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Abstract

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  • 1
    The large conductance, calcium-sensitive K+ channel (BKCa channel) is a unique member of the K+-selective ion channel family in that activation is dependent upon both direct calcium binding and membrane depolarization. Calcium binding acts to dynamically shift voltage-dependent gating in a negative or left-ward direction, thereby adjusting channel opening to changes in cellular membrane potential.
  • 2
    We hypothesized that the intrinsic calcium-binding site within the BKCa channel α subunit may contain an EF hand motif, the most common, naturally occurring calcium binding structure. Following identification of six potential sites, we introduced a single amino acid substitution (D/E to N/Q or A) at the equivalent of the -z position of a bona fide EF hand that would be predicted to lower calcium binding affinity at each of the six sites.
  • 3
    Using macroscopic current recordings of wild-type and mutant BKCa channels in excised inside-out membrane patches from HEK 293 cells, we observed that a single point mutation in the C-terminus (Site 6, FLD923QD to N), adjacent to the ‘calcium bowl’ described by Salkoff and colleagues, shifted calcium-sensitive gating right-ward by 50-65 mV over the range of 2-12 μM free calcium, but had little effect on voltage-dependent gating in the absence of calcium. Combining this mutation at Site 6 with a similar mutation at Site 1 (PVD81EK to N) in the N-terminus produced a greater shift (70-90 mV) in calcium-sensitive gating over the same range of calcium. We calculated that these combined mutations decreased the apparent calcium binding affinity ≈11-fold (129.5 μM vs. 11.3 μM) compared to the wild-type channel.
  • 4
    We further observed that a bacterially expressed protein encompassing Site 6 of the BKCa channel C-terminus and bovine brain calmodulin were both able to directly bind 45Ca2+ following denaturation and polyacrylamide gel electrophoresis (e.g. SDS-PAGE).
  • 5
    Our results suggest that two regions within the mammalian BKCa channel α subunit, with sequence similarities to an EF hand motif, functionally contribute to the calcium-sensitive gating of this channel.

Potassium channels form a large family of ion-selective pores that are found in both excitable and non-excitable cells. The large conductance, calcium-sensitive (maxi-K or BKCa) K+ channel is a unique member of this family in that channel opening probability is increased by both membrane depolarization and direct calcium binding. Although BKCa channels can be gated by voltage alone (Cui et al. 1997; Stefani et al. 1997; Horrigan et al. 1999), calcium binding is critical, as it ‘shifts’ voltage-dependent gating left-ward along the voltage axis, thereby allowing channel opening to occur at increasingly negative membrane potentials (Cui et al. 1997; Cox et al. 1997; Rothberg & Magleby, 1999, 2000; Jones, 1999). As a result of this gating behaviour, BKCa channels may act as ‘coincidence detectors’, and dampen excitatory events by hyperpolarizing the membrane potential in response to stimuli causing elevation of intracellular free calcium and membrane depolarization. At the neuromuscular junction, for example, blocking BKCa channels leads to an increase in neurotransmitter release in response to nerve stimulation (Robitaille & Charlton, 1992; Robitaille et al. 1993). It thus appears that the identification and characterization of the channel's putative calcium sensor is essential to our understanding of the molecular mechanisms by which BKCa channels contribute to the regulation of membrane excitability and cellular function.

Recent experiments combining heterologous expression and electrophysiological recordings of cloned mammalian (Bulter et al. 1993; Pallanck & Ganetzky, 1994; Tseng-Crank et al. 1994; Wallner et al. 1995; McCobb et al. 1995; Vogalis et al. 1996) and non-mammalian (Atkinson et al. 1991; Adelman et al. 1992; Wei et al. 1996; Jiang et al. 1997; Jones et al. 1998) BKCa channel α subunits have strongly suggested that the channel's calcium sensitivity is imparted by a calcium-binding site or sensor that is intrinsic to the pore-forming α subunit. Towards the goal of identification, we hypothesized that the intrinsic calcium-binding site of the BKCa channel may resemble an ‘EF hand’ motif, as found in prototypic calcium binding proteins, such as calmodulin and troponin C (Falke et al. 1994; Linse & Forsen, 1995). Recently, EF hand motifs have been identified in voltage-gated L-type Ca2+ channels (de Leon et al. 1995; Peterson et al. 2000) and twin-pore K+ channels (Czempinski et al. 1997; Salinas et al. 1999), which appear to contribute to the observed calcium sensitivity of these channels. Based on structure-function studies of calmodulin and troponin C, we introduced mutations into potential EF hand motifs within the BKCa channel α subunit that were designed to ‘impair’ calcium binding without grossly altering overall structure (Linse & Forsen, 1995). The effects of such mutations were assessed electrophysiologically in excised inside-out membrane patches over a wide range of free calcium concentrations (≈5 nm to 500 μm). Based on changes in the half-maximal voltages of activation (V1/2 values) and in the kinetics of channel gating, together with observed direct 45Ca2+ binding to a C-terminal domain, our data suggest that the BKCa channel contains two EF hand motifs, one located in the N-terminus and the other in the C-terminus, adjacent to the ‘calcium bowl’ recently identified by Salkoff and co-workers (Schreiber & Salkoff, 1997; Schreiber et al. 1999). Both motifs appear to functionally contribute to the calcium-dependent gating of this channel.

METHODS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

The cDNA constructs encoding the mouse brain mSloα subunit (Pallanck & Ganetzky, 1994) and green fluorescent protein (GFP) (Chalfie et al. 1994), along with procedures for site-directed mutagenesis and transient transfection of HEK 293 cells have been recently described (Braun et al. 2000; Ling et al. 2000). All mutations were directly confirmed by either single- or double-stranded cDNA sequencing.

Electrophysiology

Macroscopic currents were recorded at 35 ± 0.5 °C from excised inside-out membrane patches of transfected HEK 293 cells using an Axopatch 200B patch clamp amplifier and pCLAMP 6.03 software. BKCa channel currents were activated by voltage clamp pulses delivered from a holding potential of 0 mV to membrane potentials ranging from −180 to 240 mV; tail currents were recorded at +50, −80 or −120 mV. Current traces were filtered at 2-5 kHz (4-pole Bessel filter) and acquired on a Dell Pentium II-based computer at a sampling frequency of 8-10 kHz using a Digidata 1200 analog/digital interface. In some cases, on-line leak subtraction was performed using a -P/5 protocol. Recording micropipettes were pulled from thin-walled borosilicate glass capillaries (1.2 mm i.d., 1.5 mm o.d., World Precision Instruments (WPI), Sarasota, FL, USA) using a Sutter P-89 horizontal electrode puller. Micropipettes were filled with a solution containing (mm): 5 KCl, 140 KOH, 1 MgCl2, 1 CaCl2, 10 Hepes, pH adjusted to 7.3 with methanesulfonic acid, and had tip resistances of 1.5-3.5 MΩ. The bath solution contained (mm): 5 KCl, 140 KOH, 1 MgCl2, 2 EGTA or HEDTA, 10 Hepes; the pH was adjusted to 7.2 with methanesulfonic acid. Based on calculations using the WinMaxC program (Bers et al. 1994), variable amounts of a 0.1 m CaCl2 solution were added to give the desired free calcium concentrations. The level of free calcium in each solution was then independently confirmed using a calcium electrode (Orion model 93-20) with calibration standards (WPI) ranging from pCa 8 to 2. The recording chamber (≈0.3 ml volume) was perfused at a constant rate of 1-1.5 ml min−1, using a set of manually controlled solenoid valves to switch between various solutions.

Transfected HEK 293 cells seeded on coverslips were placed in a temperature-controlled recording chamber on the stage of a Nikon Eclipse TE300 inverted microscope. Individual cells expressing BKCa channels were then identified visually by co-expression of the marker protein GFP under epifluorescence using 480 nm excitation and 510 nm emission filters.

Preparation of GST fusion proteins and 45Ca2+ binding assay

A cDNA fragment, encoding ≈240 amino acids from the C-terminus of the mouse BKCa channel α subunit (747ASNFHY… GATPEL980) was excised using Nhe I and Xho I restriction sites and subcloned in frame with the glutathione-S-transferase (GST) open reading frame of the bacterial expression plasmid pGEX-KG (Guan & Dixon, 1991). Following transformation, exogenous protein expression was induced in a 500 ml bacterial culture by addition of 0.1 mm isopropylthio-β-galactose (IPTG) (final) for 3-4 h at 37 °C. Bacteria were collected by centrifugation at ≈5000 g for 10 min, then stored at −20 °C. The GST-BKCa channel fusion protein was found to be insoluble and was isolated as described by Frangioni & Neel (1993), with minor modifications. Briefly, bacteria were suspended in 10 ml of phosphate-buffered saline containing 0.5 mg ml−1 lysozyme, then incubated on ice for 30 min. The resuspension was frozen at −80 °C for 2 h, thawed at room temperature and sonicated using 10 × 15 s bursts of a probe sonicator. The lysed bacteria were centrifuged, and the pellet was suspended in 15-20 ml of buffer (10 mm Tris-HCl, pH 8.0, 150 mm NaCl, 1 mm EDTA). N-Lauroyl-sarcosine (Sarcosyl) was added to a final concentration of 1 % (w/v) and the bacterial lysate was sonicated for 1 min at 4 °C using a bath sonicator. Triton X-100 was then added to a final concentration of 2-3 % (v/v) and the soluble protein was incubated overnight with ≈0.5 ml of pre-swollen glutathione-agarose beads. Following binding, the BKCa channel domain was cleaved from GST by incubation for 1 h at room temperature in 5 ml of buffer (50 mm Tris-HCl, pH 8.0, 150 mm NaCl, 2.5 mm CaCl2, 0.1 % (v/v) β-mercaptoethanol) containing 4 μg ml−1 thrombin. Proteolysis was stopped by addition of 1 mm phenylmethylsulfonyl fluoride (PMSF) (final) and the soluble material was dialysed in 10 mm NH4HCO3, then concentrated by lyophilization. This above isolation procedure was also carried out for control bacteria expressing only the GST portion of the pGEX-KG plasmid. Protein concentrations were measured using a modified Lowry procedure (Bio-Rad Laboratories).

To perform direct calcium binding, the isolated BKCa channel C-terminal domain was resolved by SDS-PAGE and electrotransferred to 0.2 μm nitrocellulose membrane, and 45Ca2+ binding was carried out as described by Ngai et al. (1987). Briefly, nitrocellulose membranes were washed for 4 × 10 min at room temperature in 100 ml of buffer containing 60 mm KCl, 5 mm MgCl2, 10 mm imidazole HCl, pH 6.8, and then incubated for 10 min in the same buffer containing 45CaCl2 at a final concentration of ≈1 MBq l−1. Unbound 45CaCl2 was removed by washing for 5 min in 200 ml of 50 % ethanol, after which nitrocellulose membranes were dried in a fume hood and then exposed to Kodak X-Omat AR film for 3-6 days in a film cassette equipped with an intensifying screen. The amount of 45Ca2+ binding was quantified using a Gel-Doc apparatus with Quantity One version 4.1.0 image analysis software (Bio-Rad Laboratories).

Data analysis

Pairs of current-voltage relations were recorded at each concentration of free calcium, and were then averaged for subsequent analysis. Normalized conductance-voltage (G-V) relations were calculated from tail current amplitudes measured 0.2-0.3 ms following the step to the tail potential. All G-V relations were fitted with single Boltzmann functions, according to the equation:

  • image

where Vm is the experimental test potential (in volts), V1/2 is the half-maximal voltage of activation (in volts; defined as the membrane potential at which 50 % of the channels are open), z is the valency of the permeable ion, and F/RT= 37.67 V−1 at 35 °C.

The time constants (τ) of deactivation were derived from single exponential fits of macroscopic tail currents recorded at +50, −80 or −120 mV, following voltage clamp steps to potentials producing ≈50 % activation of maximal current amplitude. Exponential fits were performed over a period of 4-5 ms, beginning 0.25-0.3 ms after the step to the tail current potential. Similarly, time constants of activation were determined from single exponential fits to currents over a period of 7-10 ms, beginning 0.25 ms after the start of the voltage clamp step to the indicated membrane potentials.

To estimate equilibrium dissociation constants of calcium binding from plots of half-maximal voltages of activation versus free calcium concentrations (Fig. 7), V1/2 values over the range of 1-40 μm calcium were fitted to the following relationship by linear regression analysis:

image

Figure 7. Gating model and predictions for wild-type and ‘impaired’ BKCa channel α subunits

A, a simplified scheme of voltage- and calcium-dependent BKCa channel gating originally described by Cox et al. (1997). The channel is shown as a tetramer of α subunits which can move between the closed (squares) and open (circles) states in a voltage-dependent manner; ‘L’ represents the equilibrium constant ([Co]/[Oo]) between the closed and open states of the channel. Calcium binding (•) to any subunit in either the closed or open state is governed by the binding constants, Kc and Ko, respectively. As calcium binding to the closed channel increases, the voltage-dependent transition to the open state becomes easier. The equation describing this model is shown below:

where Kc= 12 μm, Ko= 1 μm, Q= 1.2 e, L(0) = 1500 and T= 308 K (35 °C). B, normalized conductance-voltage relations for a wild-type BKCa channel calculated using the above model and a series of free calcium concentrations ranging from 10 nm to 1 mm. The calcium binding constants for the closed and open states have been set at 12 μm and 1 μm, respectively. The values for Q, L and T are the same as those stated above. C, a similar series of calculations carried out for an ‘impaired’ BKCa channel in which calcium binding affinity has been decreased 10-fold for both the closed and open states (i.e. Kc= 120 μm and Ko= 10 μm). D, a plot of the calculated half-maximal voltages of activation (V1/2 values) derived from B and C (open symbols) versus free calcium concentrations (10 nm, 100 nm, 200 nm, 500 nm, 1 μm, 2 μm, 5 μm, 10 μm, 20 μm, 50 μm, 100 μm, 200 μm, 500 μm and 1000 μm); symbols for both wild-type (circles) and ‘impaired’ (squares) BKCa channels are connected by continuous lines. For comparison purposes, the experimental V1/2 values (taken from Fig. 6) for wild-type channels and channels containing the Sites 1 and 6 double mutation have also been plotted (filled symbols) using the same X- and Y-axes.

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  • image

(Cui et al. 1997), where δ is the electrical distance travelled by calcium into the membrane electric field; this value ranged from 0.25 to 0.4.

Values for wild-type and mutant BKCa channels were examined statistically using a one-way analysis of variance (ANOVA); differences between values were considered to be statistically significant at a level of P < 0.05.

RESULTS

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

In the present study, we hypothesized that the intrinsic calcium sensor of the mammalian BKCa channel may contain an element(s) resembling an EF hand motif, which is the most common, naturally occurring calcium binding module thus far identified (Moncrief et al. 1990; Falke et al. 1994). We further reasoned that if such a sensor were present, it would be likely to contain an ‘impaired’ or imperfect EF hand structure to account for the low affinities of calcium binding estimated from single channel activities (10-50 μm) (McManus, 1991). We therefore searched the primary sequences of cloned BKCa channel α subunits using a minimal ‘signature’ pattern of the most common residues found within the EF hand structure (G-X-I/L/V), starting with the conserved Gly residue in the middle of the calcium binding loop (present in > 95 % of identified EF hands (Marsden et al. 1990; Falke et al. 1994)). Positive sequences were then examined for the presence of negatively charged residues that could act as calcium coordinating ligands.

Based on such a screen, we identified six potential EF hand-like motifs in the BKCa channel α subunit, the positions and sequences of which are depicted in Fig. 1. Of these sites, 1 and 6 appear to have the highest similarity to bona fide calcium binding, EF hand sequences. Although Site 6 was initially identified in the Drosophila BKCa channel, we were surprised to find that the conserved Gly is replaced by a Thr residue in Site 6 of all mammalian BKCa channel α subunits (refer to green T in Fig. 1B), and by a Ser in BKCa channels from chick (Jiang et al. 1997; Rosenblatt et al. 1997), turtle (Jones et al. 1998) and nematode (Wei et al. 1996). This discrepancy may be resolved by the fact that both Thr and Ser are found as naturally occurring substitutions of this critical Gly, based on a survey of 165 bona fide EF hand-containing proteins (Marsden et al. 1990).

image

Figure 1. Sites of potential ‘EF hand’ motifs in the BKCa channel α subunit

A, cartoon of the BKCaα subunit showing the positions of potential EF hand motifs relative to the channel's predicted topology. B, alignment of BKCa channel α subunit sequences from mouse (mSlo) containing the 27 amino acid exon at splice site 4 also reported in human (Tseng-Crank et al. 1994; McCobb et al. 1995), rat (Ha et al. 2000) and Drosophila (dSlo) with the third EF hand motif from human calmodulin (CaM 84-111) and an EF hand consensus sequence (Stryer, 1995). ‘O’ and ‘n’ denote oxygen-containing (i.e. D, E, N, Q, S, T) and non-polar residues, respectively. Residues acting as calcium coordinating ligands have been highlighted in red (Stryer, 1995). The ligands are denoted by both their positions within the predicted loop (i.e. indicated by numbers 1, 3, 5, 7, 9, 12) and their calcium coordinating positions using conventional notation (i.e. x, y, z, -y, -x, -z). Note that the -z position (residue 12 of the loop) acts as a bidentate ligand and contributes two side-chain oxygens towards calcium binding. The ‘calcium bowl’ overlapping Site 6 has been underlined for both mSlo and dSlo.

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To test these predictions experimentally, we took advantage of the detailed crystallographic and biochemical information describing the importance of individual residues within the EF hand helix-loop-helix structure to calcium binding (Linse & Forsen, 1995). Thus, we reasoned that if an EF hand-like structure contributed to the intrinsic calcium sensor of BKCa channels, then mutating residues predicted to act as important calcium coordinating ligands should decrease calcium binding, and thereby impair, or possibly abolish, calcium-sensitive gating of the channel. In addition, we predicted that any such mutations should not influence voltage-dependent activation (i.e. observed in the absence of calcium), which is the primary mechanism of channel gating and appears to be separable from calcium binding (Stefani et al. 1997; Cui et al. 1997; Horrigan et al. 1999).

Figure 2 shows macroscopic currents recorded from an excised inside-out membrane patch taken from a HEK 293 cell transiently expressing a wild-type BKCa channel α subunit cloned from mouse brain (Pallanck & Ganetzky, 1994). In the presence of 2 mm EGTA (0 Ca2+, Fig. 2A), BKCa channels demonstrate largely voltage-dependent gating, in which voltage clamp steps to very positive potentials (> 180 mV) are required to achieve maximal open probability (Popen). Addition of increasing concentrations of free cytosolic calcium (≈1 to 120 μm) to these channels (Fig. 2B-E) allows opening to occur at more negative membrane voltages. In the presence of 12 and 120 μm free calcium (Fig. 2D and E), we observed inwardly directed currents at the start of negative voltage clamp steps, which then decayed during the remainder of the pulse. Due to the combined effects of the 0 mV holding potential and the high levels of free calcium, these inward currents are the result of open BKCa channels, which conduct current initially, then deactivate to a new steady state of activity during the negative pulse. The effect of free calcium is illustrated by plots of normalized conductance-voltage (G-V) relations calculated from the current families shown in Fig. 2A-E; these plots clearly demonstrate how channel gating is shifted left-ward along the voltage axis in the presence of calcium and are consistent with the results of earlier studies using cloned BKCa channels (Wei et al. 1994; Lagrutta et al. 1994; Wallner et al. 1995; DiChiara & Reinhart, 1995; Cui et al. 1997).

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Figure 2. Macroscopic currents recorded from wild-type BKCa channels in an excised inside-out membrane patch

A-E, current families recorded in response to increasing concentrations of cytoplasmic free calcium. Current traces are shown as the average of 2 independent current-voltage families recorded sequentially. F, normalized conductance-voltage relations (G/Gmaxvs. Vm) calculated from tail current measurements for the traces in A-E. Smooth lines through the data points represent the fits of single Boltzmann functions.

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It is known from detailed studies of authentic EF hand-containing proteins that substitution of the bidentate calcium coordinating ligand (e.g. Glu or Asp) at the -z position of the calcium binding loop can significantly decrease calcium binding affinity of the EF hand (Linse & Forsen, 1995). For the BKCa channel, we hypothesized that substitution of the equivalent Glu/Asp by Gln/Asn or Ala would also alter calcium-sensitive gating, if an EF hand motif contributed to calcium binding. We further anticipated that similar substitutions in domains of the channel not containing a functional EF hand would be without consequence on BKCa channel activity.

Figure 3 shows macroscopic currents recorded from an excised inside-out membrane patch containing BKCa channels with an Asp to Asn substitution at the -z position (Asp81) of Site 1 (refer to Fig. 1). In the presence of 2 mm EGTA with no added calcium, this mutant channel displays little change in gating behaviour compared to the wild-type channel, indicating that this mutation does not alter the gating of this channel by voltage alone. Furthermore, activity of this mutant channel in the presence of increasing concentrations of free calcium does not appear grossly different from wild-type channels under the same recording conditions, indicating that this single mutation does not abolish calcium sensitivity. However, closer inspection of the normalized G-V plots indicates that the gating of this mutant channel at micromolar concentrations of free calcium is shifted 30-50 mV more positive compared to wild-type channels. This effect is also illustrated in Fig. 6, which plots the half-maximal voltages of activation (V1/2 values) versus the free cytosolic calcium concentrations for wild-type and mutant BKCa channels. Conversely, equivalent substitutions within Site 2 (Glu257 to Gln), Site 3 (Glu354 to Gln) and Site 4 (Asp410 to Asn) had no apparent effect on BKCa channel behaviour (refer to Fig. 6) in the absence or presence of free calcium, indicating that neutralization of a negatively charged residue within these regions is not sufficient to alter channel gating. Substitution of Glu770 by Gln in Site 5 was found to produce non-functional channels, and therefore no further analysis was carried out on this particular mutant.

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Figure 3. Macroscopic currents recorded from Site 1 ‘impaired’ BKCa channels (see Figs 1 and 2) containing a single Asp to Asn substitution

A-E, current families recorded in response to increasing concentrations of cytoplasmic free calcium. Current traces are shown as the average of 2 independent current-voltage families recorded sequentially. F, normalized conductance-voltage relations (G/Gmaxvs. Vm) calculated for the current traces in A-E.

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image

Figure 6. Plot showing the half-maximal voltages of BKCa current activation (V1/2 values) versus the cytoplasmic free calcium concentrations

Data have been calculated from both wild-type channels and channels containing Asp to Asn substitutions at Sites 1, 2, 3, 4 and 6, as well as the combined Sites 1 and 6 double mutant (refer to Fig. 1). The number of membrane patches used to calculate the mean data points (±s.e.m.) at the individual free calcium concentrations for the wild-type and mutant channels varied as follows: wild-type, 7-12 patches; Site 1, 4-7 patches; Site 2, 3-4 patches; Site 3, 4-5 patches; Site 4, 3-5 patches; Site 6, 6-9 patches; Sites 1 and 6 double mutant, 5-11 patches.

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Examination of the various EF hand-like motifs identified in the BKCa channel (refer to Fig. 1) indicates that, in addition to Site 1, the sequence of Site 6 also displays strong similarity to that of an authentic EF hand. It is noteworthy that Site 6 overlaps with a region described by Salkoff and colleagues as the ‘calcium bowl’ and is immediately adjacent to a string of acidic residues reported to influence the calcium sensitivity of this channel (Schreiber & Salkoff, 1997). These investigators have shown that specific mutations within the calcium bowl or replacement of an extended region encompassing the calcium bowl by the equivalent domain from the calcium-insensitive mSlo3 channel (Schreiber et al. 1998) leads to reduced calcium sensitivity of BKCa channel gating (Schreiber et al. 1999). Figure 4 shows voltage clamp records from a Site 6 mutant containing an Asp to Asn substitution (Asp923) at the -z position of the presumed loop.

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Figure 4. Macroscopic currents recorded from Site 6 ‘impaired’ BKCa channels (see Fig. 1) containing a single Asp to Asn substitution

A-E, current families recorded in response to increasing concentrations of cytoplasmic free calcium. Current traces are shown as the average of 2 independent current-voltage families recorded sequentially. F, normalized conductance-voltage relations (G/Gmaxvs. Vm) calculated for the traces in A-E.

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We observed little change in voltage-dependent gating in the absence of free calcium (Fig. 4A, 2 mm EGTA) and no consistent effect on gating was detected when both Site 1 and 6 mutations were compared with wild-type. Furthermore, the raw current traces indicate that the Site 6 mutation did not grossly alter channel gating in response to increasing concentrations of calcium (Fig. 4B-E).

To examine if the behavioural effects of Asp to Asn mutations at Sites 1 and 6 occurred in an additive or non-additive fashion, we created a double mutant containing both of these substitutions. As observed for each individual mutant, voltage-dependent gating of this Sites 1 and 6 double mutant in the absence of free calcium was not different from wild-type channels (Fig. 5A). Similarly, in the presence of free calcium, the general characteristics of macroscopic currents for the Sites 1 and 6 double mutant were not grossly different from those of wild-type currents (refer to Fig. 2). However, closer examination of current traces at increasing calcium concentrations (Fig. 5B-E) suggests that the kinetics of macroscopic current activation and deactivation may be affected relative to the wild-type channels.

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Figure 5. Macroscopic currents recorded from a double mutant BKCa channel (see Fig. 1) containing Asp to Asn substitutions at both Sites 1 and 6

A-E, current families recorded in response to increasing concentrations of cytoplasmic free calcium. Current traces are shown as the average of 2 independent current-voltage families recorded sequentially. F, normalized conductance-voltage relations (G/Gmaxvs. Vm) calculated for the traces in A-E.

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Furthermore, the V1/2 values for the activation of the Sites 1 and 6 double mutant are shifted more positive (70-90 mV) compared to wild-type channels than either the Site 1 or 6 single mutant. These observations thus suggest that Asp to Asn mutations at Sites 1 and 6 have synergistic effects on the calcium sensitivity of BKCa channel gating.

Figure 6 shows a plot of the half-maximal voltages of activation (V1/2 values) for wild-type and mutant BKCa channels versus the free cytosolic calcium concentrations. For wild-type channels, this relationship has a sigmoidal appearance, in which the voltage dependence of gating is weakly affected at low calcium concentrations (< 10−6m) and approaches saturation at higher levels of free calcium (> 10−4m). This phenomenon is thus similar to that reported earlier for murine mSlo channels expressed in Xenopus oocytes (Cui et al. 1997). A number of key points can be inferred from this graph: (1) the voltage dependence of BKCa channel gating in the presence of 2 mm EGTA (i.e. plotted at a free calcium concentration of 5 × 10−9m) appears to be unaffected by mutations at any of the sites; (2) at the highest concentration of free calcium used experimentally (≈500 μm), the V1/2 values of activation for both wild-type and mutant channels converge at approximately the same membrane voltage; and (3) the V1/2 values for channels containing mutations at Sites 1 and/or 6 are right-ward shifted over the range of calcium concentrations to which the BKCa channels are most sensitive. Somewhat unexpectedly, we found that the Site 3 mutant appears to be right-ward shifted at high (100-500 μm) calcium concentrations, but behaves essentially as wild-type at lower levels of free calcium. At present, we do not have a clear interpretation of this finding, which may become more evident upon further characterization of this mutant.

To estimate the change in calcium binding affinity produced by the Sites 1 and 6 double mutant, the equilibrium dissociation constant (Kd) for calcium binding was derived from a linear regression analysis of the plotted V1/2 values over the calcium concentration range of 1-40 μm (see Methods). For wild-type channels, the derived Kd was 11.3 μm and for the Sites 1 and 6 double mutant, this value was increased to 129.5 μm, indicating that these combined point mutations decrease the apparent calcium binding affinity by ≈11-fold.

The observed changes in calcium-sensitive gating were next examined in the context of a recent mathematical model that describes well the voltage- and calcium-sensitive gating of macroscopic BKCa channel currents (Cox et al. 1997). Using this model (see Fig. 7A), we calculated conductance-voltage (G-V) relations in the presence of increasing concentrations of free calcium for a wild-type BKCa channel (Fig. 7B) and a mutant BKCa channel whose calcium binding affinity is decreased 10-fold (Fig. 7C). In Fig. 7D, the calculated half-maximal voltages of channel activation (V1/2) are plotted versus the free calcium concentrations for both the wild-type and mutant channels.

The model predicts that an equivalent decrease in the calcium binding affinity of the closed and open states of the channel will produce a parallel right-ward shift in calcium-sensitive gating along the voltage axis, without a significant change in either voltage-dependent activation in the absence of calcium or the shape of the relationship between V1/2 and free calcium. For ease of comparison, the experimentally determined V1/2 values for wild-type and Sites 1 and 6 mutant channels from Fig. 6 have been replotted in Fig. 7D. It is clear from this type of analysis that a strong similarity exists between these two data sets.

The reported calcium sensitivity of the activation and deactivation kinetics of the channels encoded by the Slo gene (DiChiara & Reinhart, 1995; Cui et al. 1997) provides a useful parameter to assess the effects of mutations on potential calcium-binding sites identified in Fig. 1. For example, close inspection of the current records for the Sites 1 and 6 double mutant (Fig. 5B-E) suggests that channel activation is slowed, while deactivation appears to be more rapid compared to wild-type channels. Cui et al. (1997) have previously shown that both the activation and deactivation kinetics of macroscopic currents arising from expressed mSlo channels are calcium sensitive; increasing concentrations of calcium speed up activation and slow deactivation over a range of membrane voltages. The observed effects of our Sites 1 and 6 mutation on current kinetics would thus be consistent with a reduction in the calcium sensitivity of the channel.

To examine whether the Sites 1 and 6 double mutation also affected gating kinetics, tail currents were recorded at −80 mV following voltage clamp steps to potentials producing ≈50 % of maximal channel activation over a range of free calcium concentrations of 1-12 μm. As shown in Fig. 8A, the time course of tail current decay for wild-type BKCa channels becomes slower as calcium is raised from 1 to 12 μm. However, for the Sites 1 and 6 double mutant, the magnitude of this slowing is significantly decreased (Fig. 8B), consistent with a reduced calcium sensitivity of this mutant channel. Fig. 8C shows a plot of the time constants of tail current deactivation derived from single exponential fits versus the concentration of free calcium for wild-type and mutant BKCa channels.

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Figure 8. EF hand mutations alter the calcium sensitivity of BK current deactivation

Macroscopic BKCa channel currents were activated in the presence of 1, 2, 4 and 12 μm free calcium by voltage clamp steps to potentials producing an open probability (Popen) of ≈0.5 and tail currents were then recorded at −80 mV. The deactivation time courses of normalized tail currents for wild-type BKCa channels and channels containing the Sites 1 and 6 double mutation are shown in A and B, respectively. Dashed lines represent single exponential fits to the decaying phase of the currents. C, time constants of deactivation for wild-type and mutant BKCa channels plotted over a range of free calcium concentrations; τ values (means ±s.e.m., n= 3-8) were determined from single exponential fits to the decaying phase of tail currents recorded at +50, −80 or −120 mV following voltage clamp steps to membrane potentials producing a Popen of ≈0.5 at each concentration of free calcium. The tail current potentials used for the various calcium concentrations are indicated above the symbols. Asterisks denote that τ values for the Sites 1 and 6 double mutant are statistically different from wild-type, P < 0.05. Explanation of symbols is as follows: •, wild-type; ⋄, Site 1 mutant; ▵, Site 6 mutant; □, Sites 1 and 6 double mutant. D, time constants of current activation in the presence of 2 mm EGTA plotted over a range of positive potentials for the wild-type channel and the Sites 1 and 6 double mutant. Macroscopic currents were recorded from a holding potential of 0 mV using voltage clamp steps to the indicated membrane potentials. The activation time constants were derived from single exponential fits to the first 7-10 ms of the current trace, beginning 0.2 ms after the voltage step. The plotted values represent the means ±s.e.m. for 4-6 individual membrane patches.

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In the absence of free calcium, tail current deactivation at +50 mV was unaffected by any of the mutations described, in agreement with the lack of effect observed on the half-maximal voltage of activation (V1/2 value) (Fig. 6). However, in the presence of increasing concentrations of calcium (1-120 μm), macroscopic tail current deactivation was significantly faster for the Sites 1 and 6 double mutant compared to the wild-type BKCa channel. The Site 6 mutation alone also increased the deactivation of tail currents, but this effect did not reach a level of statistical significance. The single Asp to Asn mutation at Site 1 had very minor effects on macroscopic current decay and similar mutations at Site 3 or 4 produced no alteration of current deactivation (data not shown).

To further characterize voltage-dependent gating of the Sites 1 and 6 double mutant, the time constants of current activation in the presence of 2 mm EGTA were compared for this mutant to those of wild-type BKCa channels. Over the voltage range of +140-240 mV, we observed no differences in the τ values for current activation between wild-type and mutant channels (Fig. 8D), consistent with the observed lack of effect of this double mutant on the half-maximal voltage of channel activation (see Fig. 6) and the predictions of the model presented in Fig. 7A.

The observations presented thus far are consistent with our original hypothesis that the intrinsic calcium sensor of the BKCa channel α subunit may contain an EF hand motif, and that mutations known to reduce calcium binding by this structure would predictably decrease the calcium-sensitive gating of the BKCa channel, without altering the process of voltage-dependent gating. The data shown in Fig. 6 suggest that the single point mutation producing the largest change in calcium sensitivity is that at Site 6 (Asp to Asn). To directly examine whether Site 6 may contain an intrinsic calcium-binding site, a C-terminal region of ≈230 amino acids encompassing Site 6 was prepared as a bacterially expressed, glutathione-S-transferase (GST) fusion protein. Following isolation, the fusion protein was cleaved by thrombin, liberating a soluble ≈31 kDa protein (Fig. 9A, right-hand lane).

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Figure 9. Direct calcium binding by a bacterially expressed fusion protein containing the Site 6 domain from the BKCa channel C-terminus

A region of ≈230 amino acids (747ASNFHY … GATPEL980) from the BKCa channel α subunit was expressed as a glutathione-S-transferase (GST) fusion protein in bacteria, purified by glutathione-agarose chromatography, and cleaved by thrombin to liberate the Site 6-containing domain. A, a Coomassie blue-stained, polyacrylamide gel of a 5 μg sample of the soluble proteins recovered following thrombin cleavage of glutathione-agarose-bound material from bacteria expressing either GST alone (lane 1) or the GST-BK domain fusion protein encompassing the Site 6 sequence (lane 2). The sizes and positions of molecular weight markers (in kDa) are shown on the left-hand side. The Site 6-containing domain (denoted by the arrow) is observed to migrate at a molecular mass of ≈31 kDa, in close agreement with its predicted molecular mass of 28.1 kDa. B, increasing amounts (7.5-20 μg) of the soluble protein fractions shown in lanes 1 and 2 of A, as well as 5 μg of purified bovine brain calmodulin (CaM), were resolved by SDS-PAGE and electrotransferred to nitrocellulose membrane. The membranes were incubated with ≈2 μm45Ca2+ for 30 min at room temperature, and 45Ca2+ binding was visualized by autoradiography. The upper and lower panels, respectively, show 45Ca2+ binding to soluble material isolated from bacteria expressing the BKCa channel Site 6 domain or bacteria expressing GST alone. 45Ca2+ binding to purified CaM served as a positive control. C, plot of direct 45Ca2+ binding to increasing amounts of soluble protein material isolated from control bacteria (GST alone, •) or bacteria expressing the BKCa channel Site 6 domain (▪). 45Ca2+ binding was quantified following autoradiography using a Gel-Doc instrument and Quanitity One image analysis software (Bio-Rad Laboratories).

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In bacteria expressing just the GST portion alone, a similar ≈31 kDa protein was not observed in the soluble fraction following thrombin cleavage (Fig. 9A, left-hand lane). Increasing amounts of both the samples shown in Fig. 9A were then resolved by SDS-PAGE and electrotransferred to a nitrocellulose membrane; the membrane was then incubated with ≈2 μm radioactive 45Ca2+. Using autoradiographic detection, the wild-type BKCa channel domain containing Site 6 was observed to bind 45Ca2+ (upper panel, Fig. 9B), whereas, no binding was detected in the protein sample isolated from bacteria expressing GST alone (lower panel, Fig. 9B). 45Calcium binding to purified bovine brain calmodulin, shown in the left-hand lane of each panel, served as a positive control. The increase in 45Ca2+ binding observed with increasing amounts of the Site 6-containing domain is plotted in Fig. 9C. This observation of direct calcium binding to a C-terminal region of a mammalian BKCa channel is in agreement with similar data recently presented by Bian et al. (2000) for the Drosophila BKCa channel.

DISCUSSION

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

In the current study, we have presented the hypothesis that the BKCa channel α subunit may contain ‘EF hand-like’ structural elements within its primary sequence that contribute to the intrinsic calcium-binding site of this channel. Our data suggest that two ‘imperfect’ EF hand structures, one in the N-terminus and the other in the C-terminus, overlapping the ‘calcium bowl’ identified by Salkoff and colleagues (Schreiber & Salkoff, 1997; Schreiber et al. 1999), contribute to the calcium sensitivity of mammalian BKCa channels.

In our strategy, we introduced point mutations known to decrease the calcium binding affinity of authentic EF hands (Glu/Asp at the -z position to Gln/Asn or Ala) (Linse & Forsen, 1995) into select regions of the BKCa channel α subunit, then screened channel mutants by examining their calcium-sensitive gating electrophysiologically. Of the six sites initially identified as containing potential EF hand motifs (see Fig. 1), we observed that mutations only at Sites 1 and 6 altered calcium-sensitive gating of BKCa channels in a manner consistent with the predictions of our strategy and those of the model of Cox et al. (1997) (see Fig. 7D). Most interestingly, combining the single Asp to Asn substitutions at both Sites 1 and 6 was found to have a synergistic effect on calcium sensitivity, suggesting that both sites may contribute to the calcium-sensitive gating of this channel. This conclusion would be consistent with the observation that EF hand motifs naturally occur in pairs, with individual EF hands separated by short (5-15 amino acids) linker sequences (Strynadka & James, 1989; Falke et al. 1994). Structurally, it has been observed that this arrangement allows adjacent EF hands to stabilize one another as they bind in an anti-parallel fashion (Strynadka & James, 1989). It is noteworthy that Site 6 from Drosophila or C. elegans is virtually identical to the mammalian sequence (refer to Fig. 1), whereas Site 1 is absent in Drosophila and only somewhat conserved in C. elegans. It is thus possible that in addition to differences observed amongst splice variants of Drosophila BKCa channels, the absence of Site 1 may also contribute to the weak calcium sensitivity reported for this channel (Lagrutta et al. 1994; Wei et al. 1994). Our observations that the mutant channels remain functional, despite their shift in apparent calcium sensitivity, would suggest that the channel's calcium-binding site(s) is not essential to the basic voltage-dependent opening and closing of the conductance pore, in agreement with the reported functionality of the mSlo1 core/mSlo3 tail chimeric channel (Schreiber et al. 1999). Rather, the intrinsic calcium sensor may act as a ‘gating modifier’, to secondarily enhance the process of channel gating by membrane voltage, which appears to be the primary stimulus promoting channel activation (Stefani et al. 1997; Horrigan et al. 1999; Rothberg & Magleby, 2000).

If Sites 1 and 6 were to form a pair of EF hands in the mammalian BKCa channel, it is possible that this may occur through either an intramolecular association, or an intermolecular arrangement, in which Site 6 of one α subunit would pair with Site 1 of an adjacent α subunit. Earlier studies have shown that a pair of EF hands within a protein may contain a ‘dead’ motif, such that the pair is capable of binding only a single calcium ion. This situation is true for cardiac troponin C (Strynadka & James, 1989), recoverin (Flaherty et al. 1993) and neurocalcin (Ladant, 1995). If only Site 6 is capable of binding calcium, then Site 1 may in fact be a ‘dead’ EF hand motif and act to stabilize calcium binding at Site 6 via a structural interaction. Such a conclusion would be further consistent with the estimated stoichiometry of calcium binding by the BKCa channel of two to five calcium ions per channel (McManus, 1991), or approximately one ion per α subunit, given the presence of four α subunits per holo-channel (Shen et al. 1994).

How might our observations of a putative EF hand motif fit within the framework of the C-terminal ‘calcium bowl’ described by Salkoff and colleagues. One possible interpretation is that the string of negative charges adjacent to Site 6 may act as a ‘magnet’ for cytosolic calcium, thereby creating a higher local concentration in the vicinity of Site 6 and promoting calcium binding to the low affinity EF hand-like structure. Given the lack of direct structural data describing a calcium-binding site composed of five to six consecutive negative charges, our data support the idea that at least one EF hand motif acts in concert with a series of acidic residues to form an intrinsic calcium sensor. Direct structural studies examining the calcium binding properties of this region will be necessary to resolve such speculation.

Finally, it is interesting that in some calcium-sensitive ion channels lacking identifiable EF hand motifs, such as small (Xia et al. 1998; Keen et al. 1999) and intermediate conductance (Fanger et al. 1999), calcium-activated K+ channels and ryanodine receptors (Meissner, 1994), a constitutively bound form of calmodulin is utilized to impart calcium sensitivity. It would thus appear that the EF hand motif, whether it be a part of the primary structure, as in twin-pore K+ channels (Czempinski et al. 1997; Salinas et al. 1999), or contributed by exogenous calmodulin, is the module of choice in calcium-sensitive membrane ion channels. The results of our study describing a role for putative EF hand motifs in the calcium sensitivity of BKCa channels are thus consistent with this trend.

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Acknowledgements

  1. Top of page
  2. Abstract
  3. METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements

The authors would like to thank Dr Howard Schulman (Stanford University) for his support and encouragement during the initial phase of this study and his critical reading of the manuscript. Discussions and suggestions provided by Dr R. W. Aldrich (Stanford University) and members of his laboratory have been most helpful and appreciated. This work was supported by a MRC operating grant and an AHFMR Establishment grant to A.P.B. Research Scholarships to A.P.B. from The Alberta Heritage Foundation for Medical Research and The Heart and Stroke Foundation of Canada are gratefully acknowledged.