNA was in about two- to threefold molar excess over the core domain cRNA.
Xenopus laevis oocytes were separated enzymatically using collagenase as previously described (15 ng of each cRNA; total of
210 ng/oocyte) or mSlo1 core and mSlo3 tail (
525 ng of each cRNA; total of
1050 ng/oocyte). We found that the mSlo3 tail channels typically did not express as well as the mSlo1 tail channels, and, therefore, required microinjection of a larger amount of cRNA. In experiments examining the properties of wild-type channels, oocytes were microinjected with 0.8 ng for wild-type mSlo1or 4070 ng for wild-type mSlo3. Coinjection of mSlo3 core domains with mSlo1 tail domains does not produce functional channels (
Although we have not demonstrated directly that all channels studied were comprised of both core and tail domains, several lines of evidence suggest that this was the case. First, no functional BK channels were detected in patches from oocytes injected with only mSlo1 core cRNA (75 ng/oocyte). This observation is consistent with results of
Single-channel Recording
Currents were recorded from wild-type mSlo1, wild-type mSlo3, mSlo1 tail, and mSlo3 tail channels expressed separately in Xenopus oocytes 24 d after injection using the inside-out configuration of the patch-clamp technique (
In all experiments, the extracellular solution contained 150 mM KCl, 5 mM TES [N-Tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid] pH buffer and 100 µM GdCl3 to block endogenous mechanosensitive channels (
Analysis of Single-channel Recordings
In experiments measuring specific kinetic parameters that are highly sensitive to differences in filtering (such as mean open time and the mean number of openings per burst), current records were low-pass filtered with a 4-pole Bessel filter to give a final effective filtering of 78 kHz (-3 dB). In experiments in which the parameters of interest were relatively insensitive to the level of filtering (such as single-channel conductance and open probability [PO]), current records were low-pass filtered to give a final effective filtering of 38 kHz. Single-channel currents were first recorded on a digital data recorder (DC-37 kHz), and after additional filtering (as described in this paragraph) were sampled by computer at a rate of 200 kHz.
The methods used to measure interval durations with half-amplitude threshold analysis and to use stability plots to identify data with stable channel activity for analysis and also to exclude data during mode shifts have been described previously (96% of the detected intervals in BK channels from cultured rat skeletal muscle (
The methods used to log-bin the intervals into one dimensional (1-D) dwell-time distributions, fit the distributions with sums of exponentials using maximum likelihood fitting techniques, 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 define bursts is detailed in
For all mSlo1 tail channels and mSlo3 tail channels, burst analysis was performed on data from patches containing a single channel in which the PO < 0.75, as it became increasingly difficult to define gaps between bursts as PO increased above this value. For mSlo1 and mSlo3 wild-type channels, burst analysis was performed on single-channel patches and also on some multichannel patches when the channel activity was sufficiently low so that only one channel was open at a time. In these 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 were estimated in multichannel patches by determining the mean gap between bursts as if the data were from a single channel, and then multiplying the mean durations of the gaps by the numbers of channels in the patch. There was no need to correct estimates of the mean burst duration, mean open time, or mean number of openings per burst, since only one channel was open at any given time during a burst.
Two dimensional (2-D) dwell-time distributions were generated as detailed in
Dependency plots were constructed from the 2-D dwell-time distributions as described in
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(1) |
where Nobs(to,tc) is the observed number of interval pairs in bin (to,tc), and Nind(to,tc) is the calculated number of interval pairs in bin (to,tc) if adjacent open and closed intervals pair independently (at random). The expected number of interval pairs in bin (to,tc) for independent pairing is:
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(2) |
where P(to) is the probability of an open interval falling in the row of bins with a mean open duration of to, and P(tc) is the probability of a closed interval falling in the column of bins with a mean closed duration of tc. P(to) is given by the number of open intervals in row to divided by the total number of open intervals, and P(tc) is given by the number of closed intervals in column tc divided by the total number of closed intervals.
Single-channel current amplitudes were obtained by plotting histograms of the number of observations versus current amplitude and then measuring the distance between the peaks, which represents the closed and open current levels, and also by visually fitting cursor lines to closed and open single-channel current levels in displayed current records. Similar results were obtained using both methods.
For the TEA experiments, the concentrations of TEAi that reduced the single-channel current amplitude by 50% (Kd) at membrane potentials ranging from +30 to +90 mV were estimated from fits of a Langmuir function to plots of percent current versus TEAi. The fraction of current remaining, 1 - y, is:
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(3) |
Kd values at 0 mV were estimated using the
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(4) |
where z, Bi, Kd (0 mV) are the blocker valence, concentration, and zero-voltage dissociation constant, respectively, and d is the fraction of voltage drop at the blocking site measured from the intracellular side of the membrane (
Statistics
Results are presented as the mean ± SEM. Unless otherwise indicated, differences among group means were tested for significance using the t test.
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RESULTS |
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Fig 1 presents a schematic diagram of the wild-type and chimeric channels studied in this paper to investigate the contribution of the tail domain of BK channels to the gating and conductance properties of the channel. The proposed membrane topology of the Slo family of channels consists of seven transmembrane segments S0S6 plus a pore (P)-forming region, together with four hydrophobic intracellular regions S7S10 (56% identity and the tail regions have
38% identity. As detailed in Fig 1, our paper studies wild-type mSlo1, wild-type mSlo3, a chimera expressed from the core of mSlo1 plus the tail of mSlo1 (mSlo1 tail channels), and a chimera expressed from the core of mSlo1 plus the tail of mSlo3 (mSlo3 tail channels). Functional channels are not expressed from the core of mSlo3 and the tail of mSlo1 (
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Comparison of Kinetic Properties between Wild-type mSlo1 and Wild-type mSlo3
Fig 2 presents single-channel currents recorded from mSlo1 and mSlo3. In later sections of this paper, we study a chimera made from the core domain (S0S8) of mSlo1 and the tail domain (S9S10) of mSlo3 to examine the contribution of the tail domain to the gating. To interpret the chimeric data, it is first necessary to know how the properties of the two wild-type channel types differ. Some of the differences have already been characterized by 16 mV/e-fold change in PO) was similar to the voltage sensitivity of mSlo1 (
106 pS) was considerably less than that of mSlo1 channels (
270 pS;
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To characterize the difference in gating kinetics between mSlo3 and mSlo1, an experimental condition was found that gave similar PO's for the two channels so that the kinetics could be compared. Comparison at similar PO's is necessary, as the mean open times and burst duration of BK channels such as mSlo1 change with PO (0 µM Ca2+i and +80 mV, mSlo3 (PO
0.05) had much briefer open times, shorter burst durations, and a lower single-channel conductance than mSlo1 (PO
0.04). These differences are readily apparent in Fig 2 B, which shows the currents on a faster time base.
To quantify the difference in bursting kinetics between these two channel types, we measured mean burst duration, mean open time, mean number of openings per burst, and the mean duration of the gaps (closed intervals) between bursts. Bursts were identified by using 1-D closed dwell-time distributions to define a critical gap. Closed intervals longer than the critical gap were then taken as gaps between bursts (MATERIALS AND METHODS). Fig 2 (D and F) presents the 1-D closed dwell-time distributions for the channels presented in Fig 2A and Fig B. The arrows indicate the mean gap durations. The dashed line in Fig 2 D indicates the distribution of effective gap durations after correcting for the three channels in the patch (MATERIALS AND METHODS). For these two experiments, the mean burst duration of mSlo3 (0.42 ms) was approximately sevenfold briefer than that of mSlo1 (2.8 ms). The briefer burst duration of the mSlo3 channel was largely due to the approximately sixfold decrease in mean open time of 0.55 ms for mSlo1 to 0.09 ms for mSlo3. The difference in mean open times can be seen by comparing the open dwell-time distributions in Fig 2 (C and E), where the mean open times are indicated by arrows. In spite of the briefer burst duration for mSlo3, the PO's for the two channels were similar because the mean gap duration for mSlo3 (4.2 ms) was approximately sixfold briefer than the mean gap duration for mSlo1 (24.2 ms).
Burst parameters from experiments such as those shown in Fig 2 were obtained from a total of five patches with mSlo1 (PO range: 0.010.04) and five patches with mSlo3 (PO range: 0.010.05). Results are summarized in Fig 3. Mean burst duration, mean open time, and the mean duration of gaps between bursts were 4-fold,
3-fold, and
2.3-fold less, respectively, for mSlo3 than for mSlo1. There was no significant difference in the mean numbers of openings per burst. In these same experiments, the single-channel conductance was 100 ± 4 pS for mSlo3 and 271 ± 4 pS for mSlo1, which is consistent with values reported previously for these channels (
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Replacing the mSlo1 Tail with the mSlo3 Tail Greatly Decreases Ca2+ Sensitivity and Increases Open Probability in the Absence of Ca2+
To examine the effect of replacing the mSlo1 tail with the mSlo3 tail on the Ca2+ sensitivity of PO and on the basal activity in the absence of Ca2+i, single-channel currents were recorded from channels comprised of the mSlo1 core domain and either the mSlo1 tail domain (mSlo1 tail channels) or the mSlo3 tail domain (mSlo3 tail channels), as diagramed in Fig 1. Fig 4 A presents currents recorded at +30 mV with 0, 8.7, and 67 µM Ca2+i from a single mSlo1 tail channel and from a single mSlo3 tail channel. Three observations are immediately apparent from these current records: (1) the level of activity in 0 Ca2+i was higher for the mSlo3 tail channel than for the mSlo1 tail channel; (2) the effect of Ca2+i on increasing PO (Ca2+ sensitivity) was far less for the mSlo3 tail channel than for the mSlo1 tail channel; and (3) the high Ca2+i (67 µM) only drove the PO of the mSlo3 tail channel to 0.2 compared with almost complete activation for the mSlo1 tail channel.
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To examine further this marked difference in Ca2+ sensitivity, PO was plotted against Ca2+i for four mSlo1 tail channels in Fig 4 B and seven mSlo3 tail channels in Fig 4 C (+30 mV). Unlike the mSlo1 tail channels that all had a very low PO in the absence of Ca2+i (0.0005 ± 0.0003), four of the mSlo3 tail channels showed a relatively high level of activity in the absence of Ca2+i, with PO's ranging from 0.14 to 0.34, and three showed lower PO's, ranging from 0.0001 to 0.01. (The reason for the wide range in activity for mSlo3 tail channels in the absence of Ca2+i is not known, but a wide range of activity was observed in two separate series of experiments, and will be addressed in the DISCUSSION.) Also, unlike the mSlo1 tail channels, which had PO's approaching 0.96 at 67 µM Ca2+i, the mSlo3 tail channels were not fully activated at +30 mV, even with 1,000 µM Ca2+i, where the mean PO was 0.43 ± 0.08. This lack of maximal activation is not because mSlo3 tail channels have reached some type of inherent limit, as depolarization of mSlo3 tail channels can drive them to high levels of activity (see Fig 6).
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All four mSlo1 tail channels showed a steep increase in PO with increasing Ca2+i (Fig 4 B). The mean Hill coefficient was 3.2 ± 0.2 (range: 2.93.5) and the mean Ca2+i required to achieve a PO of 0.5 (Kd) was 11.8 ± 1.5 µM (range: 8.314.2). These results are within the range of previous observations for wild-type mSlo1 (
In contrast to the high Ca2+ sensitivity of the mSlo1 tail channels, the mSlo3 tail channels showed a much smaller increase in PO with increasing Ca2+i. Accurate estimates of the Hill coefficients could not be obtained for the mSlo3 tail channel because, except for one channel that appeared to reach a maximum PO, it was unclear whether maximum activation was reached for the other channels, even with 1,000 µM Ca2+i. Higher concentrations than this were not examined because of the possibility of effects of Ca2+i on low affinity Mg2+ sites (2045% of those for the mSlo1 tail channels, and only apply over a limited range of PO. The Kds for the mSlo3 tail channels were 13, 34, 140, 340, 500, 700, and 9,200 µM when the maximum PO was set to 0.5, and were 440, 740, 1,200, 1,500, 24,000, and 29,000 µM when the maximum PO was set to 0.96. The wide variability in the estimated Kds indicates that some unknown factor is not under control, but in any case, the Kds for mSlo3 tail channels are typically considerably greater than for mSlo1 tail channels.
Since the activity of wild-type mSlo3 channels is pH-dependent, the question arises whether the small effects of Ca2+i might be due to a small but systematic change in the pH of the solutions below the resolution of the pH electrode. This is unlikely to be the case, as the mSlo3 tail channels showed little pH sensitivity in preliminary experiments in which the pH was changed from 6.5 to 8.0 (unpublished data), and
Replacing the mSlo1 Tail with the mSlo3 Tail Decreases the Ca2+ Dependence of Both Mean Open and Mean Closed Times
To gain insight into the mechanism of the difference in Ca2+ sensitivity between mSlo1 tail channels and mSlo3 tail channels, mean open and closed interval durations were measured and plotted versus Ca2+i for the same channels presented in Fig 4. Increasing Ca2+i from 0 to 67 µM increased the mean open time of mSlo1 tail channels approximately eightfold (Fig 5 A), with little or no increase in the mean open time of mSlo3 tail channels (Fig 5 B). Over the same range of Ca2+i, the mean closed time of mSlo1 tail channels decreased 2,300-fold (Fig 5 C), compared with an
14-fold decrease in the mean closed time of mSlo3 tail channels (Fig 5 D). Thus, the greatly decreased Ca2+ sensitivity of mSlo3 tail channels reflects a greatly decreased Ca2+ dependence of both mean open time and mean closed time.
Replacing the mSlo1 Tail with the mSlo3 Tail Decreases the Voltage Dependence
To examine whether replacing the tail of mSlo1 with the tail of mSlo3 altered the voltage dependence of mSlo1, currents were recorded from a single mSlo1 tail channel and a single mSlo3 tail channel at +20, +50, and +80 mV. As shown in Fig 6 A, both channels had a low activity at +20 mV (PO 0.04), which then increased dramatically with depolarization, with a suggestion that the increase for the mSlo3 tail channel was less than for the mSlo1 tail channel. To examine the voltage dependence further, we plotted PO versus membrane potential for five mSlo1 tail channels in Fig 6 B and five mSlo3 tail channels in Fig 6 C. The mSlo1 tail channels were studied in 0 µM (Fig 6, closed circle and closed diamond), 8.7 µM (Fig 6, closed inverted triangle and closed square), and 25.6 µM (closed triangle) Ca2+i. The mSlo3 tail channels were studied in 0 µM (open triangle, open square, and open circle), 8.7 µM (open diamond), and 15.2 µM (open hexagon) Ca2+i. For these particular experiments with mSlo1, the data obtained from different channels at the same Ca2+i essentially overlap. For mSlo3 channels, there were no consistent relationship between Po and Ca2+i.
The lines are Boltzmann fits to the data. Only three of the five fits are readily apparent in Fig 6 C because of the overlap of fitted curves. To facilitate comparison between the channels, the Boltzmann fits to the mSlo3 tail channels in Fig 6 C are plotted in Fig 6 B as dotted lines on the plots from the mSlo1 tail channels. A shallower slope for the mSlo3 tail channels is seen, indicating a decreased voltage dependence for the mSlo3 tail channels (17.3 ± 0.9 mV per e-fold change in PO; range: 15.819.5 mV) compared with the mSlo1 tail channels (12.3 ± 0.3 mV; range: 11.713.4 mV), and this difference was significant (P < 0.001). The estimated effective gating charge (in units of electronic charge, eo) for mSlo3 tail channels was 1.49 ± 0.06 (range: 1.311.62), and for mSlo1 tail channels was 2.08 ± 0.05 (range: 1.912.18). Both these estimates are within the ranges of values reported for wild-type mSlo1 and also other wild-type BK channels (
Replacing the mSlo1 Tail with the mSlo3 Tail Decreases Burst Duration
The single-channel currents in Fig 4 and Fig 6 suggest that, in addition to decreasing both Ca2+ sensitivity and voltage dependence, replacing the tails of mSlo1 channels with the tails of mSlo3 channels may also decrease burst duration. To examine this possibility, we compared mSlo1 tail channels and mSlo3 tail channels at similar PO's, to control for the fact that the burst duration of mSlo1 channels increases with PO, independent of whether the channels are activated by Ca2+i or depolarization (
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To quantify the difference in bursting kinetics between these two channels, we measured several burst parameters including mean burst duration, mean open time, mean number of openings per burst, and mean duration of the gaps (closed intervals) separating bursts. As in the burst analysis of the wild-type channels, bursts were identified by using 1-D closed dwell-time distributions to define a critical gap to separate bursts. Fig 7 (D and F) presents the 1-D closed dwell-time distributions, and the arrows indicate the mean gap durations. Burst analysis of the single-channel currents indicated that replacing the mSlo1 tail with the mSlo3 tail decreased burst duration 3.4-fold, from 8.6 ms for the mSlo1 tail channel to 2.5 ms for the mSlo3 tail channel. The decreased burst duration of the mSlo3 tail channel was largely due to a decrease in the mean number of openings per burst, from 6.1 for the mSlo1 tail channel to 2.9 for the mSlo3 tail channel, as well as a decrease in mean open time, from 1.2 ms for the mSlo1 tail channel to 0.79 ms for the mSlo3 tail channel. This 34% decrease in mean open time can be seen by comparing the 1-D open dwell-time distributions in Fig 7 (C and E), where the arrows indicate mean open time. For the same PO, replacing the mSlo1 tail with the mSlo3 tail also decreased the mean duration of gaps between bursts fourfold, from 17.4 to 4.3 ms (Fig 7D and Fig F, arrows).
To characterize the differences in bursting kinetics between mSlo1 tail channels and mSlo3 tail channels over a range of PO, bursting parameters were measured and plotted against PO for 10 datasets from three mSlo1 tail channels and 10 datasets from three mSlo3 tail channels (membrane potential: +30 mV; 067 µM Ca2+i). As shown in Fig 8 A, the mean burst duration of mSlo3 tail channels was three- to ninefold briefer than that of mSlo1 tail channels over the range of PO examined (0.00050.75). The briefer burst duration was associated with a two- to threefold decrease in mean open time (Fig 8 B) and a decrease in the mean number of openings per burst (Fig 8 C). In addition, the gaps between bursts for the mSlo3 tail channels were four- to fivefold briefer than those for mSlo1 tail channels (Fig 8 D). Thus, replacing the mSlo1 tail with the mSlo3 tail resulted in a consistent and pronounced modulation of bursting kinetics over an 1,500-fold range of PO.
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If the tail domain of the Slo family of channels modulates the bursting kinetics, then it might be expected that transferring the tail of mSlo3 to mSlo1 would also transfer the bursting kinetic properties of mSlo3. To test this hypothesis, the parameters defining the bursting kinetics of wild-type mSlo1 and wild-type mSlo3 channels are plotted on Fig 8 (large circles containing a plus sign) for comparison to the kinetics of mSlo1 tail channels and mSlo3 tail channels. In general, replacing the tail of mSlo1 with the tail of mSlo3 transferred the bursting kinetic properties of wild-type mSlo3 channels.
Replacing the mSlo1 Tail with the mSlo3 Tail Does Not Change the Number of Detected Kinetic States Entered during Gating
BK channel gating has been well-described by schemes in which the channel enters multiple kinetically distinct open and closed states (
Fig 9 (A and B) plots estimates of the number of significant exponential components (number of detected states) required to describe the open (A) and closed (B) 1-D dwell-time distributions for 15 datasets from five patches, each containing a single mSlo1 tail channel, and for 15 datasets from five patches, each containing a single mSlo3 tail channel. Multiple datasets were obtained from each channel by obtaining data at different Ca2+i. The estimates are plotted against the number of intervals analyzed, since the ability to resolve exponential components increases with increasing numbers of intervals (
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Estimates of the minimum number of open states ranged from two to four for both types of channels. The mean number of detected open states for mSlo1 tail channels (2.9 ± 0.2) was not significantly different (P < 0.05, Mann-Whitney test) from the mean number of detected open states for mSlo3 tail channels (2.9 ± 0.2). Estimates of the number of detected closed states ranged from four to eight for mSlo1 tail channels and from three to seven for mSlo3 tail channels. The mean number of detected closed states for mSlo1 tail channels (5.6 ± 0.3) was not significantly different (P < 0.05, Mann-Whitney test) from the mean number of detected closed states for mSlo3 tail channels (5.1 ± 0.3). Thus, each channel type typically entered a minimum of three or more open and five to six closed kinetic states during normal activity.
These results suggest that the pronounced difference in bursting kinetics between mSlo1 tail channels and mSlo3 tail channels does not reflect a major difference in the number of detected kinetic states entered during gating. However, we cannot rule out the possibility that mSlo1 tail channels and mSlo3 tail channels do differ in the number of kinetic states entered during gating and that these differences were not detected due to overlapping time constants and/or small areas of some of the exponential components.
mSlo1 Tail Channels and mSlo3 Tail Channels Have Similar Kinetic Structures
Replacing the mSlo1 tail with the mSlo3 tail resulted in a pronounced change in bursting kinetics. Such differences in kinetics could arise from a fundamental change in the gating mechanism or a change in the transition rates among states. The observation that the numbers of detected states did not change would argue against a fundamental change in the gating mechanism, but this is not clear. One way to help distinguish between these two possibilities is to examine the effective connections (transition pathways) among the various open and closed states. 2-D dwell-time distributions, which plot the relative number of occurrences (frequency) various pairs of adjacent open and closed intervals of specified durations are observed in the single-channel record, contain correlation information that can help define these connections (
Fig 10 (A and C) presents the 2-D dwell-time distributions for the mSlo1 tail channel and the mSlo3 tail channel, whose single-channel currents were shown in Fig 7. The y- and x-axes plot the log of the durations of adjacent open and closed intervals, respectively, and the z axis plots the square root of the number of observations per bin. To facilitate comparison, the PO's were similar for both channel types (0.30 and 0.34). The general shapes of the plots were similar, but with differences in the relative frequency of the various interval pairs. For example, for both channel types, the components of interval pairs that occurred most frequently were long open intervals adjacent to brief closed intervals (Fig 10A and Fig C, position 3). These interval pairs typically give rise to openings separated by brief closings within bursts. A major difference in the two plots is that the mSlo3 tail channel shows a higher frequency of brief open intervals adjacent to brief closed intervals (Fig 10 C, arrow). These interval pairs would contribute to the briefer mean open time and briefer burst duration of mSlo3 tail channels. Also notice that the long closed intervals of the mSlo3 tail channel are shifted toward briefer durations (positions 2 and 4). This shift reflects the briefer duration of gaps between bursts for mSlo3 tail channels. Fig 10 (E and G) presents 2-D dwell-time distributions for another pair of channels studied at a lower PO (0.19 and 0.14), where the relative frequency of brief open intervals is much greater for the mSlo3 tail channel than for the mSlo1 tail channel (arrow and position 2). Thus, as would be expected from their differences in bursting kinetics, mSlo1 tail channels and mSlo3 tail channels have differences in the relative frequencies of interval pairs.
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In addition to providing a profile of the relative frequencies of interval pairs, the 2-D dwell-time distributions also contain correlation information which gives information about the connections between different open and closed states (and compound states) of different mean lifetimes (
Dependency plots provide a means to display this correlation information (
Fig 10B and Fig D and Fig F and Fig H, presents dependency plots derived from the 2-D dwell-time distributions in Fig 10A and Fig C, and Fig E and Fig G, respectively. Although mSlo1 tail channels and mSlo3 tail channels differ kinetically, a comparison of the four dependency plots suggests that these channels have similar kinetic structures, since the plots for both channel types have the characteristic saddle shape that is typical of dependency plots for BK channels (
The saddle shape in the dependency plots arises from excesses and deficits of specific interval pairs. In particular, plots from both mSlo1 tail channels and mSlo3 tail channels show a deficit of brief open intervals adjacent to brief closed intervals (position 1), an excess of brief open intervals adjacent to long closed intervals (position 2), a deficit of long open intervals adjacent to long closed intervals (position 4), and an excess of long open intervals adjacent to brief closed intervals (position 3).
Such a saddle-shaped dependency plot indicates that for each channel type, there is a general inverse relationship between the durations of open and closed states and compound states. That is, brief open states (or compound states in each comparison) tend to be adjacent to long closed states, and long open states tend to be adjacent to brief closed states. Thus, the data suggest that the effective connections among the various open and closed states (kinetic structure) are similar between mSlo1 tail channels and mslo3 tail channels, consistent with the idea that the difference in bursting kinetics arises from differences in the transition rates among states rather than from differences in the fundamental gating mechanisms.
Replacing the mSlo1 Tail with the mSlo3 Tail Decreases the Single-channel Conductance
In addition to modulating gating properties, replacing the mSlo1 tail with the mSlo3 tail decreased single-channel conductance. Fig 11 (A and B) presents single-channel currents recorded from a mSlo1 tail channel and a mSlo3 tail channel at a membrane potential of +70 mV at two different time bases. The dashed lines denote the maximum current amplitude observed for each channel. The mSlo1 tail channel had a maximum amplitude of 20.9 pA, compared with
17.3 pA for the mSlo3 tail channel.
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To estimate the slope conductances of these two channels, we plotted single-channel current amplitude versus membrane potential. As shown in Fig 11 C, the difference in current amplitude was observed over a wide range of voltages, with no obvious rectification for either channel. The single-channel conductances were 285 pS for the mSlo1 tail channel and
243 pS for the mSlo3 tail channel.
Estimates of slope conductances from plots such as those shown in Fig 11 C for 20 patches with mSlo1 tail channels and 24 patches with mSlo3 tail channels are presented as histograms in Fig 11 D. A clear shift toward lower conductances for mSlo3 tail channels was observed. The superimposed lines are fits of Gaussian functions to the distributions, yielding mean slope conductances of 276 ± 4 pS for the mSlo1 tail channels and 238 ± 4 pS for the mSlo3 tail channels. Thus, replacing the mSlo1 tail with the mSlo3 tail results in a 14% reduction in single-channel conductance (P < 0.001).
Replacing the mSlo1 Tail with the mSlo3 Tail Increases the Sensitivity to Block by Internal TEA
The lower single-channel conductance observed when the mSlo1 tail is replaced with the mSlo3 tail suggests that the mSlo3 tail alters channel structure or charge distributions in a way that reduces the movement of K+ through the pore. TEA, which can reduce single-channel conductance of K+ channels, has been a useful tool for exploring the vestibules of K+ channels (
Fig 12 A presents current recordings from an mSlo1 tail channel and an mSlo3 tail channel with internal TEA concentrations (TEAi) of 0, 10 and 50 mM (+70 mV), showing that TEA reduced the single-channel current amplitude for each channel type. The reduction in current is referred to as a fast block, as discrete blocking events are not observed. Fig 12 (B and C) plots single-channel current amplitude against membrane potential for the mSlo1 tail channel and mSlo3 tail channel shown in Fig 12 A. For both channel types, TEA reduced single-channel current amplitude at all potentials examined. The block was voltage-dependent, becoming greater at more positive potentials. Comparison of the degree of block at positive potentials between the two plots suggests that the mSlo3 tail channels are more sensitive to block by internal TEA. For example, at +100 mV, 10 mM TEA reduced the amplitude of currents from the mSlo1 tail channel by 5% compared with a 20% reduction for the mSlo3 tail channel.
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To further characterize this apparent difference in TEA sensitivity, we examined doseresponse relationships for each channel type. Fig 12 D plots the current versus TEAi for the two channels shown in Fig 12 (AC). The solid lines are fits of a Langmuir function (Equation 3), which yielded Kd values of 77 and 55 mM for the mSlo1 tail channel and mSlo3 tail channel, respectively. The dotted lines in Fig 12 D show graphically that the Kd for each channel type corresponds to the TEAi that reduces single-channel current amplitude to 50% of the control value. Kd values were also estimated for data obtained at +30, +50, and +70 mV using the same method, and at 0 mV using the
The
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DISCUSSION |
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In this study, we have shown that replacing the COOH-terminal tail domain region (S9S10) of mSlo1 channels with the corresponding tail domain of mSlo3 channels conferred many of the properties of wild-type mSlo3 channels to the chimeric channels comprised of the mSlo1 core domains and mSlo3 tail domains. Compared with channels expressed from mSlo1 cores and mSlo1 tails (mSlo1 tail channels), the channels expressed from mSlo1 cores and mSlo3 tails (mSlo3 tail channels) were much less Ca2+ sensitive, had a higher open probability in the absence of Ca2+, were less voltage-dependent, had briefer mean open times, briefer burst durations, a lower single-channel conductance, and an increased sensitivity to block by internal TEA. These observations suggest that the tail domains (S9-S10) of BK channels modulate the kinetic, conductance, and activation properties of the channel. This modification could involve both direct and indirect actions of the tail domain.
Variability among Channels
One difficulty with single-channel studies, is that there can be considerable variability in the PO and kinetics among channels of the same apparent type under constant experimental conditions. The variability can be divided into two types: (1) variability in the PO and/or kinetics over time for individual channels; and (2) stability in the PO and kinetics over time for individual channels, but differences in the PO and/or kinetics among separate channels of the same apparent type. The first type of variability has been termed wanderlust kinetics (
The second type of variability, that among channels of the same type, is compensated for in our study by presenting separate plots of data from a number of single-channel patches so that the range of variability among channels is apparent. The mean and the SEM of the kinetic parameters determined from the individual channels are also presented to give the average response. Although the variability in the PO and kinetics among mSlo3 tail channels under constant experimental conditions could be considerable, this variability does not alter the conclusions of our study, because the kinetics of the mSlo1 tail and mSlo3 tail channels are so characteristically different.
The S9S10 Tail Domain of mSlo1 Is a Major Determinant of Ca2+ Sensitivity
Replacing the mSlo1 tail with the mSlo3 tail converted the channel from a highly sensitive Ca2+- activated channel (Fig 4 B) to a channel that was only weakly modulated by Ca2+ over a narrow range of PO (Fig 4 C). These observations are consistent with those of
However, it cannot be excluded that some of the residual effects of Ca2+ and Cd2+ observed with mSlo3 tail channels may arise from the region of the mSlo3 tail replacing the calcium bowl, as this region still contains two negative charges (compared with the eight negative charges in the calcium bowl) that would be available to coordinate divalent cation binding, with negative charges located elsewhere in the channel. However, this possibility seems unlikely since wild-type mSlo3 channels show little Ca2+ sensitivity (
Replacing the mSlo1 tail with the mSlo3 tail reduced the Hill coefficient from 3.2 to <40% of this value, and limited the Ca2+-dependent change in PO to a small fraction of the range of Po accessible by varying voltage. A reduced Hill coefficient could arise from a decreased number of Ca2+ binding sites or from a decreased efficacy of action of Ca2+. Since BK channels are tetramers (
subunits of mSlo1, for a total of four primary sites, plus perhaps four additional secondary binding sites. The simplest explanation for the reduced Hill coefficient of the mSlo3 tail channels is that the number of effective Ca2+ binding sites is reduced due to the absence of calcium bowls in the mSlo3 tails, and that the remaining Ca2+ binding sites are less effective in activating the channel.
In addition to decreasing the Hill coefficient, replacing the mSlo1 tail with the mSlo3 tail increased the PO in 0 µM Ca2+i (+30 mV) from 0.0005 for mSlo1 tail channels to, on average,
0.15 for mSlo3 tail channels. Although the average PO of mSlo3 tail channels was orders of magnitude greater than for mSlo1 tail channels under these experimental conditions, there was a wide range in the PO among mSlo3 tail channels under these conditions, ranging from 0.0001 to 0.34. Although a number of factors could lead to differences in PO among channels (as discussed on p. 727), an additional explanation for this variability among mSlo3 tail channels is that different numbers of mSlo3 tails may have been associated with the cores. Although excess cRNA for the mSlo3 tails, when compared with that for the core, was injected into the oocytes to reduce this possibility (MATERIALS AND METHODS), it cannot be excluded that different numbers of mSlo3 tails may have associated with some of the mSlo3 tail channels. Since functional channels cannot form from cores alone (
Our observations that the activity of mSlo3 can be considerable in 0 µM Ca2+i at voltages (+30 mV) where the activity of mSlo1 tail channels is very low, is consistent with the results of
Replacing the Tail of mSlo1 with the Tail of mSlo3 Reduces the Voltage Sensitivity
The decreased voltage dependence of mSlo3 tail channels was indicated by a 28% reduction in the estimates of the effective gating charge determined from Boltzmann fits. However, mSlo1 tail channels and mSlo3 tail channels have an identical S4 transmembrane segment, which is thought to be the primary voltage sensor (
Replacing the Tail of mSlo1 with the Tail of mSlo3 Transfers the Bursting Properties of mSlo3
Replacing the mSlo1 tail with the mSlo3 tail resulted in a pronounced change in bursting kinetics, including decreases in mean burst duration, mean open time, mean number of openings per burst, and the mean duration of gaps between bursts (Fig 8). These are the changes that would be expected, based on the differences in properties between wild-type mSlo1 and wild-type mSlo3 (Fig 2 and Fig 3), provided that the tail of Slo channels contributes to the bursting kinetics. Our observations together with previous observations (
Two lines of evidence suggest that the transfer of bursting kinetics with the transfer of the tail does not arise from fundamental changes in the gating kinetics, but rather more subtle changes in the transitions rates among the states involved in the gating. First, we found no significant difference in the number of detected open or closed states after replacement of the mSlo1 tail with the mSlo3 tail (Fig 9). Second, whereas the relative magnitudes of the various components of the dependency plots could be quite different for the two channel types, the characteristic saddle shape of the dependency plots (
The transfer of bursting kinetics by replacing the mSlo1 tail with the mSlo3 tail could arise because the tail directly contributes to the gating machinery, or alternatively, because the tail serves to modulate the gating machinery in the core of the channel. The proposal of subunit stabilizes the gating kinetics of the channel, limiting the number of kinetic modes that are available for gating.
For mSlol channels, replacing the incorrect mSlo3 tail with the correct mSlo1 tail increases mean burst duration, the mean number of openings per burst, the mean open time, and the mean gap duration for data compared at the same PO over a wide range of PO (Fig 8). Interestingly, the addition of the auxiliary ß1 subunit to mSlo1 channels increases all these parameters even further (compare Fig 8 with Fig 6 in
Why Doesn't the Elimination of the Calcium Bowl Decrease the Number of States Detected during Gating?
Since Ca2+ activates BK channels, it would be expected that the binding of each additional Ca2+ to the channel would further change its properties, creating an additional state. If the calcium bowl contributes a major Ca2+ binding site to each of the four tails, as proposed (
This question can be addressed in terms of kinetic models that describe the gating of BK channels. In these discrete state Markov models, the voltage sensor in each of the four subunits of the channel can enter two conformations, and each subunit can bind a Ca2+, leading to two-tiered 50-state allosteric models for the gating (
For a first approximation, this is found to be the case. For analysis of 1-D distributions with >500 intervals, wild-type mSlo1 channels gated among two to three detected open and three to five detected closed states in 0 Ca2+i (Fig 7, open symbols, in
This greater than predicted number of closed states could arise for channels with absent calcium bowls for several reasons: first, mSlo3 tail channels do display a weak Ca2+ dependence, so they retain some secondary Ca2+ binding sites that could contribute additional states; and, second, the 50-state models are too simple and, thus, under predict the numbers of states the channel can gate in. Some observations suggest that the 50-state models may need an additional tier of flicker closed states (
An apparent paradox concerning the 50-state models is not the numbers of states observed in the absence of the calcium bowl, but why so few of the potential 50 (or more) states are detected in the presence of Ca2+i for channels with functional calcium bowls. The maximum numbers of detected states for BK channels are typically three to four open and five to seven closed states (Fig 9, mSlo1 tail channels;
Replacing the Tail of mSlo1 with the Tail of mSlo3 Decreases Single-channel Conductance and Facilitates the Fast Block by Internal TEA
Two observations in our study suggests that the tail domain of BK channels influences the structure of the conducting pore of the channel. Replacing the mSlo1 tail with the mSlo3 tail both reduced the single-channel conductance 14% (Fig 11), and also facilitated the fast block of the channel by internal TEA, decreasing the apparent Kd for TEA binding by 30% without changing the apparent electrical distance into the field at which the binding occurs (Fig 12).
The tail domain of the channel could alter the conducting pore directly if it contributes to the intracellular part of the pore, or it could alter the access to the pore. Perhaps the tail domain is involved in forming structures at the inner part of the pore in a manner similar to the cytoplasmic ß subunit-T1 assembly in voltage-dependent K+ channels (
It is also possible that the differences in conductance and TEA block when the mSlo3 tail replaces the mSlo1 tail may be associated with differences in the gating. Perhaps the movement of the gate is restricted for mSlo3 tail channels so that the channels do not open fully, but open to a subconductance level with a conductance 14% less than the fully open mSlo1 tail channels. For Drk1 channels, openings to subconductance levels may arise from partially activated channels in which some, but not all, subunits have undergone the conformational changes required for wild-type channel opening (
Modular Construction of BK Channels
Long before it was known that the structure of the core domain of BK channels is similar to the structure of the superfamily of voltage-dependent K+ channels (INTRODUCTION),
Consistent with this idea,
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Footnotes |
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The present address of Dr. Moss is Department of Biological Sciences, Purdue University, West Lafayette, IN 47907.
* Abbreviations used in this paper: 2-D, two dimensional; BK channel, large-conductance Ca2+-activated K+ channel; MWC, Monod-Wyman-Changeux; PO, open probability.
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Acknowledgements |
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We thank Dr. Lawrence Salkoff and Dr. Matthew Schreiber for providing the cDNAs encoding wild-type mSlo1, wild-type mSlo3, the mSlo1 core, the mSlo1 tail, and the mSlo3 tail. We thank Xiang Qian for contributing some experiments for the paper.
This work was supported in part by National Institutes of Health grant AR32805 and a grant from the Muscular Dystrophy Association to K.L. Magleby.
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