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Mechanism of Increased Open Probability by a Mutation of the BK Channel

¹Departments of Neurology, ²Molecular Biophysics and Biochemistry, and ⁴Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; ³Department of Neurology, Cleveland Clinic, Cleveland, Ohio; and ⁵Veteran's Affairs Medical Center, West Haven, Connecticut

Submitted 1 May 2006; accepted in final form 25 May 2006

	ABSTRACT

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A missense mutation (D434G) has recently been identified in the subunit of the human large-conductance calcium-activated potassium (BK) channel. Interestingly, although the mutation causes an increase in open probability, individuals that carry the mutation have epilepsy and/or paroxysmal dyskinesia, disorders of increased brain excitability. To define the mechanisms of the mutation, we have used recordings from single channels and measurement of macroscopic conductances to examine the gating of the subunit, modulation by the regulatory 4 subunit, and the effect of Mg²⁺ on channel properties. Although there was relatively little difference in open dwell times for the mutant and wild-type subunits, the mutant channel spent less time in a long-lived closed state. Co-expression of the 4 subunit caused the wild-type channel to be less sensitive to calcium at low Ca²⁺ concentrations but had little effect on the mutant channel, further accentuating the difference between the wild-type and the mutant channels. In the absence of Ca²⁺, there was no difference in Mg²⁺ or voltage sensitivity of the mutant and wild-type channels, whereas in 2 mM Ca²⁺, the mutant channel had greater open probability at every Mg²⁺ concentration tested. We conclude that the D434G mutation modifies Ca²⁺-dependent activation, but we find no evidence of a direct effect on activation by Mg²⁺ or voltage. The resulting enhancement of BK channel function leads to an increase in brain excitability, possibly due to more rapid repolarization of action potentials.

	INTRODUCTION

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The large-conductance calcium-activated potassium channel (BK, Maxi-K, or slo channel) is a potassium-selective ion channel, the activation of which is increased in response to membrane depolarization and/or increased intracellular calcium (Toro et al. 1998). BK channels are highly conserved throughout evolution and are widely expressed in a variety of mammalian cells including smooth muscle and inner ear hair cells and in neurons throughout the brain (Knaus et al. 1996). These channels are formed by a combination of an or pore-forming subunit, of which there is only a single gene (KCNMA1) with at least nine splice variants in human brain (Tseng-Crank et al. 1994), and one of four isoforms of the or regulatory subunit (KCNMB1-4). Recently, a mutation in the subunit of the BK channel was identified in a family whose members have epilepsy, paroxysmal dyskinesia, or both (Du et al. 2005). The basepair substitution of A for G at codon 434 results in the replacement of a negatively charged aspartic acid residue with the neutral amino acid glycine (D434G). This mutation leads to an increase in single-channel open probability of the subunit expressed alone. This was the first mutation of the BK subunit identified in a human disease and the only mutation reported in the subunit that causes an increase in sensitivity of the channel to calcium.

The initial report on the D434G mutation did not fully characterize the functional properties of the mutant channel (Du et al. 2005). The mutation occurs in a part of the long cytosolic carboxyl terminus of the subunit known as the RCK domain in the interloop connecting the A helix and the B strand (Jiang et al. 2001). The RCK domain contains two regulatory sites: one contributes to the channel's sensitivity to micromolar concentrations of Ca²⁺ and the other may mediate some of the effects of millimolar concentrations of divalent cations including Mg²⁺ (Xia et al. 2002). The aspartic acid residue at 434 is highly conserved in BK channels across species (Du et al. 2005) and is substituted in the Drosophila BK channel with a glutamic acid residue, which is also negatively charged. Site-directed mutagenesis led to the conclusion that this region, which contains D434 (AC region), is involved in Ca²⁺-dependent activation (Krishnamoorthy et al. 2005). Thus it is possible that the D434G mutation leads to altered affinity for Ca²⁺ or modifies the coupling between Ca²⁺ binding and channel opening.

Four subunits can form a functional channel when expressed alone, but co-expression of regulatory subunits changes the biophysical and pharmacological properties of the channel (Dworetzky et al. 1996; McManus et al. 1995). Four different subunits (1–4) have been identified and each modifies the properties of the channel uniquely (Behrens et al. 2000; Brenner et al. 2000; Knaus et al. 1994; McManus et al. 1995; Meera et al. 2000; Uebele et al. 2000; Wallner et al. 1999; Weiger et al. 2000; Xia et al. 1999). Of these, 4 is highly expressed in the CNS (Behrens et al. 2000; Brenner et al. 2000; Meera et al. 2000; Weiger et al. 2000). Co-expression of the human and 4 subunits decreases channel openings at low intracellular Ca²⁺ concentrations when compared with expression of the subunit alone but increases channel openings at high Ca²⁺ concentrations (Brenner et al. 2000). The 4 subunit also changes the pharmacological properties of the channel. For example, when the 4 subunit is present, the BK channel is not blocked by 100–300 nM charybdotoxin or iberiotoxin, but it is blocked when the subunit is expressed alone or with other subunits (Behrens et al. 2000; Weiger et al. 2000). Because BK channels in the brain may be composed largely of + 4 subunits, the D434G mutation may alter the ability of the subunit to modulate calcium sensitivity, or alternatively, the subunit may reduce or eliminate the functional effects of the mutation on the subunit. Because initial studies on the D434G mutation were done with the subunit alone (Du et al. 2005), it is unclear whether co-expression with the 4 subunit alters the effect of the mutation on channel function.

Here we investigated the function of the D434G BK mutant in detail and compare it with the wild-type (WT) protein. First, we analyzed the kinetics of the WT and mutant subunit alone to elucidate differences in the open and closed times at different Ca²⁺ concentrations and voltages. We then co-expressed the 4 subunit with the WT and mutant subunit to determine whether the mutation alters the effect of the regulatory subunit. Finally, we asked whether the mutation may exert its effects on activation through alteration of either voltage- or Mg²⁺-dependent gating by determining the effect of different intracellular Mg²⁺ concentrations (from 0 to 20 mM) on the mutant and WT subunit in the absence of intracellular Ca²⁺ and at a Ca²⁺ concentration (2 mM) sufficient to saturate the high-affinity Ca²⁺ binding sites of the RCK domain. Our results indicate that the effect of the mutation is even greater when channels are composed of both and 4 subunits and are consistent with enhanced coupling between Ca²⁺ binding and channel opening, without affecting Mg²⁺- or voltage-dependent activation.

	METHODS

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BK channel expression

Chinese hamster ovary cells (CHO cells) were grown in Iscoves's modified Dulbecco's medium supplemented with 10% fetal bovine serum, HT (sodium hypoxanthine and thymidine), and penicillin/streptomycin (all from Invitrogen). Cells were split when they were confluent and plated onto glass coverslips in 12-well Falcon plates 1 day before transfection. cDNA for WT and mutant human BK subunits was subcloned into a pIRES2-EGFP vector (Clontech). CHO cells were transfected with 1.6 µg DNA/well using lipofectamine (4 µl/well; Life Technologies) 6–24 h before recording. For co-expression of the 4 and subunits, cDNA for the human 4 subunit was subcloned into a pIRES2-EGFP vector and cDNA for the WT and mutant subunit was subcloned into a pCDNA3 vector. CHO cells were then transfected with 1.6 µg total DNA per well in a ratio of 3 :1 4. This ratio increased the chance that every cell expressing the 4 subunit (visualized by GFP fluorescence) would also express the subunit. GenBank accession numbers: subunit variant 2 (KCNMA1), NM_002247; 4 subunit (hKCNMB4), AF215891.

Solutions

Coverslips with transfected CHO cells were placed in a recording chamber on an inverted light microscope (Axiovert 100, Zeiss) and superfused with Ringer’s solution at 2 ml/min. Transfected cells were identified using epifluorescence to visualize GFP. Patch-clamp recordings were made using borosilicate glass electrodes fabricated with a microelectrode puller (P-97; Sutter Instruments). Microelectrodes (5–100 M) were filled with a solution containing (in mM) 144 KCl, 2 MgCl₂, 2 TES, 11 glucose, 0.065 CaCl₂ (20 µM free Ca²⁺), and 0.08 EGTA (pH 7.2). Inside-out patches were obtained using standard techniques (Hamill et al. 1981) after which the electrode tip was moved into a separate minichamber fabricated from microelectrode glass (Barrett et al. 1982). The intracellular face of the patch was exposed to a solution (flowing at a rate of 1 ml/min) that was the same as the microelectrode solution, except that the amount of MgCl₂ was varied to give a free [Mg²⁺] of 0, 2, 5, 10, or 20 mM, and the amount of CaCl₂ was varied to give a free [Ca²⁺] of 0.4, 0.7, 10, 20, 70, or 100 µM or 2 mM. For recordings in calcium-free solution, the same solution was used, except with ultrapure (HPLC) water, 5 mM EGTA, and 0 mM Ca²⁺ (Shi et al. 2002). Free [Ca²⁺] was calculated using the program Webmaxc (www.standford.edu/cpatton/maxc.html), and verified using a calcium-sensitive electrode (Thermo Electron). Free Ca²⁺ concentrations obtained using this electrode varied from those obtained previously using calculations alone (Du et al. 2005), which accounts for the slight difference in the relationship between [Ca²⁺] and open probability reported here compared with that previous paper.

Electrophysiological recording and analysis

Single-channel recordings were made in the inside-out configuration from patches with 1–6 channels. These recordings were performed at room temperature (22°C) in voltage-clamp mode with a holding potential of 0 mV and test potentials from –100 to +100 mV (steps +20 mV for 3 s each). For macroscopic current recordings, inside-out patches were held at –100 mV and step depolarizations were applied from –60 to +210 mV (0 mM Ca²⁺) or from –150 to –30 mV (2 mM Ca²⁺). Recordings of current were amplified, low-pass filtered at 2 kHz, and digitized at 10 kHz using an Axopatch 1D amplifier, Digidata 1322a A/D converter and PClamp software (Axon Instruments). Data analysis was performed using Clampfit (Axon Instruments) and Origin (OriginLab Corp) software. For the single-channel recordings, the number of channels in each patch was determined using all points histograms from recordings performed at every level of calcium and voltage. Recordings were included only if these histograms revealed distinct peaks that changed appropriately in amplitude as the pipette potential changed and the number of channels could be unambiguously assigned. The open probability was analyzed and the data were fit to the Boltzmann [P_o (or G/G_max) = 1 – [1 + e^{(V_m–V_0.5)/k}]^-1] or Hill [P_o = [Ca²⁺]ⁿ_*(K_0.5ⁿ+[Ca²⁺]ⁿ)^-1] (where P_o is open probability, V_m is membrane potential, V_0.5 is half-maximal activation voltage, k is the slope factor, n is the Hill coefficient, and K_0.5 is the half-maximal Ca²⁺ concentration.) equations as described in the figures and text. Macroscopic steady-state conductances were calculated for individual patches and normalized to the maximum value (G/G_max). Data were then averaged and fit with the Boltzmann equation as shown in Fig. 5. For analysis of the open and closed dwell times of the channel, patches with a single channel (as determined above) were held at +20 or +60 mV for 3 min at two different Ca²⁺ concentrations: 2 µM and 10 µM. Dwell-time histograms were fit with one or two exponential functions as described in Table 1. Where indicated, statistical significance was evaluated with a Student's t-test. Data are expressed as means ± SE.

View larger version (24K): [in this window] [in a new window]   FIG. 5. Effect of Mg2+ (0, 5, and 20 mM) on the G-V relations of the WT (solid squares fit with solid lines) and mutant (open circles fit with dotted lines) subunits expressed alone. Macroscopic currents were recorded from excised inside-out membrane patches at the indicated free Mg2+ concentrations, in either the absence (0 mM Ca2+, A–C, n = 14–23) or the presence (2 mM, D–F, n = 4–8) of cytosolic Ca2+.

	RESULTS

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We reported previously that the D434G mutation leads to a higher single-channel open probability compared with the WT channel at Ca²⁺ concentrations from 1 to 100 µM, and at voltages ranging from –100 to +100 mV (Du et al. 2005). In this work, we further explore the relationship between channel opening and Ca²⁺, Mg²⁺, and voltage to better define the nature of the changes caused by the D434G mutation. In Fig. 1, we show single-channel recordings from WT (A) andD434G (B) expressing CHO cells in the inside-out patch configuration at 10 and 70 µM Ca²⁺ and at +60, +20, –20, and –60 mV. Depolarization and an increase in intracellular Ca²⁺ led to increased open probability for both WT and D434G channels. However, under each condition, the D434G mutant channel spent more time in the open state than the WT channel. We analyzed the open and closed dwell times by obtaining recordings for longer durations (3 min) at two voltages and Ca²⁺ concentrations. Dwell-time histograms are shown for open and closed events for WT (Fig. 2, A–C) and D434G mutant (Fig. 2, D–F) subunits at 2 µM Ca²⁺ and +60 mV and at 10 µM Ca²⁺ and +20 or +60 mV. Both open and closed dwell times were well fit with either single or double exponentials as described in the legend of Table 1. Time constants (₁ and ₂) and the relative distribution of the data (P₁ and P₂) for the WT and mutant subunits, as well as the open probability (P_o) of the channels are shown in Table 1. Raising [Ca²⁺] from 2 to 10 µM at a potential of +60 mV led to an increase in open probability for the WT channel from 0.032 to 0.74 (Fig. 2, A and C). This was associated with a shift in the distribution of closed dwell time events from one with a bimodal distribution of short and long time constant events to one with only short time constant events. There was less of an effect on the open dwell time events with a small shift of open events to a longer time constant (Table 1). At a voltage of +20 mV and a [Ca²⁺] of 10 µM (Fig. 2B), the closed dwell-time histogram had an intermediate distribution, with a minority of closed events (34%) having a long time constant.

View larger version (45K): [in this window] [in a new window]   FIG. 1. Single-channel recordings of wild-type (WT) and mutant subunit alone at 2 different Ca2+ concentrations (10 or 70 µM) measured at +60, +20, –20, or –60 mV (C and O indicate the level where the channel is closed and open, respectively).

View larger version (44K): [in this window] [in a new window]   FIG. 2. Open and closed dwell time histograms of single channels in WT (A–C) and mutant (D–F) subunits expressed alone. Excised inside-out membrane patches were held either at +20 or +60 mV and either at 2 or 10 µM intracellular [Ca2+]. The histograms of both open (left) and closed (right) dwell times were obtained from recordings for 3 min. Histograms were plotted in log-bin timescales and fit with either 1 or 2 exponential functions (solid line). In each condition, the open probability (Po) for the WT and the mutant subunits is indicated. Values of n for each histogram range from 3 to 5.

The major result is that an increase in open probability in the D434G mutant is associated primarily with a shift away from the deeper closed state with relatively little effect on the open dwell times. Additionally, the mutation does not appear to alter the distribution of dwell times beyond what would be expected for the WT channel in response to an increase in voltage or [Ca²⁺] as evidenced by the similarity in histograms at comparable open probabilities.

Effect of the 4 subunit

It is not known which specific neurons in the brain are involved in expression of the neurological phenotype seen in patients carrying the D434G mutation, and thus the exact composition of the BK channels (including splice variant and stoichiometry) in those neurons is undefined. However, because the subunit of BK channels is co-expressed in the brain with the 4 regulatory subunit (Behrens et al. 2000; Brenner et al. 2000), we focused on this combination. It has previously been reported that the human 4 subunit decreases channel openings at low calcium concentrations but increases channel openings at higher calcium concentrations, resulting in a channel with steeper calcium dependence (Brenner et al. 2000). It is possible that the D434G mutation alters the ability of the 4 subunit to modulate calcium sensitivity of the subunit or alternatively that the presence of the 4 subunit is dominant and hides the effect of the mutation on the open probability. To determine how the 4 subunit affects D434G channels, we measured the open probability of the mutant and WT subunits either expressed alone or co-expressed with the human 4 subunit at several voltages and calcium concentrations. Recordings were made at voltages ranging from –100 to +100 mV and at Ca²⁺ concentrations ranging from 0.4 to 100 µM.

As expected, the 4 subunit decreased the open probability of the WT subunit at Ca²⁺ concentrations from 0.7 to 20 µM (Fig. 3A). There was little effect at 70 µM Ca²⁺, and at 100 µM Ca²⁺, the 4 subunit led to a trend toward an increase in open probability (P > 0.05). These data are similar to those reported previously by Brenner et al. (2000) (see preceding text). When these experiments were repeated with the mutant subunit, the 4 subunit had little or no effect (Fig. 3B). Thus when co-expressed with the 4 subunit, as occurs in native neurons, the difference in calcium sensitivity between the WT and mutant channel (Fig. 3D) appeared even larger than in the absence of the regulatory subunit (Fig. 3C).

View larger version (34K): [in this window] [in a new window]   FIG. 3. Effect of co-expression of the 4 subunit on single channel open probability of the WT or mutant human subunit of the BK channel. The open probability was measured between the voltages of –100 and +100 mV and at several Ca2+ concentrations: 0.7, 10, 20, 70, and 100 µM Ca2+. The data were fit with the Boltzmann equation. A: single-channel open probability of the WT subunit expressed alone (solid symbols fit with solid lines, n = 10–11) or co-expressed with the 4 subunit (open symbols fit with dotted lines, n = 7–8) measured at different voltages. B: comparison of the open probability of the D434G subunit expressed alone (solid symbols fit with solid lines, n = 10–12) or co-expressed with the 4 subunit (open symbols fit with dotted lines, n = 10–11). C: comparison of the open probability of the WT (solid symbols fit with solid lines) and D434G (open symbols fit with dotted lines) subunits expressed alone. D: comparison of the open probability of the co-expressed mutant + 4 (open symbols fit with dotted lines) and WT + 4 (solid symbols fit with solid lines). Key for symbols for [Ca2+]: circle, 0.7 µM; triangle, 10 µM; inverted triangle, 20 µM; diamond, 70 µM; and hexagon, 100 µM.

View larger version (17K): [in this window] [in a new window]   FIG. 4. Comparison of BK channel open probability expressing the subunit alone (squares, fit with solid lines) or co-expressed with the 4 subunit (circles, fit with dotted lines) at different Ca2+concentrations (0.4, 0.7, 10, 20, 70, and 100 µM) in WT (A, shown in black in all panels) or mutant (B, shown in red in all panels) measured at +40 mV. C: comparison of the WT (squares fit with black solid lane) and D434G (circles fit with red solid line) subunits alone. D: comparison of the WT (squares fit with black dotted lane) and the mutant (circles fit with red dotted line) + 4 subunits. Data are fit to the Hill equation. The Hill coefficients are in WT: 2.8 ± 0.08 subunit; 5.3 ± 0.1 + 4 subunits and in mutant: 5.5 ± 0.2 subunit; 5.1 ± 0.09 + 4 subunits; n = 7–12.

Voltage- and Mg²⁺-dependent gating

It has previously been shown that Mg²⁺ increases the open probability of the BK channel in the presence of Ca²⁺ and can also open the channel in the absence of Ca²⁺ (Bringmann et al. 1997; Shi and Cui 2001; Zhang et al. 2001). Because the region of the subunit that contains the D434G mutation lies in the RCK domain near a regulatory site that may contribute to the effect of Mg²⁺ (Xia et al. 2002), we examined whether the mutation may affect sensitivity to Mg²⁺. To accomplish this, we recorded macroscopic currents and looked at the effect of Mg²⁺ on steady-state conductance of the subunit alone, first in a solution that did not contain Ca²⁺ and then in a solution that contained a Ca²⁺ concentration (2 mM) sufficient to saturate the high-affinity Ca²⁺ binding site.

In the absence of Ca²⁺ (Fig. 5, A–C), the normalized conductance of channels composed of either the WT mutant subunits expressed alone increased with increasing [Mg²⁺] ranging from 0 to 20 mM. This was evident as a progressive leftward shift of the G-V curves in increasing [Mg²⁺]. There was no difference in steady-state conductance of the mutant and WT channels at any voltage with Mg²⁺ concentrations of 0 or 5 mM and only minor changes in 20 mM Mg²⁺, with a slightly greater open probability of the mutant channel at +60 and +70 mV (P < 0.05) but not at any of the other voltages tested (Fig. 5C). In contrast, when recordings were made in 2 mM Ca²⁺ (Fig. 5, D–F), the normalized conductance was significantly greater in the mutant than in the WT for each of the Mg²⁺ concentrations tested.

Intracellular Mg²⁺ has also been shown to cause a decrease in single-channel conductance (Bringmann et al. 1997; Ferguson 1991), probably by blocking the pore. We examined whether the D434G mutation influenced the ability of Mg²⁺ to alter single-channel conductance of the subunit in the absence of Ca²⁺. There was no difference in single-channel conductance of the mutant and WT channels at each [Mg²⁺] tested from 0 to 10 mM (Fig. 6; Table 2).

View larger version (24K): [in this window] [in a new window]   FIG. 6. Representative single-channel currents of the WT (top) and mutant (bottom) subunits at different intracellular Mg2+ concentrations (0, 2, 5, and 10 mM) in 0 Ca2+ concentration recorded at +100 mV.

	DISCUSSION

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Increased calcium sensitivity of the D434G mutation

The D434G mutation was the first mutation of the subunit of the BK channel identified in a human disease. Many other mutations of the subunit of the BK channel have been generated experimentally using site-directed mutagenesis in efforts to identify the locations of Ca²⁺ binding. Some of them decrease calcium sensitivity (Bao et al. 2002; Shi et al. 2002; Xia et al. 2002), but none have been reported to increase calcium sensitivity. To our knowledge, the only mutations of the subunit previously reported to lead to a significant gain of function of BK was the deletion of eight amino acids in the D-D linker (Zhang and Horrigan 2005) and the R207Q mutation (Cui and Aldrich 2000) both in the mslo1 gene. These mutations led to increases in open probability, but this was not due to modulation of calcium sensitivity, because the changes occurred in the absence of calcium, and instead was due to a shift in voltage sensitivity. The D434G mutation led to an increase in open probability due to an increase in calcium sensitivity. Compared with the WT channel, the mutation only induced an increase in open probability in the presence of calcium but not in its absence (Fig. 5), providing no evidence for an independent effect of the mutation on voltage sensitivity. At levels of calcium expected to saturate the high-affinity calcium binding sites (Zhang et al. 2001), we still recorded a difference in open probability (Fig. 5). Although it is possible that there is a contribution from changes in calcium binding per se, this result suggests that there is a change in the regulation of channel opening after calcium binds to the high-affinity Ca²⁺ binding site (see following text).

Dwell times were analyzed to provide insight into the mechanisms of the effect of the mutation (Fig. 2). There were some differences between the WT and mutant channel in the time constants (₁ and ₂) of open and closed dwell times, but these differences were small and generally not statistically significant (Table 1). For the open dwell times, the relative distributions of the data (P₁ and P₂) were similar in the WT and the mutant. However, for the closed dwell times, the distributions were different. The closed dwell time analysis revealed that the mutation reduced the amount of time the channel spent in a deeper closed state. These changes are similar to those that occur in the WT channel in response to changes in calcium and voltage. Thus in this regard the behavior of the mutant channel was altered in a way that is consistent with a shift in sensitivity to calcium.

Allosteric models of Ca²⁺-dependent activation describe a mechanism by which Ca²⁺ binding leads to increased occupancy of states that are more permissive for opening (Horrigan and Aldrich 2002). The distribution of channels among these states is dependent on [Ca²⁺] and the relative affinities of these states. The decreased occupancy of a deep closed state in the mutant subunit, which we describe in Fig. 2, may reflect a decrease in affinity of this state. This is functionally similar to increasing the affinity of an open state, and is consistent with the leftward shift and increase in cooperativity of Ca²⁺ shown in Fig. 4C.

Difference in open probability between mutant and WT channels is greater with co-expression of the 4 subunit

For the WT channel, the 4 subunit shifted the Po-V relationship at low Ca²⁺ concentrations (0.7, 10, and 20 µM) to more depolarized voltages, had no effect at 70 µM Ca²⁺, and led to a trend toward a shift to more hyperpolarized voltages at 100 µM Ca²⁺ (Fig. 3A). These results are similar to those of Brenner et al. (2000). The [Ca²⁺] at which reversal occurred in their study (between 10 and 50 µM) was somewhat lower than found here (70–100 µM). This difference could be due to differences in the expression system (oocytes vs. CHO cells) or the specific splice variant of the subunit that was used. Our results are different from those reported by Lippiat et al. (2003), Weiger et al. (2000), and Ha et al. (2004), but each of these studies had methodological differences from ours, including expression system, splice variant, species used and/or methods for determination of calcium concentrations (electrode versus calculations). We also cannot rule out the possibility that there are differences in assembly of and 4 subunits between studies, resulting in differences in stoichiometry of the channel.

When the mutant subunit was co-expressed with the 4 subunit, there was little or no effect on open probability at any [Ca²⁺] compared with the mutant subunit alone (Fig. 3B). For both the mutant and the WT channel, the 4 subunit also had no effect on single-channel conductance. Thus co-expression of the 4 subunit actually accentuated the differences in open probability between the WT and mutant channels. This suggests that in native neurons that express the 4 subunit, the effect of the D434G mutation might be greater than the effect first detected in subunits expressed alone (Du et al. 2005).

The 4 subunit induced an increase in cooperativity of the WT channel without appreciably shifting the half-maximum concentration for calcium (Fig. 4A), whereas the mutation induced both an increase in calcium cooperativity and a leftward shift in calcium sensitivity (Fig. 4C). The effects of the mutation appear dominant over the effects of 4, as co-expression of the 4 subunit produced no apparent change in function of the mutant subunit. Although we find it unlikely, our data leave open the possibility that the 4 subunit and the D434G subunit do not co-assemble properly, given the lack of effect of 4 on the mutant channel. However, in native neurons, the WT subunit does co-assemble with 4 and it is the comparison of the properties of this form of the channel with the co-expressed mutant + 4 that is likely to be physiologically relevant in the etiology of the disease.

The 4 subunit increased the cooperativity of Ca²⁺ sensitivity for the WT channel (Fig. 4A), increasing the Hill coefficient from 2.8 to 5.3. This same effect has been previously reported for the rat BK channel (Ha et al. 2004), where the 4 subunit increased the Hill coefficient from 3.2 to 6.5. Interestingly, the mutant subunit already had a high Hill coefficient when expressed alone, and this was not changed with co-expression of the 4 subunit (Fig. 4B). Thus the D434G mutation itself increased Ca²⁺ cooperativity in the absence of the 4 subunit compared with WT.

Mechanism of increased Ca²⁺ sensitivity by the D434G mutation

The D434G mutation caused little or no change in single-channel conductance, either of the subunit alone or of the subunit co-expressed with the 4 subunit. It also did not alter the effect of Mg²⁺ on single-channel conductance. Instead, the results presented here indicate that amino acid 434 is important for Ca²⁺-dependent activation of the BK channel. The evidence for this includes the observation that in the absence of Ca²⁺ the mutant and WT channel were activated equally by voltage and/or Mg²⁺ (Fig. 5, A–C). These data suggest that the voltage sensor, the magnesium binding site, and linkage of these two regions to the gating apparatus are all unaffected by the mutation. Additional evidence in favor of an effect on high-affinity Ca²⁺-dependent gating is that there was a difference in open probability of the WT and mutant channels at Ca²⁺ concentrations of 0.4 µM (data not shown) and 0.7 µM, when there would be little or no binding to the low-affinity divalent cation binding sites. These conclusions are consistent with previous data localizing the voltage sensor to the S4 segment (Diaz et al. 1998) and the low-affinity divalent cation site to a different region of the RCK domain between B and C (Shi et al. 2002; Xia et al. 2002). L. Hu et al. (2003) showed that Ca²⁺-dependent activation is separate from activation by Mg²⁺ or voltage and that alteration of Ca²⁺-dependent activation does not necessarily affect either voltage- or Mg²⁺-dependent activation. The D434G mutation leads to a decrease in [Ca²⁺] required to activate the BK channel as well as an increase in cooperativity of Ca²⁺ binding. This could theoretically be due either to alteration of the affinity of the high-affinity binding site for calcium in one or more states of the channel or alteration of the conformational changes that lead to channel opening after Ca²⁺ binding. Krishnamoorthy et al. (2005) have presented evidence that the region of the channel encompassing the D434G mutation (the AC region) is involved in allosteric coupling between Ca²⁺ binding and opening but that the efficacy of this coupling is not related to the presence of a Ca²⁺ binding site in this region. Our data are consistent with this interpretation. We found that at 2 mM Ca²⁺, when the high-affinity Ca²⁺ binding sites would be expected to be completely occupied in the WT channel (Zhang et al. 2001) and the mutant channel would thus not be expected to bind more calcium, there was still a significant difference in open probability between the mutant and WT channel. Our results suggest that the differences in Ca²⁺ dependence between the WT and mutant channel are not due to direct effects on Ca²⁺ binding, but instead favor the idea that the mutation interferes with some step between Ca²⁺ binding and conformational changes permissive for channel opening. Although we cannot formally rule out an effect on the Ca²⁺ affinity of one or more states of the channel, such a change is not necessary to explain our results.

The D434G mutation is located in the A-B loop of the RCK domain of the BK channel. The crystal structures of the RCK domain in MthK (Jiang et al. 2002) and the Escherichia coli K⁺ channel (Jiang et al. 2001) have revealed that the analogous loop (which is shorter than in BK) is located at the top of the octameric gating ring, close to the membrane where it could potentially interact with an intracellular loop between adjacent transmembrane segments or the gate. Alternatively, there could be an interaction with the linker between the transmembrane core and the RCK domain itself. This linker appears to act as a spring, and shortening the linker results in a channel that is easier to open (Niu et al. 2004). The mutation is unlikely to exert its effect by increasing tension on the linker because shortening the linker, effectively increasing its tension, results in a channel with shifted voltage dependence, which we did not observe in the mutant in the absence of calcium.

Implications for the role of the BK channel in brain function

The D434G mutation enhances BK channel function and is associated with brain hyperexcitability as manifest by epilepsy and paroxysmal dyskinesia. At the time of the first report of this mutation (Du et al. 2005), several possible explanations were suggested for this effect: enhanced repolarization of action potentials and more rapid removal of inactivation of Na⁺ channels on hyperpolarization, induction of rebound spikes after hyperpolarization, greater activation of I_h due to hyperpolarization, and inhibition of GABAergic interneurons with disinhibition of thalamocortical circuitry. Any or all of these mechanisms could contribute to neuronal hyperexcitability, and several are particularly relevant to the generation of absence seizures by the thalamus (Von Krosigk et al. 1993). However, since the time of that publication another likely possibility has been proposed to explain why an increase in brain excitability could occur as a result of enhancement of BK channel activity.

Brenner et al. (2005) generated 4 knockout mice that were found to have electrographic seizures as their only abnormality. They found that absence of the 4 subunit resulted in a gain of function of the BK channel, which caused more rapid action potential repolarization in hippocampal dentate gyrus neurons. This led to a decrease in calcium influx during each action potential, secondarily resulting in less activation of SK-type calcium-activated K⁺ channels. The end result was that the neurons fired at a higher sustained rate due to loss of the spike frequency adaptation normally mediated by SK channels.

The work presented here and that of Brenner et al. (2005) are consistent with the concept that activation of BK channels leads to reduction of action potential width (Adams et al. 1982; Lancaster and Nicoll 1987; Shao et al. 1999). The sensitivity of the channel to relatively high [Ca²⁺], the strong activation by depolarization and the rapid deactivation on repolarization all suggest that the BK channel can act in some neurons to control action potential width rather than producing slow inhibition. Thus instead of inhibiting neuronal firing, the role of this channel in some neurons may be to allow sustained firing at a high rate.

Implications for human disease

Although BK channels are widely expressed throughout the human body and are considered important regulators of neuronal function, their specific role in human disease is still largely unknown. A mutation in the 1 subunit of the BK channel has been associated with reduced risk for hypertension through a mechanism of increased sensitivity to calcium in smooth muscle cells (Fernandez-Fernandez et al. 2004). Preliminary data suggest the 3 subunit of the BK channel may also play a role in seizures and epilepsy (S. Hu et al. 2003; Riazi et al. 1999), but to date no other human mutations have been described in BK channel or subunits. The subunit mutation studied here is associated with a syndrome of co-existent generalized seizures and paroxysmal dyskinesia. This work delineates an interesting new mechanism by which BK channel mutations lead to neuronal hyperexcitability and highlights new targets for pharmacological intervention in the treatment of epilepsy and paroxysmal movement disorders.

	GRANTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
This work was supported by the Veteran’s Affair Medical Center to G. B. Richerson, Postdoctoral Fellowship from the American Heart Association to W. R. Silverman, and National Institutes of Health to J. F. Bautista.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION GRANTS ACKNOWLEDGMENTS REFERENCES

 
cDNA for the 4 subunit in the pIRES2-EGFP vector was kindly supplied by Dr. Irwin B. Levitan.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Address for reprint requests and other correspondence: A. Díez-Sampedro, Neurology, LCI-704, Yale University School of Medicine, 15 York St., PO Box 208018, New Haven, CT 06520-8018 (E-mail: ana.diez-sampedro{at}yale.edu)

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