Characterization of Gating and Peptide Block of mSlo, a Cloned Calcium-Dependent Potassium Channel

Deirdre A. Sullivan, Mats H. Holmqvist, and Irwin B. Levitan

Department of Biochemistry and Volen Center for Complex Systems, Brandeis University, Waltham, Massachusetts 02254

    ABSTRACT
Abstract
Introduction
Methods
Results
Discussion
References

Sullivan, Deirdre A., Mats H. Holmqvist, and Irwin B. Levitan. Characterization of gating and peptide block of mSlo, a cloned calcium-dependent potassium channel. J. Neurophysiol. 78: 2937-2950, 1997. The 20 amino acid Shaker inactivation peptide blocks mSlo, a cloned calcium-dependent potassium channel. Changing the charge and degree of hydrophobicity of the peptide alters its blocking kinetics. A "triple mutant" mSlo channel was constructed in which three amino acids (T256, S259, and L262), equivalent to those identified as part of the peptide's receptor site in the S4-S5 cytoplasmic loop region of the Shaker channel, were mutated simultaneously to alanines. These mutations produce only limited changes in the channel's susceptibility to block by a series of peptides of varying charge and hydrophobicity but do alter channel gating. The triple mutant channel shows a significant shift in its calcium-activation curve as compared with the wild-type channel. Analysis of the corresponding single amino acid mutations shows that mutation at position L262 causes the most dramatic change in mSlo gating. These results suggest that the three amino acids mutated in the mSlo S4-S5 loop may contribute to, but are not essential for, peptide binding. On the other hand, they do play a critical role in the channel's calcium-sensing mechanism.

    INTRODUCTION
Abstract
Introduction
Methods
Results
Discussion
References

Large conductance calcium-dependent potassium channels play important roles in neurons, including determining action potential duration and influencing neurotransmitter release (Robitaille and Charlton 1992). These channels serve as molecular integrators between the cellular signals of intracellular calcium and membrane potential, although the mechanisms by which these signals influence channel activity are not well understood. Members of this class of potassium channel have been cloned from a number of sources, including the Drosophila mutant slowpoke (dSlo) (Adelman et al. 1992; Atkinson et al. 1991), mouse (mSlo) (Butler et al. 1993), and human (hSlo) (Dworetzky et al. 1994; Pallanck and Ganetzky 1994; Tseng-Crank et al. 1994). These channels are thought to resemble voltage-gated potassium channels in their overall protein topology, with the notable difference that calcium-dependent potassium channels have a much longer C-terminal domain (Atkinson et al. 1991). Although these two types of potassium channels are regulated in different ways, some regions of their amino acid sequences are well conserved. The area of highestsequence conservation is the putative pore region (H5),and the S4-S5 cytoplasmic loop is also well conserved(Fig. 1).


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FIG. 1. Location of critical residues in Shaker and mSlo. Voltage-gated and calcium-dependent potassium channels have a similar topology with 6 membrane-spanning regions (S1-S6) and a pore loop (H5). Sequences of the S4-S5 cytoplasmic loop regions of the Shaker B and mSlo channels are aligned using Genetics Computer Group alignment software. Using a gap weight of 0.05 and a length weight of 0.005, the sequences show a 73.3% similarity and a 53.3% identity in this region. We constructed 4 mSlo S4-S5 loop mutants (*): T256A, S259A, L262A, and a triple mutant (T256A + S259A + L262A).

In the Shaker B voltage-gated potassium channel, the S4-S5 loop region has been identified as a receptor site for the N-type inactivation gate (Isacoff et al. 1991), which is composed of the N-terminal 20 amino acids of the channel protein (Hoshi et al. 1990). Mutation of five amino acids in the S4-S5 loop of Shaker affects the stability of the inactivated state and alters channel conductance (Isacoff et al. 1991). Cysteine substitution followed by chemical modification also identifies this region as being critical for N-type inactivation (Holmgren et al. 1996). These experiments suggest that the S4-S5 loop forms at least part of a receptor for the inactivation gate and lies near the channel's ion permeation pathway. Most calcium-dependent potassium channels do not display the rapid N-type inactivation that is a characteristic of voltage-gated potassium channels, although rat adrenal chromaffin cells do have an inactivating calcium-dependent potassium channel (Solaro et al. 1995). When inactivation is removed from this channel, by treating its cytoplasmic side with protease, inactivation can be mimicked by applying a 20 amino acid synthetic Shaker inactivation peptide to the cytoplasmic side of the channel (Solaro and Lingle 1992). A similar phenomenon is observed with mutant noninactivating Shaker channels (Murrell-Lagnado and Aldrich 1993a,b; Zagotta et al. 1990).

The Shaker inactivation peptide blocks a number of other channels including native calcium-dependent potassium channels from rat brain (Foster et al. 1992) and porcine coronary artery smooth muscle (Toro et al. 1992), the cloned calcium-dependent potassium channel, dSlo (Perez et al. 1994), and a voltage-gated potassium channel in basolateral membranes of Necturus enterocytes, (Dubinsky et al. 1992). The 20 amino acid inactivation peptide is composed of an N-terminal hydrophobic domain and a positively charged C-terminal domain. Experiments with the Shaker B potassium channel and the calcium-dependent potassium channel from porcine coronary artery smooth muscle have shown that the positive charge of the peptide affects its association rate, whereas its degree of hydrophobicity affects its dissociation rate (Murrell-Lagnado and Aldrich 1993a; Toro et al. 1994). These experiments suggest that the voltage-gated and calcium-dependent potassium channels share a common receptor site for the peptide.

We are studying the activity of the cloned calcium-dependent potassium channel, mSlo. Compounds that inhibit the conductance of or influence the gating properties of calcium-dependent potassium channels may serve as valuable tools for understanding how these channels function in vivo. We have found that the wild-type Shaker inactivation peptide blocks the mSlo channel and have used mutant peptides to investigate which features of the peptide are important for its ability to block mSlo. We also tested whether or not the receptor site for the peptide is conserved between Shaker and mSlo. Three out of five amino acids, identified as being part of a putative peptide receptor site in the Shaker B channel, are conserved in mSlo. Mutational analysis indicates that peptide block is altered but not eliminated by mutation of these three S4-S5 loop amino acid residues. In addition, these residues play a critical role in the calcium- and voltage-dependent gating of the channel.

    METHODS
Abstract
Introduction
Methods
Results
Discussion
References

cDNA expression and site-directed mutagenesis

A cDNA encoding mSlo (mbr5) (Butler et al. 1993), a gift from the laboratory of Dr. Larry Salkoff, was subcloned into expression vectors: pMT3 (for COS cell expression) and pcDNA3 (Invitrogen, San Diego, CA) (for HEK293 cell expression). pMT3, a derivative of pMT2 (Sambrook et al. 1989), was a gift from the laboratory of Dr. Daniel Oprian. The pMT3-mSlo channel was expressed using the COS cell system for membrane preparations and use in bilayer studies. pcDNA3-mSlo was used for expression in HEK293 cells that were used for patch recordings. Channel mutants were generated using polymerase chain reaction (PCR) mismatch mutagenesis (Kammann et al. 1989). DNA from positive clones was isolated using the Qiagen Miniprep Kit (Qiagen, Chatsworth, CA). Mutants were sequenced throughout the PCR-amplified region using the PRISM DyeDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems) at the Brandeis sequencing facility.

Cell culture and transfection procedures

HEK293 cells were grown and maintained according to established procedures (Holmes et al. 1996). Specifically, cells were plated in the center of 35-mm dishes such that they could be transfected with a minimal volume, 200 µl, containing 0.2 µg of total plasmid DNA using the lipofectamine method according to the manufacturer's instructions (GibcoBRL, Grand Island, NY). Individually transfected cells were identified using a bead identification method (Invitrogen) (Jurman et al. 1994).

COS cells were maintained and transfected, using the DEAE-Dextran method (Oprian et al. 1987). Specifically, cells were transfected at 50-80% confluency with 2 µg of plasmid DNA per plate. Ten 10-cm plates were used per membrane preparation as described previously (Muller et al. 1996; Sun et al. 1994). Before use, an aliquot of the membrane preparation was pelleted by centrifuging in an Eppendorf 5415C Centrifuge at 14,000 rpm for 30 min at 4°C. The resulting pellet was resuspended in approximately twice the starting volume of cis bilayer setup solution (see following text), and the resuspended membranes were frozen and thawed three times using a dry ice-ethanol bath. The membranes then were sonicated briefly (2 s) before use. This preparation was stored at 4°C. All chemicals were obtained from Sigma Chemical (St. Louis, MO) unless otherwise specified.

Bilayer recording

Single channel activity was observed using a planar lipid bilayer setup (Miller 1986). Planar lipid bilayers were formed using a mixture of lipids (Avanti Polar Lipid, Birmingham, AL): 1-palmitoyl-2-oleyl-sn-glycero-3-phosphatidylethanolamine and L-alpha -phosphatidylserine (brain, sodium salt) (3:1) in n-decane. Membranes and peptides were always added to the cis side of the chamber, which corresponds to the cytoplasmic face of the channel. The cis solution contained (in mM) 10 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 400 KCl, 2 MgCl2, 1.2 CaCl2, 1 ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), pH 7.2; free calcium was 200 µM as determined with a calcium electrode (Microelectrodes, Bedford, NH). The trans side of the chamber, corresponding to the extracellular side of the channel and defined as zero voltage, contained (in mM) 10 HEPES, 50 KCl, and 0.1 EGTA, pH 7.2. After insertion of a single channel into the bilayer the cis and trans potassium concentrations were equalized, and all experiments were performed under conditions of symmetrical 400 mM potassium. Iberiotoxin was a gift from Dr. C. Miller, Brandeis University. NSOO4 was a gift from Dr. V. Gribkoff, Bristol-Myers Squibb Pharmaceutical Research Institute.

Channel currents were amplified with an Axopatch 200 patch-recording amplifier (Axon Instruments, Foster City, CA), filtered at 1 kHz with a low-pass Bessel filter (80 dB/decade) and stored on VHS video tapes. Data were digitized, using a DAP-800 acquisition board (Microstar Laboratories, Bellevue, WA) interfaced with a Dell 486DX/33 computer, at 500- or 1,000-µs intervals using steady state patch recording software (SSPAT) written in our laboratory by Manuel Esquerra. Two software programs were employed to generate dwell time histograms, AXGOX written by Noel Davies (University of Leicester) (Davies 1993), and DVIEW, written by Alex Stewart (Brandeis University). Using the AXGOX program, a 50% threshold was used to detect open and closed events, with linear regressions joining pairs of data points on either side of the threshold giving the estimated time of crossing. The events were log-binned at 25 bins per log10 unit (Sigworth and Sine 1987). DVIEW generates event lists from a digitized trace and was used when it was necessary to set a limit for the minimum block time. Event lists generated by this program were then imported into Origin Version 3.5 (Microcal Software, Northampton, MA) where they were binned and plotted as dwell time histograms.

Patch recording

Patch recordings of HEK293 cells were done 1-4 days after transfection. Cells were viewed using a Zeiss inverted microscope. The pipette solution contained (in mM) 150 KCl, 10 HEPES, 2 MgCl2, and 0.5 bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA), pH 7.4. The bath solutions had the same composition as the pipette solution except for different amounts of CaCl2. Free calcium concentration was established as described (Vopio et al. 1994). The purity and Kd of the calcium chelater, BAPTA, in the bath solution was determined using a Scatchard plot. Based on the BAPTA concentration, a calculated amount of calcium was added to produce the different concentrations of free calcium. Finally, the free calcium concentration was verified using a calcium-sensitive electrode.


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FIG. 2. Wild-type and triple mutant mSlo are blocked by the Shaker inactivation peptide. Single channels were incorporated into the bilayer under identical conditions of +20 mV and 200 µM free calcium: o, open channel current level; c, closed channel current level. Increasing amounts of calcium and peptide were added to the cis/cytoplasmic face of the channel. A, top: single wild-type mSlo channel at +20 mV in the presence of 200 µM free calcium (Po = 0.96, 6,592 events in 300 s). Middle: same channel in the presence of 100 mM calcium (Po = 0.98, 6,990 events in 300 s). Bottom: same channel in the presence of 100 mM calcium plus 100 µM Shaker inactivation peptide (Po = 0.89, 30,303 events in 600 s). B, top: single triple mutant mSlo channel incorporated into the bilayer at +20 mV in the presence of 200 µM calcium (Po = 0.01, 1,634 events in 300 s). Middle: same channel in the presence of 100 mM calcium (Po = 0.71, 63,625 events in 600 s). Bottom: channel in the presence of 100 mM calcium plus 100 µM Shaker inactivation peptide (Po = 0.56, 48,475 events in 400 s).

Patch electrodes were pulled from Jencons glass and coated with silicone elastomer (Sylgard; Corning, Midland, MI) before fire polishing. Pipette resistances were between 2 and 4 MOmega . Macroscopic currents were recorded from inside-out patches at room temperature, using an EPC-7 amplifier (LIST-Medical, Darmstadt, Germany) (Hamill et al. 1981). The current signal from the EPC-7 was filtered with a corner frequency of 5 kHz using an eight-pole Bessel filter (Frequency Devices 902PF, Haverill, MA) before acquiring it on a DAP800/2 acquisition board (Microstar Laboratories). Data were analyzed and voltage protocols were generated using Matlab software (Mathworks, Natick, MA). Inside-out patches were tested with depolarizing pulses from a holding potential of -150 or -100 mV. The depolarizing pulse potential ranged from -140 to +200 mV, and the pulse was 10-100 ms in duration. Most patches were tested with >10 different voltage protocols at each calcium concentration. Solutions containing different calcium concentrations were applied to the patches by gravity perfusion through a linear array of six microcapillary tubes (Drummond Microcaps, Fisher Scientific). The perfusion system allowed for repeated application of solutions per patch. Liquid junction potential correction was not made because pipette and bath solutions were identical except for free calcium concentration. The ratio between seal resistance (1-10 GOmega ) and the magnitude of calcium-dependent current (0.5-5 nA) was large, therefore no leak subtraction was made. The half activation point and the slope factor were determined by fitting normalized conductances, calculated from peak tail currents at each test potential, to a Boltzmann function: G = {1 + exp[-(Vm - V0.5)/K]}-1, where G = conductance, Vm = membrane potential, V0.5 = half-maximal activation voltage, and K = slope factor, the voltage required for an e-fold change in conductance.

Peptides

All peptides were synthesized by a commercial facility, either the Brandeis University peptide facility (Waltham, MA) or Bio-Synthesis (Lewisville, TX). Before use, the peptides were purified using reverse phase high-performance liquid chromatography. Purified peptides were resuspended in 20 mM HEPES and 100 mM NaCl, pH 7.4.

    RESULTS
Abstract
Introduction
Methods
Results
Discussion
References

Shaker inactivation peptides block wild-type mSlo and an S4-S5 loop "triple mutant" mSlo

The Shaker inactivation peptide blocks the mSlo channel in a concentration dependent manner and produces two blocks, a short block (tau  = 2.6 ms) with a Kd of 412 µM and a long block (tau  = 15 ms) with a Kd of 1.4 mM. These blocks can be distinguished further by their voltage dependence and sensitivity to tetraethylammonium (data not shown). To investigate a possible role for the S4-S5 loop as a peptide binding site, we simultaneously mutated to alanines the three conserved amino acids in mSlo (T256, S259, and L262) that would correspond to a Shaker-like receptor site (Fig. 1). The T256A+S259A+L262A mSlo construct will be referred to as the "triple mutant" mSlo channel throughout this paper. The resulting channel has the same single channel conductance as wild-type mSlo (~300 pS at +20 mV and 400 mM symmetrical potassium), is sensitive to NSOO4, a compound that specifically opens "maxi K" channels (McKay et al. 1994), and is blocked by nanomolar amounts of Iberiotoxin (Galvez et al. 1990) added to the external face of the channel. However, its gating characteristics are very different from the wild-type mSlo channel. Figure 2, top, shows a wild-type mSlo channel (A) and a triple mutant mSlo channel (B) reconstituted under control conditions (single channels, +20 mV, and 200 µM free calcium). The wild-type channel has a very high open probability (Po) under these conditions, >0.95. Under the same conditions, the triple mutant channel has a Po of 0.01. At such a low Po, channel block of the triple mutant channel would be difficult to observe and analyze. However, in the presence of millimolar concentrations of calcium, the triple mutant channel's open probability increases (Fig. 2B, middle). Figure 2, bottom, shows the effect of adding 100 µM Shaker inactivation peptide to the cytoplasmic side of a single wild-type (A) or triple mutant (B) mSlo channel at +20 mV and in the presence of 100 mM calcium. The wild-type mSlo channel shows a decrease in Po, from 0.98 to 0.89, after the addition of 100 µM peptide (Fig. 2A, bottom), and the mean open time decreases from 82 to 18 ms. These values are similar to those observed at 200 µM calcium, indicating that millimolar calcium concentrations do not inhibit the peptide's ability to block the channel and therefore the calcium and peptide binding sites do not overlap. After the addition of 100 µM peptide, the triple mutant channel's Po decreases from 0.71 to 0.56. An estimate of the Kd of the inactivation peptide for the triple mutant channel of 373 µM was made from the change in Po with peptide concentration. Dwell time histograms (data not shown) show that application of 100 µM wild type peptide to a triple mutant channel produces a short blocking event of ~3 ms, as seen by a 30% increase in the number of events contributing to this time constant, and that the mean open time decreases from 6.56 to 4.44 ms. Using time constants obtained from the dwell time histograms to estimate kon and koff for the short blocking event, the peptide's affinity for the triple mutant channel is estimated to be 456 µM (see Table 1 for parameters).

 
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TABLE 1. Kinetic parameters of peptide block


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FIG. 3. Triple mutant mSlo is blocked by the increase in charge peptide. A: single channel current traces. Top: from a single wild-type mSlo channel (left) and a single triple mutant channel (right) incorporated into the bilayer. Wild-type channel is exposed to 200 µM calcium on its cytoplasmic side, and the triple mutant channel is exposed to 100 mM calcium. Both experiments were done at +20 mV. Bottom: effects of adding 20 µM increase in charge peptide to the cytoplasmic face of the channels. B: effect of increasing peptide concentration on channel open probability (Po). Experimental points from 1-4 experiments (means ± SE) were fitted to the equation: Po = Kd/(Kd + [peptide]N), where Kd is the dissociation constant of the peptide, N is the Hill coefficient and [peptide] is the concentration of the peptide. Following values for the concentration of peptide needed to obtain half-maximal blockade were obtained: Kd = 12 µM for the wild-type (bullet ) channel and Kd = 25 µM for the triple mutant (black-diamond ) channel. C: concentration dependence of association and dissociation rates. 1/tau open increases with increasing peptide concentration and the slope of the line gives an on rate for the wild-type (bullet ) channel of 4.8 ± 0.47 µM-1 s-1 and for the triple mutant (black-diamond ) channel of 4.7 ± 0.3 µM -1 s-1. 1/tau block does not change significantly with increasing peptide concentration; slope = -0.0003 ± 0.0001 for wild-type (open circle ) and 0.00002 ± 0.0007 for the triple mutant (diamond ) channel. Time constants were obtained from dwell time histograms from 1-4 experiments.


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FIG. 4. Increase in charge and hydrophobicity peptide blocks wild-type and triple mutant mSlo. A: top line shows a single wild-type mSlo channel current trace recorded at +20 mV in the presence of 200 µM calcium. Subsequent traces show the effect of adding 2 µM increase in charge and hydrophobicity peptide to the cytoplasmic side of the channel. Note that these traces have a longer time scale than previous figures, displaying 30 s of data in each line. B: top trace shows 2 triple mutant channels incorporated into the bilayer at +20 mV and 100 mM calcium. Subsequent traces show the same channels in the presence of 2 µM peptide.

Experiments with mutant peptides have shown that changing the charge or hydrophobicity of the peptide can affect its blocking characteristics. It has been reported that increasing the charge of the peptide increases its affinity for potassium channels by increasing its association rate (Murrell-Lagnado and Aldrich 1993a; Toro et al. 1994). We synthesized a peptide (MAAVAGLYGLGKKRQHRKKQ) that contains a greater degree of positive charge (more than +6) than the wild-type peptide (more than +2) and also found that it had a greater affinity for mSlo. Figure 3A, bottom, shows the effect of adding 20 µM of this peptide to the cytoplasmic face of a wild-type (left) or triple mutant (right) mSlo channel. A dose response curve for this peptide's effect on the open probability of both wild-type and triple mutant mSlo channels is included in Fig. 3B. Fitting the data in Fig. 3B shows that the increase in charge peptide blocks the wild-type channel with a Kd of 12 µM and the triple mutant channel with a Kd of 25 µM. The on rate (kon) for the most abundant blocking event produced by this peptide for both the wild-type and triple mutant channels, obtained from a plot of 1/tau open versus peptide concentration (Fig. 3C), is 4.8 µM-1/s. 1/tau block (koff) does not change with increasing peptide concentration (Fig. 3C) for either the wild-type or triple mutant channel, indicating that the peptide binds to both channels by a simple bimolecular reaction. The peptide does exhibit a slightly faster off rate from the triple mutant channel than from the wild-type channel (Table 1). Defining Kd as koff/kon, using the values obtained from Fig. 3C, the increase in charge peptide blocks the wild-type channel with an affinity of 19 µM and the triple mutant channel with an affinity of 26 µM (values similar to those obtained from the plot of Po vs. peptide).

The degree of hydrophobicity of the peptide influences its dissociation rate (Murrell-Lagnado and Aldrich 1993a; Toro et al. 1994). Toro et al. (1994) described a peptide that incorporates both an increase in positive charge and hydrophobicity, producing long-blocking events of the calcium-dependent potassium channel from porcine coronary artery smooth muscle. We synthesized a similar peptide (MVVVVGLFGLGKKRQHRKKQ). Figure 4A shows the activity of a single wild-type mSlo channel in the presence of 2 µM increase in charge and hydrophobicity peptide. Increasing both the charge and the hydrophobicity of the peptide yields a peptide that produces very long blocks with a duration of ~15 s, ~50-fold longer than any of the other blocking events we observed. This peptide does not produce any type of short flickery block that can be distinguished from normal channel gating (at concentrations as high as 15 µM). The on rate for the increase in charge and hydrophobicity peptide is 0.015 µM-1 s-1, indicating that these blocks are rare. The Kd for the long block produced by this peptide is 4 µM. The increase in charge and hydrophobicity peptide also blocks the triple mutant channel, and as shown in Fig. 4B and Table 1, its effects are similar to those on wild-type mSlo. Thus we have tested three different peptides with very different properties and have found that all of them block both wild-type and triple mutant channels. The results suggest that the S4-S5 loop region may play some subtle role in the channel's interactions with the peptides, but more detailed kinetic analysis and further mutational experiments are needed to determine its precise role.


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FIG. 5. Effect of calcium on triple mutant mSlo. A: calcium increases the open probability of triple mutant mSlo. Top line shows a current trace from a single, triple mutant mSlo channel, initially incorporated into the bilayer at +20 mV, 200 µM free calcium (Po = 0.01). Following traces show the same channel in the presence of 10, 50, and 100 mM calcium (Po = 0.25, 0.62, and 0.71, respectively). B: calcium increases mean open time. Mean open times (tau open) at the indicated calcium concentrations were obtained from dwell time histograms from 1-4 experiments fit with a single exponential function. C: closed dwell time histograms. Left: closed time distribution at 200 µM calcium; 1,419 events were obtained in 300 s and used to construct the histogram. Po was 0.003 and the mean closed time 203 ms. Right: closed time distribution at 50 mM calcium. Under these conditions, 24,258 events were obtained in 600 s for the histogram. Po was 0.88 and the mean closed time 1.74 ms.

Effect of calcium and voltage on triple mutant mSlo

In view of the very different gating properties of the wild-type and triple mutant channels (Fig. 2, A and B, top traces), we examined the effects of different calcium concentrations on channel gating. Figure 5A shows a single channel current trace of a triple mutant channel incorporated into the bilayer under control conditions (+20 mV and 200 µM free calcium). At 200 µM calcium, the channel's Po is 0.01. The Po increases to 0.25 at 10 mM calcium, then to 0.58 at 50 mM and to 0.75 at 100 mM. Kinetic analysis of the triple mutant channel shows that increasing calcium causes an increase in the channel's mean open time (Fig. 5B). Figure 5C shows closed dwell time histograms from a triple mutant channel obtained at 200 µM (left) and 50 mM (right) calcium. The closed dwell time histograms are fit with four exponentials at all of the calcium concentrations tested. The distribution of the four time constants changes with increasing amounts of calcium. Increasing calcium concentration increases the channels' Po by increasing the number of the shortest closed events while decreasing the number of longer closed events. Similar experiments with the wild-type channel show that increasing the calcium concentration from 200 µM to millimolar levels does not significantly change the gating kinetics of the channel at +20 mV. Dwell time histograms from wild-type channels at 200 µM calcium are quantitatively similar to those obtained from triple mutant channels at 100 mM calcium.


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FIG. 6. Effect of calcium on macroscopic mSlo currents. Macroscopic currents from inside-out patches of HEK293 cells transiently transfected with wild-type (A) or triple mutant (B) mSlo. Currents were elicited by 100-ms depolarizing voltage steps from a holding potential of -100 mV to pulse potentials from -80 mV to +80 mV, in 20-mV increments. Inside-out patches were exposed to intracellular free calcium concentrations ranging from 0.3 to 10 µM.

The calcium sensitivity of the triple mutant channel also was investigated at the macroscopic current level using inside-out patches detached from pcDNA3-mSlo transfected HEK293 cells. Figure 6 shows the effects of increasing amounts of calcium on macroscopic current traces from patches containing similar numbers of wild-type (A) or triple mutant (B) mSlo channels. Wild-type mSlo shows a rapidly activating current in response to depolarization, which is enhanced at higher calcium concentrations. Using the same voltage protocol, the triple mutant mSlo channels require ~10-fold higher calcium concentrations to elicit a similar magnitude of steady state macroscopic current.

One possible explanation for this difference between the wild-type and triple mutant channels is a perturbation of the normal activation or deactivation mechanisms of channel gating. Figure 7, A and B, shows the activation time constants obtained from both wild-type and triple mutant channels at 10 (A) and 50 µM (B) calcium. The activation time constants show a similar voltage dependence, decreasing with increasingly depolarized membrane potential, although the triple mutant activation time constants consistently show a rightward shift. Figure 7C shows the deactivation time constants derived from tail currents of wild-type channel recordings. The deactivation time constants increase as calcium is increased as would be expected for a calcium dependent channel. However, as seen in Fig. 7D, the deactivation time constants for the triple mutant channels do not show a similar calcium dependence. The deactivation time constant is independent of calcium concentration and is comparable with that seen with wild-type channel currents at nominally zero calcium.


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FIG. 7. Activation and deactivation parameters for the wild-type and triple mutant mSlo channels. A and B: activation kinetics of wild-type and triple mutant mSlo macroscopic currents. Macroscopic currents were obtained at 10 µM (A) and 50 µM (B) calcium as described in Fig. 6, in the voltage range of -10 to +100 mV for wild-type (bullet ) and +60 to +100 mV for triple mutant (black-diamond ) channels. Activation time constants (tau activation) were derived from exponential fits of the current traces obtained by stepping to the indicated voltages. Each point represents the mean ± SE for 3-20 values. C: wild-type channel deactivation kinetics are calcium dependent. Macroscopic currents were elicited at +100 mV, as described in Fig. 6, at 0, 1, 10, and 50 µM calcium. Deactivation time constants (tau deactivation) were derived by exponential fitting of tail currents. Points represent the means ± SE obtained for 334-550 values. D: triple mutant channel deactivation kinetics are not affected by calcium. Deactivation time constants were obtained from fits to tail currents after steps to +100 mV with 1, 10, 50 and 200 µM calcium. Points represent the means ± SE for 248-337 values.

mSlo channels observed in the two different recording configurations consistently showed a different calcium sensitivity. The mSlo channels expressed in COS cells and observed in the bilayer configuration needed ~100-fold more calcium for activation than the mSlo channels expressed in HEK293 cells and observed in the detached patch configuration. This shift in calcium sensitivity also is seen for the mSlo mutant channels. Figure 8 compares the voltages required for half-maximal activation of the wild-type and triple mutant mSlo channels in the two different recording configurations, as a function of calcium concentration. In the bilayer configuration (open symbols in Fig. 8), the calcium concentration at which activation is half-maximal differs by ~100-fold between the wild-type and triple mutant channels. A similar trend is seen in patch recordings (closed symbols in Fig. 8). Despite the differences in calcium sensitivity in the two different recording configurations, the triple mutant channel has a decreased calcium sensitivity in both configurations, which is observed as an increase in the voltage required for half-maximal activation at each calcium concentration tested.


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FIG. 8. Half-maximal activation voltage is shifted in the triple mutant mSlo channel. Voltage required for half-maximal activation (V1/2) of both the wild-type and triple mutant mSlo channel types, in the 2 different recording configurations, is shown. Steady state V1/2 values from macroscopic currents were obtained from plots of G/Gmax vs. voltage for the wild-type (bullet ) and the triple mutant (black-diamond ) mSlo channels. V1/2 values from single channel data, obtained in the bilayer configuration, were obtained from plots of Po vs. voltage for the wild-type (open circle ) and triple mutant (diamond ) channels, at the calcium concentrations indicated. Points represent means ± SE.

Single S4-S5 loop mutants

To investigate how each of the mutated amino acids in the triple mutant channel contributes to its phenotype, we constructed the corresponding single amino acid mutants (T256A, S259A, and L262A). Figure 9A shows single channel current traces from the mSlo wild-type and mutant channels reconstituted in the bilayer configuration under conditions of +20 mV and 200 µM free calcium. Under these conditions, the wild-type and S259A mSlo channels (bottom 2 traces in Fig. 9A) look very similar. Kinetic analysis could not distinguish any significant differences in their time constants and distributions under control conditions (Table 2). The L262A and triple mutant channels are also phenotypically similar under these conditions (top 2 traces in Fig. 9A), and Table 2 shows that the durations and distributions of the time constants used to fit the data from these two channels are very similar. The L262A mutation shows that changing a single amino acid in the S4-S5 loop can have dramatic effects on the channel's gating. The T256A mSlo channel has a phenotype between the two extremes. This single-channel analysis indicates that the amino acid at position L262 contributes the most to the triple mutant channel's phenotype. Figure 9B shows macroscopic current traces from the five different channels in the presence of 1 µM free calcium. These traces reflect the same trend for channel activity that is observed in the bilayer configuration.


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FIG. 9. S4-S5 loop mutant channels. A: single channels, from COS cell membrane preparations, were incoporated into the bilayer under identical conditions (+20 mV, 200 µM free calcium). Top line shows a single channel current trace from a triple mutant mSlo channel; subsequent lines show a current trace from a L262A mSlo channel, a T256A mSlo channel, a S259A mSlo channel, and a wild-type mSlo current trace, respectively. B: examples of macroscopic current responses elicited from triple mutant mSlo, the 3 single mutants (L262A, T256A, and S259A), and wild-type mSlo in patches from transfected HEK293 cells. Currents were elicited by a depolarizing pulse to +50 mV from a holding potential of -100 mV, at an intracellular calcium concentration of 1 µM. Current traces were normalized by dividing them by the maximum current level of each patch. Maximum current levels were calculated using the mean total conductance for each patch during a depolarization to +50 mV. Mean total conductance of each patch was determined by fitting Boltzmann equations to G-V curves at saturating conductance.

 
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TABLE 2. Time constants and distributions for the wild-type and mutant mSlo channels

The effects of calcium and voltage were measured for all of the channels using the bilayer recording configuration (Fig. 10). Figure 10A shows how the channels' Po values change with increasingly depolarized voltage in the bilayer configuration. The wild-type and S259A channel activities are measured at a concentration of 10 µM free calcium, and the other mutant channel activities were observed at a concentration of 200 µM free calcium. At these calcium concentrations, the different mutant channels have similar maximum open probabilities. The channels have voltage dependences ranging from 9.2 mV for the S259A channel to 16.7 mV for the triple mutant channel per e-fold change in Po. The slope factor originally reported for the mSlo channel was ~15 mV per e-fold change in Po at 100 and 10 µM calcium (Butler et al. 1993). Slope factor values have been reported from 8 mV, for the rat brain channel incorporated into bilayers and channels of a pituitary cell line (Lang and Ritchie 1990; Reinhart et al. 1991), to 39 mV for "maxi K" channels in myocytes (Markwardt and Isenberg 1992). Figure 10B shows the effect of increasing calcium concentration on the channels' Po. At a holding voltage of +20 mV, the wild-type and S259A mSlo channels show a similar calcium dependence, with a calcium concentration of ~40 µM necessary for half-maximal activation and a Hill coefficient of ~3. The triple mutant channel calcium activation curve is shifted ~1,000-fold to the right at +20 mV as compared with that of the wild-type channel. Fitting the data for the triple mutant channel shows that a calcium concentration of 14.6 mM is needed for half-maximal activation and a Hill coefficient of <1 is observed. The activation curves of the T256A and L262A mutant channels are shifted ~10- and 100-fold to the right as compared with the wild-type channel and give Hill coefficients between the wild-type and triple mutant channels (for T256A, n = 1.8 and for L262A, n = 0.9). Hill coefficients for calcium binding to maxi K type, calcium-dependent potassium channels have been reported ranging from 1-2 (Moczydlowski and Latorre 1983) to 3-6 (Magleby and Pallotta 1983). The mutation at position L262 contributes the most to the triple mutant channel's phenotype, although calcium and voltage activation curves indicate that the simultaneous mutation of all three amino acids does have an additive effect. The dramatic shift in the calcium activation curve of the triple mutant channel suggests that the S4-S5 loop plays an important role in the calcium-sensing machinery of the channel.


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FIG. 10. Effect of calcium and membrane potential on mSlo channels. A: open probability was measured in the range of +20-50 mV at 10 µM free calcium for the wild-type (bullet ) and S259A (black-square) channel types and at 200 µM free calcium for the T256A (black-triangle), L262A (black-down-triangle ), and triple mutant (black-diamond ) channel types. Po of the channel can be related to its voltage dependence by the Boltzmann relationship: Po = [1 e-K(V-V0.5)]-1 such that the voltage sensitivity, K, is given by the slope of the line: ln [Po/(1 - Po)] vs. voltage. Fitting the data with a straight line, the following values were obtained for the slope factor: K = 10.2 mV per e-fold change in Po for the wild-type channel, 9.2 mV for the S259A channel, 13.9 mV for the T256A channel, 11.9 mV for the L262A channel, and 16.7 mV for the triple mutant channel. B: S4-S5 loop mutant channels show a decreased calcium sensitivity. Po was measured at the indicated calcium concentrations at +20 mV in the bilayer configuration. Experimental points were fit with the equation Po = Pmax/{1 + [(K/[calcium])N]}, where K is the equilibrium binding constant and N is the Hill coefficient for calcium binding. Following parameters were obtained, using Pmax = 1.0: n = 2.7 and K = 44 µM for wild-type (bullet ) mSlo; n = 3.5 and K = 35 µM for S259A (black-square) mSlo; n = 1.8 and K = 150 µM for T256A (black-triangle) mSlo; and using Pmax = 0.8, n = 0.89 and K = 2.2 mM for L262A (black-down-triangle ) mSlo and n = 0.97 and K = 14.6 mM for triple mutant (black-diamond ) mSlo.

    DISCUSSION
Abstract
Introduction
Methods
Results
Discussion
References

We have found that the Shaker inactivation peptide can block mSlo, a cloned calcium-dependent potassium channel. The blocking events that we describe for mSlo include a short block with a time constant of 2.6 ms and a Kd of 412 µM, and a less frequently occurring long block with a time constant of 15 ms and a Kd of 1.4 mM. The peptide also produces two types of block of the Type I calcium-dependent potassium channel from rat brain (Foster et al. 1992). The short block described for the rat brain channel has a time constant of 16 ms and a Kd of 8 µM, and the longer block has a time constant of 160 ms and a Kd of 275 µM. The peptide also blocks the calcium-dependent potassium channel from porcine coronary artery smooth muscle (Toro et al. 1992), with a time constant of 4 ms and a Kd of 95 µM. Another cloned, calcium-dependent potassium channel, dSlo, has been expressed in oocytes and its activity observed using the planar lipid bilayer configuration (Perez et al. 1994). Under these conditions, the Shaker peptide blocks dSlo with a Kd of 480 µM, an affinity similar to that described here for the short block of mSlo. Thus the wild-type Shaker peptide is able to produce a number of blocking events of differing affinities with various types of calcium-dependent potassium channels, ranging from 8 µM for the short blocking event with the rat brain channel to 412 µM for mSlo---a difference of ~50-fold. All of the experiments described in the preceding text were done using the planar lipid bilayer setup. The primary difference among these experiments is the source of the channels---native preparations from rat brain and porcine coronary artery versus cloned channels expressed in heterologous expression systems. beta  subunits for calcium-dependent potassium channels have been described (Dworetzky et al. 1996; Garcia-Calvo et al. 1994). Coexpression of mSlo (a pore forming alpha  subunit) with the cloned bovine beta  subunit produces channels that show an increased sensitivity to calcium and voltage and an increased sensitivity to a drug, DHS-1 (McManus et al. 1995). Thus it is possible that the presence or absence of auxiliary channel subunits or cytoskeletal elements could account for some of the differences in the Shaker peptide's affinity for calcium-dependent potassium channels from different sources. The differing affinities of the peptide for the various calcium-dependent potassium channels suggest that the precise structural characteristics of the receptor for the peptide may vary among different channels.

To investigate further the interaction between the peptide and the channel, we mutated amino acids that might contribute to the peptide's receptor site. Several residues in the S4-S5 loop of the Shaker channel have been implicated as being important for N-type inactivation and ion conduction (Holmgren et al. 1996; Isacoff et al. 1991). Five amino acids were identified that, when mutated, abolished or slowed fast inactivation [T388, S392, E395, L396 (Isacoff et al. 1991), and A391 (Holmgren et al. 1996)]. These results implied that these amino acids might be part of the receptor site for the fast inactivation domain. The T, S, and L residues are conserved in the S4-S5 loop of mSlo, athough two critical residues identified as being part of the receptor in Shaker, A391 and E395 (Holmgren et al. 1996; Isacoff et al. 1991), are not conserved in mSlo. Experiments with peptides of varying charge and hydrophobicity also suggested that the characteristics of the binding site would be similar between the Shaker and calcium-dependent channels, consisting of a negatively charged site and a hydrophobic pocket (Murrell-Lagnado and Aldrich 1993a,b; Toro et al. 1994). However, mutating the amino acids conserved in the S4-S5 loop of mSlo produces only subtle changes in the channel's susceptibility to peptide block, suggesting the involvement of other amino acids or regions of the channel in the receptor site.

In mutating residues in the S4-S5 loop of the mSlo channel, we found that simultaneously changing three amino acids (T256, S259, and L262) to alanines had dramatic effects on the channel's properties. Our experiments show that the triple mutant channel has a decreased calcium sensitivity, suggesting that the S4-S5 loop plays a role in the calcium-sensing or calcium-signal transduction mechanisms of the mSlo channel. In our experiments, the wild-type mSlo channel has a single channel conductance of ~300 pS at 400 mM symmetrical potassium (data not shown) and is open >95% of the time at +20 mV and 200 µM free calcium. Under identical experimental conditions, the triple mutant channel displays a similar single channel conductance, but has very different gating kinetics and is open <1% of the time. A similar shift in channel activity is seen at the macroscopic current level in membrane patches, even though the mSlo channels observed in the patch configuration show a higher calcium sensitivity than those observed in the bilayer configuration. Our single mutant analysis showed that changing only one amino acid, L262, was sufficient to dramatically alter the gating characteristics of the channel. However, all three amino acids need to be mutated to account for the full shift in Hill coefficient and voltage dependence, suggesting that the combination of these three amino acids makes an important contribution to the channel's ability to respond to calcium. Fifty to 100-fold higher calcium concentrations are needed to activate the triple mutant channel, suggesting that the S4-S5 loop plays an integral part in relaying the message between calcium binding and channel gating. Similar observations, of a 100-fold shift in calcium sensitivity resulting from mutations in the S4-S5 loop, have been reported for the hSlo channel (Krause et al. 1996).

The location of the calcium binding site on calcium-dependent potassium channels is still unknown, although there is evidence that the C-terminal portion of the Slo channels confers calcium sensitivity (Wei et al. 1994). Our experiments are consistent with a simplified model for mSlo gating as proposed previously (Blatz and Magleby 1987)
C ⇄ CCa<SUP>2+</SUP>⇄ CCa<SUP>2+</SUP><SUB>2</SUB>⇄ CCa<SUP>2+</SUP><SUB>3</SUB>⇄ OCa<SUP>2+</SUP><SUB>3</SUB>⇄ OCa<SUP>2+</SUP><SUB>4</SUB>
where multiple calcium ions bind to the channel in a cooperative manner and calcium binds to and stabilizes the open state of the channel. Such a scheme can account for our observations for the wild-type channel where the Hill coefficient for calcium binding is >3. This model assumes that the channel does not open without calcium bound, and that the open channel can bind additional calcium ions that stabilize the open state of the channel. The Hill coefficient for the triple mutant channel is <1, which implies that the calcium binding sites are decreased in number or now act independently. Furthermore, with the wild-type channel, deactivation is a calcium-dependent process. Calcium stabilizes the open state, as observed by the increase in the deactivation time constant with increasing calcium concentration. However, the triple mutant channel's deactivation kinetics are independent of calcium. This channel closes at a constant rate, regardless of the calcium concentration, suggesting that the state to which calcium normally binds in the open configuration is no longer available. These data are consistent with the idea that one of the mSlo calcium binding sites is available only when the channel is open and that this site is missing or remains unavailable in the triple mutant channel. The S4-S5 loop is well situated to participate in channel gating because it is adjacent to the S4 region of the channel, and amino acids within the S4-S5 loop have been shown to affect the voltage-dependent gating of the Shaker channel (McCormack et al. 1994).

The relation between calcium and voltage in activating calcium-dependent potassium channels remains unclear. DiChiara and Reinhart (1995) showed recently that the effects of calcium and voltage on dSlo and hSlo can be separated. In these experiments, they showed that calcium and voltage modulate distinct kinetic states and that calcium is the predominant signal that regulates activation and deactivation kinetics. Chemical modifications of calcium-activated potassium channels have been reported that seem to disrupt the interactions of calcium and voltage on these channels (Cornejo et al. 1987; Pallotta 1985; Salomao et al. 1992). Recent experiments have suggested that Slo channels can be activated independently of calcium at very depolarized voltages (Cox et al. 1997; Cui et al. 1997; Meera et al. 1996; Stefani et al. 1997). Experiments focusing on hSlo gating currents suggest that these channels actually can operate in two modes, a calcium-independent mode in which channel opening is strictly a voltage-dependent process and a calcium-dependent or modulated mode in which calcium acts to switch the channel into a conformational state where less voltage is required for opening (Stefani et al. 1997). Furthermore, mSlo channels can be activated to the same maximal open probability over a number of calcium concentration ranges indicating that calcium is not the limiting factor for the channels to be activated by voltage (Cox et al. 1997). Other experiments using mSlo show that most of the voltage dependence of channel activation can be attributed to movement of charges intrinsic to the channel and not to voltage dependent calcium binding (Cui et al. 1997). These experiments support a model in which at low or zero calcium, Slo channels can open in response to strong depolarization, and at higher calcium concentrations, calcium acts to facilitate channel opening. The mSlo mutants described in this paper show a decreased sensitivity to calcium and hence may serve as valuable tools for discriminating the effects of voltage and calcium on these channels.

    ACKNOWLEDGEMENTS

  We are grateful to L. Salkoff for providing the mSlo cDNA, A. Stewart for computer programming, and D. Naranjo for helpful discussions.

  This research was supported by a National Institutes of Health grant to I. B. Levitan.

    FOOTNOTES

   Present address of D. A. Sullivan: Department of Neurobiology, Harvard Medical School, Boston, MA 02115.

  Address reprint requests to I. B. Levitan.

  Received 23 May 1997; accepted in final form 1 August 1997.

    REFERENCES
Abstract
Introduction
Methods
Results
Discussion
References

0022-3077/97 $5.00 Copyright ©1997 The American Physiological Society