The Brain-Specific Beta4 Subunit Downregulates BK Channel Cell Surface Expression

The large-conductance K+ channel (BK channel) can control neural excitability, and enhanced channel currents facilitate high firing rates in cortical neurons. The brain-specific auxiliary subunit β4 alters channel Ca++- and voltage-sensitivity, and β4 knock-out animals exhibit spontaneous seizures. Here we investigate β4's effect on BK channel trafficking to the plasma membrane. Using a novel genetic tag to track the cellular location of the pore-forming BKα subunit in living cells, we find that β4 expression profoundly reduces surface localization of BK channels via a C-terminal ER retention sequence. In hippocampal CA3 neurons from C57BL/6 mice with endogenously high β4 expression, whole-cell BK channel currents display none of the characteristic properties of BKα+β4 channels observed in heterologous cells. Finally, β4 knock-out animals exhibit a 2.5-fold increase in whole-cell BK channel current, indicating that β4 also regulates current magnitude in vivo. Thus, we propose that a major function of the brain-specific β4 subunit in CA3 neurons is control of surface trafficking.

The BK channel is a tetramer of a subunits [15,16] that assemble with an auxiliary b subunit in up to a 1:1 stoichiometry [17,18]. The four identified b subunit genes show tissue-specific expression (reviewed by [19]), where the most abundant CNS isoform is b4 [20,21]. Coexpression of b4 in heterologous cells slows activation kinetics of BK channel currents [20,22,23,24], generally increases the amount of Ca ++ and depolarization required for channel gating [21,23,25] but see [26], and confers resistance to the specific peptide antagonists iberio-and charybdotoxin [25,27]. Indeed, genetic knock-out of b4 results in larger BK channel currents gated by action potential (AP) firing and is associated with increased firing activity and spontaneous seizures in mice [1].
In contrast to the many investigations into how b4 influences the biophysical properties of BK channels, a role for this subunit in controlling the cellular location of BK channels has not been systematically investigated [28], although other b subunits can modulate trafficking of the channel [29,30]. Regulated trafficking of BK channels in neurons is particularly interesting, as the number of endogenous channels at the plasma membrane may be small [31,32], in the tens to hundreds range, in contrast to Na + channels or glutamate receptors that are 100 to 1000-fold more densely distributed on the cell surface. Modest changes in the number of plasma membrane BK channels may profoundly influence the magnitude of whole-cell BK channel currents and, consequently, firing output and network excitability. Interestingly, although BK channels have been extensively studied in the CNS, few studies have detected BK channels with pharmacological or biophysical properties consistent with b4-containing channels [20,21,22], despite the broad expression of this subunit across the brain.
To investigate the role of b4 in regulating cell-surface trafficking of BK channels, we used a novel fluorogen activating protein (FAP) [33] to genetically tag the extracellular N-terminus of the BKa subunit. Cotransfection of tagged BKa with b4 in HEK-293 cells led to a 70% reduction in cell-surface channel fluorescence, assessed by application of a membrane-impermeant dye. In the presence of b4, BKa was sequestered in the ER, and mutation of a C-terminal ER retention/retrieval motif in b4 was sufficient to restore high levels of BKa at the plasma membrane.
How does b4 regulate BK channel currents in the CNS? Both BKa and b4 are highly expressed in CA3 pyramidal neurons.
However, whole-cell BK channel currents from CA3 neurons in wild-type mice do not appear to contain b4, based upon their biophysical or pharmacological properties, consistent with a role for b4 in intracellular sequestration of the channel. Importantly, whole-cell BK channel currents in CA3 neurons from b4 knockout mice are 2.5-fold larger than in wild-type or heterozygote animals. Taken together, our data indicate that the b4 subunit can regulate cell-surface BK channel localization.

Materials and Methods
All animal experimental protocols were approved by Carnegie Mellon University's Institutional Animal Care and Use Committee (Animal welfare assurance number A3352-01).

Cloning
Cortical and hippocampal tissues were collected from 3 C57BL/6 mice aged P14. RT-PCR was performed on RNA isolated from hippocampal tissue homogenates using gene-specific primers designed from annotated sequences (NCBI reference sequences NM_010610.2 for KCNMA1/BKa and NM_021452.1 for KCNMB4/b4), and PCR products were cloned and sequenced. The BKa variant isolated lacked the STREX exon. The b4 variant isolated was identical to published sequence (NM_021452.1 for KCNMB4/b4). The L5 FAP was affinitymatured using published procedures (Szent-Gyorgi 2008), and a novel variant (L91S) with a 5-fold improved quantum yield was isolated. The L5-FAP was fused in-frame to the BKa sequence beginning at the amino acids MDAL at the N-terminus. The FAP-BKa construct was synthesized according to the RT-PCR-derived BKa sequence in a pUC57 vector (Genscript Corporation), and the BKa and b4 constructs were subcloned into the pCS2+ using a CMV promoter to drive expression in mammalian cells. Mutant b4 constructs were created by replacing C-terminal lysines in the last six amino acids to alanines for the b4-Ala and by adding three serines to the C-terminal for the b4-polySer construct.

Cell culture and transfection
HEK-293FT (Invitrogen) cells were grown in complete DMEM medium with FBS at 37uC at 5% CO 2 and 75% humidity. Cells were seeded in 35 mm dishes and grown to ,70% confluence for 24 hours. HEK-293 cells were transfected using Neofectin (Mid Atlantic Biolabs) according to manufacturer's specifications. One mg BKa DNA was transfected, and an equimolar amount of b4 was included where indicated. Cells were cultured for another 24 hours post transfection and were either imaged live at 37uC in a humidified chamber or fixed for immunocytochemistry.

Live-cell imaging and analysis
Images were acquired on a LSM 510 Meta confocal microscope (Carl Zeiss). Immediately before imaging, media was replaced with complete DMEM without FBS with either cell impermeable MG-2p (100 nM) or cell permeable MG-ester dyes (100 nM). Cells were maintained at 37uC, 5% CO 2 and 75% humidity throughout imaging. Fields for imaging and analysis were selected using only GFP fluorescence to identify transfected cells and were collected 3-10 minutes after dye addition.
Exported tiff files were analyzed in ImageJ. To determine surface fluorescence levels, mean cell membrane pixel intensities were determined as follows. A 250-pixel, circular ROI was placed on different regions of the plasma membrane of expressing cells (identified by both MG and GFP fluorescence) in a north-southeast-west orientation.
Because transient transfections with multiple plasmids does not guarantee expression of all plasmids (for example, some cells may not have taken up the b4 plasmid), analysis was limited to cells that exhibited both GFP fluorescence as well as MG-2P surface signal. This method may have led to an overestimation of surface localization of FAP-tagged BKa in the BKa+b4 transfected cells, since some cells may not have expressed the b4 construct. Adjacent membranes from two transfected cells were rejected from analysis. ROIs for 2-4 cells were analyzed per image field, and 20 fields per transfection experiment were evaluated. Thus, ,60 cells per condition were examined for each experiment. Experiments were repeated at least three times. Mean, minimum and maximum pixel intensities for each ROI were calculated, averaged for each cell, and then averaged across cells for each separate transfection. To account for potential differences in expression levels across different transfection experiments, all transfection datasets were normalized to the value of the mean surface fluorescence for the BKa-alone transfection for that specific experimental day. A histogram for the normalized fluorescence intensity values across all experimental days was generated (see Figure 1).

In situ hybridization
Digoxigenin (DIG)-labeled probes were prepared using the Roche DIG RNA Labeling Kit. The b4 probe isolated from our RT-PCR experiments spanned the entire transcript. Brains were collected from C57BL/6 animals of either sex, perfused with 4% paraformaldehyde (PFA) and fixed overnight in PFA. The tissue was sunk in 30% sucrose and sectioned at 50 mm thickness on a cryostat. Sections were washed in PBS and MOPS buffer then placed in a hybridization solution overnight before incubating with the probe at 57uC for 5 days. Sections were then washed in MAB solution and blocked with 2% block (Roche) for 15 minutes at room temperature followed by overnight incubation with 1:2000 alkaline phosphatase conjugated anti-DIG antibody (Roche) in MAB+2% block. Hybridization signal was detected with an alkaline phosphatase reaction at 37uC for several hours.

Immunocytochemistry and analysis
Twenty-four hrs post-transfection, HEK-293 cells were fixed in 4% PFA+4% sucrose for five minutes at room temperature. Cells were washed with PBS and permeabilized using a solution containing 0.5% Triton X-100, 5% 1 M glycine and 5% normal goat serum for 30 minutes at room temperature. Appropriate primary antibodies were diluted in this blocking solution and incubated with the transfected cells for 1 hour at room temperature. After washing with PBS, the cells were incubated with the appropriate secondary antibody for 30 minutes at room temperature. For experiments with non-permeabilized labeling, Triton X-100 was replaced with PBS in all solutions. Primary antibodies used were anti-BKa (Neuromab) and anti-b4 (Neuromab for single labeling or Alomone labs for double-labeling) at a 1:500 dilution. Secondary antibodies used were Alexa 488conjugated goat anti-rabbit (Invitrogen) 1:750, Alexa488-conjugated goat anti-mouse (Invitrogen) 1:750, Alexa594-conjugated goat anti-mouse (Invitrogen) 1:750 and Cy5-conjugated goat antimouse (Jackson Immuno Research) 1:750.
Quantitation of overlapping expression for BKa or b4 with KDEL-mRFP in fixed cells was done as follows. A line was drawn from the center of the nucleus to a point just outside the cell membrane, for portions of the plasma membrane that were not abutting another cell, and pixel intensities in both channels along this line were recorded for 10 cells from two separate transfections. For all cells the values for each channel were normalized to their highest intensity, and distance was normalized to the maximum distance for that cell. The normalized values across all cells were averaged and plotted as shown in Figure 2.
To estimate the magnitude of steady-state BK channel currents in CA3 neurons, cells were held at 280 mV and a 40 ms prepulse of 240 mV followed by an 800 ms voltage step to +100 mV was applied before and after the application of paxilline or iberiotoxin. Currents from after drug application were subtracted from the pre-application currents to yield the paxilline-sensitive or iberiotoxin-sensitive BK channel current. The current from the voltage step was determined from an average of 4 sweeps. Capacitive current was subtracted for both the pre-and post-drug application currents by the P/4 method.
In comparison to recordings from heterologous cells, estimating BK channel currents in neurons is considerably more difficult. We are aware that our measurements may be dominated by BK channels that lie close to the soma, due to inadequate voltage clamp in the distal portions of the cell. However, because the critical comparisons for this study were of the steady-state currents isolated by two different drugs, and all other recording parameters were the same (cell type, cell age, input resistance), we expect that these uncontrolled variables should cancel out.

b4 exerts a dominant negative effect on cell-surface trafficking of BK channels
Tracking the surface distribution of ion channels, a technically challenging issue in cellular neuroscience, has long been a subject of great interest. Although intrinsically fluorescent protein tags such as synaptophluorin have been used to examine surface expression of other membrane proteins, intracellular fluorescence is incompletely quenched at near-neutral pH in some intracellular compartments such as the ER [34]. Since many ion channels show enormous intracellular reservoirs, this fluorescence makes it difficult to resolve or quantitate lower levels of cell-surface staining. To bypass this, a FAP [33] was used to genetically tag the a subunit of the BK channel at the extracellular N-terminus [35] ( Figure 1A). Upon small-molecule dye binding to the FAP, fluorescent output is enhanced 20,000-fold, providing outstanding signal-to-noise ( Figure 1B). Because well-characterized antibodies against extracellular regions of BKa have not been generated, FAP-tagging of the protein enabled direct comparison of cellsurface channel localization using a cell-impermeable dye, malachite green diethylene glycol (MG-2p; [33]), in the presence and absence of b4. Furthermore, this analysis could be carried out in living cells under normal growth and temperature conditions. This is particularly important, because channel trafficking can be acutely altered by temperature and mechanical stress [29,30,36].
The genes encoding BKa and b4 were isolated from mouse brain tissue, and cloned into a mammalian expression vector. Sequences matched the BKa STREX-lacking isoform [37] and the canonical b4 transcript (Figures S1, S2). The L5 FAP tag was fused in frame to the N-terminus of BKa, at the amino acid sequence beginning MDALIIPV. In HEK-293 cells transfected with FAP-tagged BKa alone, application of the impermeable MG-2p dye revealed a clear outline of the plasma membrane ( Figure 1C). Cotransfection of FAP-BKa with unlabeled b4 revealed a marked reduction in cell-surface fluorescence in the presence of MG-2p dye ( Figure 1D). This was not due to suppression of BKa-protein in cotransfected cells, since application of a membrane-permeable MG-ester dye revealed FAP-BKa in intracellular compartments under both transfection conditions ( Figure 1E, F).
Quantitative analysis of cell-surface fluorescence was carried out for FAP-BKa-alone and FAP-BKa+b4 transfected cells. Because HEK-293 cells can show a range of values for surface fluorescence, a phenomenon that is likely to depend upon different levels of gene expression in the transfected cells, a histogram was constructed for fluorescence values across multiple cells for each transfection condition. A significant difference in the across-cell distribution of fluorescence intensity was observed for the two experimental groups ( Figure 1G; n = 54 regions of interest (ROIs) for each condition, averaged over five independent transfections, 270 ROIs total, p,10 23 by two-sample K-S test). Overall, the presence of b4 induced a nearly 70% reduction in mean surface fluorescence (normalized mean surface intensity, FAP-BKa-alone, 160.03, versus FAP-BKa+b4, 0.3460.02, p,10 25 by paired t-test; see also Figure 3). Similar intracellular but different cell-surface expression of BKa in the absence/presence of b4 are schematized in Figure 1H,I.
As an additional method to evaluate b4-regulated surface expression of BKa, dual-label immunohistochemistry on transfected, fixed HEK-293 cells was performed and analyzed by confocal microscopy of a thin z-section through the cell ( Figure  S3). These experiments took advantage of small differences in the level of b4 expression in different transfected cells and allowed comparison of surface to internal BKa on a cell-by-cell basis. Surface BKa was tightly coupled to the amount of b4 expressed, with cells expressing little b4 showing the greatest surface:internal ratio. Conversely, cells that expressed high levels of b4 exhibited the lowest ratio of surface: internal BKa immunofluorescence ( Figure S3A-F). This conclusion was further supported by live cell imaging experiments using spectrally-distinct cell-permeable and impermeable dyes. In BKa+b4 transfected cells, surface labeling of the FAP-tagged BKa was notably lower compared to the BKaalone although intracellular levels of BKa appeared grossly similar (data not shown).
b4 concentrates BK channels in the ER b4 expression might reduce surface BKa levels by facilitating degradation of the pore-forming channel, or by interacting with BKa subunits to reduce cell-surface trafficking. To determine whether BKa and b4 are concentrated in the same intracellular compartment and to identify this structure, HEK-293 cells were co-transfected with FAP-BKa, b4, and monomeric RFP (mRFP) tagged with an N-terminal prolactin signal sequence and a Cterminal sequence (KDEL) that leads to protein retention in the endoplasmic reticulum (ER; mRFP-KDEL; gift of T. Lee and E.L. Snapp; [38]). Importantly, untransfected HEK-293 cells exhibited no BKa or b4 immunoreactivity, indicating that these cells do not express endogenous BK channel subunits that might alter trafficking results [39] (and data not shown).
In FAP-BKa and mRFP-KDEL transfected and fixed cells, clear colocalization of immunolabeled BKa and mRFP was observed, indicating that the bulk of BKa subunits reside in the ER (Figure 2A). This is similar to what has been observed for other K + channel proteins that also show large intracellular reserves [40,41,42]. By immunohistochemistry, BKa signal was not pronounced at the margins of the cell, highlighting the benefits of the FAP tag that allows visualization of cell-surface protein without spectral contamination from intracellular stores.
In FAP-BKa+b4-expressing cells, BKa immunolabeling showed similar overlap with mRFP-KDEL ( Figure 2C). However, in both FAP-BKa-alone and b4 cotransfected cells, some BKa could be observed outside of the mRFP-KDEL ER compartment, indicating that at least some fraction of the BKa subunit may escape the ER ( Figure 2B,D; both distributions are significantly different, p,0.001 by K-S test).
b4 immunostaining in fixed transfected cells confirmed both b4 subunit expression and b4 colocalization with BKa and KDEL-mRFP ( Figure 2E-G). Localization analysis indicated that b4 and mRFP-KDEL distributions were not significantly different ( Figure 2F, p.0.1), suggesting that the b4 subunit is largely retained within the ER. Based upon the finding that b4 coexpression is sufficient to reduce surface FAP-BKa, as well as that immunofluorescence for both proteins show substantial overlap with mRFP-KDEL and with each other, we propose that b4 directly interacts with the BKa subunit to retain the channel complex in the ER.
Since the epitope for the b4 antibody is within the extracellular loop of the two-transmembrane domain protein (see Figure 1A), surface localization of b4 can be determined by immunocytochemistry in non-permeabilized cells. Under these conditions, b4 immunoreactivity was not observed at the cell surface ( Figure 2H) even though significant b4 expression can be seen after permeabilization ( Figure 2G). These data show that although transfected HEK-293 cells indeed express b4 protein, this b4 protein is undetectable at the plasma membrane.

Identification of an ER retention motif in b4
The amino acid determinants of proteins that reside in the ER have been well-defined [41,43,44,45]. Canonical amino acid motifs for ER retention/retrieval of both lumenal and transmembrane proteins are KDEL or KKXX at the carboxy (C)-terminus (where XX are the last two amino acids of the protein) respectively. Although b4 lacks either of these canonical motifs, the last six amino acids, KKRKFS, may also serve as an ER retention sequence under some conditions [44] (Figure 3A).
To test whether these amino acid residues in b4 were sufficient to exclude co-expressed FAP-tagged BKa from the plasma membrane, these lysine residues were mutated to alanines (b4-Ala; Figures 3A, S2). Because it has also been demonstrated that the KKXX motif must be at the C-terminus of the protein [44], we also investigated whether addition of three amino acids (serines) to the C-terminus would disrupt the ER retention/retrieval function of b4 (b4-polySer; Figures 3A, S2).
Cotransfection of FAP-BKa with b4-Ala or FAP-BKa with b4-polySer, followed by application of the impermeable MG-2p dye, exhibited robust surface fluorescence for both co-transfected b4 mutants ( Figure 3B-E). Quantitation of surface fluorescence intensity across cells indicated that mean surface fluorescence was similar or even greater than FAP-BKa-only transfected cells (normalized mean surface intensity: b4-Ala 1.1760.06, b4-polySer 1.1260.04; significantly different from BKa-alone by ANOVA; n = 54 ROIs per transfection experiment over 5 experiments total for each mutant; Figure 3F). In addition, the distribution of FAP-BKa fluorescence surface intensity values for the two mutant b4 proteins was fully overlapping with that observed for FAP-BKaalone transfected cells (p = 0.10 by two-sample K-S test for b4-Ala and p = 0.13 for b4-polyser versus BKa alone; Figure 3G). Thus, the b4 C-terminal motif KKRKFS is required for ER retention of the BKa channel subunit in transfected HEK-293 cells.
An alternate explanation for the increased surface localization of BKa is that mutant b4 constructs are not efficiently expressed in transfected cells. To verify that the b4-Ala and b4-polySer constructs are present in cells expressing BKa, b4-immunocytochemistry was carried out in the FAP-BKa and mutant b4 transfected HEK-293 cells. Strong intracellular immunoreactivity for both b4-Ala ( Figure 4A) and b4-polySer was observed, indicating that these mutations do not markedly suppress b4 protein levels.
Immunocytochemistry using a b4 antibody in non-permeabilized cells revealed robust cell-surface staining for both b4-Ala and b4-polySer cotransfections with FAP-BKa ( Figure 4B,D). This demonstrates that our experimental conditions are sufficient to detect not just FAP-tagged BKa, but also b4 protein, at the cell surface. The presence of both FAP-tagged BKa and mutant b4 at the cell surface suggest that modifications of the C-terminal sequence of b4 are sufficient to liberate BKa+b4 channels from the ER.

b4-containing channels have a minor contribution to whole-cell BK channel currents in hippocampal neurons
Although many trafficking studies are carried out in heterologous cells, protein overexpression may alter steady-state distributions of channels. A more appropriate test of whether b4containing BK channels are present and functional at the cell surface is to examine BK channel properties in CNS neurons that endogenously express high levels of b4. Because the majority of BK channels in neurons, as in HEK-293 cells, reside in intracellular stores [46], cell-surface immunolocalization of BK channels in brain tissue is difficult to resolve. However, BK channel currents can be electrophysiologically isolated using whole-cell patch clamp recording, a direct method to analyze BK channel function in living neurons.
To identify brain areas that express elevated b4 in control tissue, in situ hybridization using a b4 anti-sense probe was performed. Hybridization in hippocampal area CA3 was substantially greater than in almost any other brain area ( Figure 5A, B). If b4containing BK channels at the cell-surface play a major role in regulating neuronal BK currents, we reasoned that this would be prominent in these neurons.
To probe for the presence of BKa+b4-containing channels, whole-cell patch-clamp recordings from CA3 pyramidal neurons were carried out and BK channel currents were identified using a pharmacological subtraction method ( Figure 5E). The pan-BK antagonist paxilline, which blocks all BK channels, was used to isolate the whole-cell BK current, and this value was compared to the amplitude of the iberiotoxin-sensitive current. If the majority of current is carried by BKa+b4 channels, we reasoned that the amplitude of the iberiotoxin-sensitive current would be significantly smaller than the paxilline-sensitive current (schematized in Figure 5F). Alternatively, if there was little or no current carried by BKa+b4 channels, there should be no difference between the amplitude of the paxilline-sensitive and the iberiotoxin-sensitive current (schematized in Figure 5G).
Whole-cell K + currents were isolated before and after antagonist application and subtracted offline. Because b4-containing channels are slowly activating and may take hundreds of milliseconds to reach peak current [20,21,23], current amplitude was calculated at the end of an 800 ms pulse from 280 to +100 mV ( Figure 5H-J).
Using the pan-BK channel antagonist paxilline, the magnitude of steady-state BK channel current was 1.4060.22 nA (10 nM paxilline; n = 7 cells; Figure 5H). Although these experiments typically used a concentration of paxilline that reflects the low IC 50 established for this drug [47,48], similar current amplitudes were also obtained using drug concentrations that were 100-fold higher (1 mM paxilline 1.3560.26 nA, n = 6 cells; p = 0.9 versus 10 nM paxilline; Figure 5H,J). Thus, it is unlikely that we systematically underestimated the amplitude of BK channel currents.
Surprisingly, the magnitude of iberiotoxin-sensitive BK channel currents was nearly identical to that obtained using the pan-BK channel antagonist paxilline (50 nM iberiotoxin; 1.5960.62 nA, n = 6 cells; p = 0.8 versus 10 nM paxilline; Figure 5I,J). These data suggest that there is little contribution of b4-containing BK channels to whole-cell BK channel currents in CA3 pyramidal neurons ( Figure 5G). It is alternatively possible that BK channels in CA3 neurons may contain b4 with a reduced stoichiometry that is sensitive to iberiotoxin blockade.
The influence of b4 on the biophysical properties of BK channel currents has been well-studied [1,20,21,22,23,24,25,26,27]. In HEK-293 cells, where voltage clamp can be excellent, b4 typically slows the activation kinetics (t) of BK currents [1,20,23,26]. Indeed, these effects of b4 have been postulated to strongly influence channel function in vivo [1]. Estimating t for BK channel currents in neurons is problematic, in part because voltage clamp is imperfect in these cells. Activation time constants for iberiotoxin and paxilline-sensitive currents were comparable (t = 2.0160.56 ms for 10 nM paxilline; 2.8961.22 ms for 50 nM iberiotoxin; p = 0.29).
Some previous studies, including those where recordings from CNS neurons were carried out, have identified iberiotoxin-resistant BK channel currents. For example, in neurons from the dentate gyrus, single-channel recordings clearly have shown iberiotoxinresistant BK channels from membrane patches pulled from the cell soma [1]. To resolve this contradiction and determine whether our inability to observe b4-containing channels might be due to our whole-cell recording approach, we also compared paxilline and iberiotoxin-sensitive whole-cell BK channel currents from dentate gyrus neurons. The magnitude of the iberiotoxin-sensitive current was more than 10-fold smaller than the paxilline-sensitive current (iberiotoxin 0.11+0.2 nA; n = 5 cells versus paxilline 1.6+0.06 nA; n = 3 cells). The activation kinetics of the pharmacologically isolated BK channel current was similar between the iberiotoxin and paxilline-sensitive current, suggesting that this measurement in neurons is an unreliable indicator of the molecular composition of the channel (t for iberitoxin-sensitive current 8.41+1.8 ms versus paxilline 7.26+1.38 ms). These data show that the pronounced effect that b4 has on BK channel trafficking can be influenced by neuron cell type.

BK channel currents are larger in b4 knock-out animals
A critical test of the hypothesis that b4 can regulate whole-cell BK channel current is to examine the amplitude of BK current in CA3 neurons where no b4 is expressed. Consistent with our in situ hybridization results, hippocampal area CA3 showed strong expression of the GFP reporter that had been knocked-in to the b4 expression locus ( Figure 6A). Pharmacological isolation of wholecell BK channel current revealed that CA3 neurons in b4 knock-out animals showed a highly significant, more than 2.5-fold increase in BK channel current amplitude compared to heterozygote littermates (paxilline-sensitive current in b4 +/2 mice: 1.6260.32 nA; n = 11 cells versus b4 2/2 mice 3.5160.59 nA; n = 12 cells; p = 0.01, Figure 6C,D). BK channel current amplitude in heterozygote animals was comparable to values from wild-type animals (see Figure 5J). We saw no effect on the activation kinetics for the pharmacologically subtracted currents, which were comparable between the heterozygote and knock-out groups (t = 2.2060.46 in b4 +/2 mice versus 1.6160.32 in b4 2/2 animals, p.0.1).
These data are in concordance with the negative-regulatory function of b4 on BK channel surface expression that were obtained via live-cell imaging after channel overexpression in heterologous cells. Discussion BK channels, because of their large single-channel conductance and co-activation by depolarization and intracellular Ca ++ , play an important role in regulating neuronal firing. BK channels disproportionately contribute to neuronal whole-cell K + channel currents, where they can carry more than half the total voltagegated K + current. We have shown that expression of cell-surface BK channels is controlled by the presence of the brain-specific b4 subunit. Using a novel FAP to track the location of tagged BK channels at the cell surface and cytoplasm, we find that coexpression of b4 significantly reduces BKa channel protein at the plasma membrane, a function that is dependent upon a Cterminal ER retention/retrieval motif.
In CA3 neurons, which exhibit the highest levels of b4 expression compared to almost any neurons within the rodent CNS, pharmacologically-isolated whole-cell BK channel currents display none of the expected characteristics for BKa+ b4 channels, suggesting that these channels may not be present at the cell surface. Furthermore, genetic ablation of the b4 subunit was sufficient to significantly increase whole-cell BK channel currents in knock-out animals. Thus, we propose that an important function of the CNS-specific BK channel accessory subunit b4 in CA3 neurons is control of cell-surface trafficking of the BK channel complex.
These studies employed a FAP tag to track the location of BK channels in living cells. This methodology has significant advantages compared to more standard techniques, such as GFP protein tags, where the surface fluorescence signal can be overwhelmed by the much larger intracellular stores of channel protein. Although the GFP-based Phluorin [49], a fluorescence protein tag whose signal is quenched in acidic cell compartments, has been useful for studying vesicle fusion [50,51], the nearly neutral pH of the ER has been associated with breakthrough fluorescence that complicates analysis. Advantages of the FAP system are use of membrane permeable and impermeable dyes, high signal-to-noise due to low fluorescence of unbound dye, and the potential to carry out real-time imaging experiments. Since unbound dye has essentially no fluorescence, a specific signal is generated without washing off excess dye, a property that will enable imaging in more complex tissue environments. Additionally, because the fluorescence signal from a single-dye-FAP interaction can be so bright (5 to 20-fold brighter than GFP), this technology may be particularly well-suited to studying the localization and dynamics of individual molecules, such as single BK channels at the plasma membrane.
Much experimental effort has gone into understanding the biophysical consequences of b4 on BK channel function, with little attention to how this auxiliary subunit can control channel localization. Using live-cell imaging and immunolocalization in heterologous cells, as well as electrophysiological measurements in CA3 neurons that express high levels of b4, we find that b4 expression is associated with a dramatic reduction of BK channels at the cell surface. In addition, we find that genetic ablation of b4 is sufficient to significantly enhance whole-cell BK channel currents. This may explain the paradoxically broad expression of b4 in the CNS (Figure 6 and [20,21,22]) despite iberiotoxinpharmocology indicating that BK channels lack b4 in many neurons [52,53,54,55,56,57]. Taken together, our results suggest that the effect of b4 on channel function may be much more indirect than previously imagined.
Nevertheless, a few studies report the presence of iberiotoxininsensitive BK currents in the CNS, specifically in posterior pituitary nerve terminals and dentate gyrus granule neuronal soma and their mossy fiber terminals [1,2,58,59]. Further, the b4 subunit was found to promote surface expression of the related slo3 channel in Xenopus oocytes [60]. How can the present findings be resolved with this? Our comparison of iberiotoxin and paxilline-sensitive currents in dentate gyrus neurons indicates that in some neurons, b4 is not sufficient to reduce cell-surface channel expression. Thus, there may be additional factors, including activation of signaling pathways, which can regulate channel localization in a cell-type specific manner.
Some studies have also suggested that the b4 subunit may regulate subcellular distribution of the BK channels into axonal or dendritic compartments [6,59,61,62]. Because of the limitations of voltage clamp in neurons (i.e. poor voltage control in the distal processes of the cell), such b4-containing channels might be hard to detect in CA3 neurons. It is also possible that BK channels can be redistributed to the plasma membrane under some circumstances, for example, following mechanical dissociation of cells prior to analysis [36,62,63,64], or during periods of high firing. Redistribution could occur over very short time intervals (100 s of ms), during firing bursts, or might be regulated over the course of many minutes. Such regulation has been observed for K v 2.1 type K + channels [46]. Our finding that b4 expression is associated with the regulation of surface levels of BK channels suggests a new mechanism for dynamic control of channel activity.
A caveat of the present study is that direct association of the BKa subunit with b4 was not directly demonstrated in transfected cells. However, the rescue of surface expression with cotransfection of the b4 mutant construct suggests that there is some interaction between the two proteins. Furthermore, our preliminary recordings from transfected HEK-293 cells indicate that the increase in surface expression observed in BKa+b4-Ala expressing cells is linked to iberiotoxin-resistant whole-cell currents. Further studies will be required to unambiguously demonstrate the interaction of BKa and b4 in our experimental preparation.
Enhanced BK channel currents have been linked to neuronal hyperexcitability and epilepsy in cortical neurons [1,7,9,65]. Indeed, BK channel antagonists can reduce bursting in abnormally active tissue [4,7] and have a profound anticonvulsant effect in vivo [10]. The large contribution of BK channels to the total K + channel current in CA3 neurons suggests that these channels may play a particularly important role in controlling the excitability of these cells.
The data presented here characterize the detailed molecular mechanisms that control cellular BK channel trafficking. We have identified a new role for the BK b4 channel subunit -ER retention -as well as the specific amino acid residues that are necessary to direct this function. Additionally, we have shown that BK channels in CA3 neurons are not associated with the pharmacological and biophysical hallmarks of b4-containing channels as described in previous studies using heterologous cells, and that the absence of b4 is sufficient to significantly enhance whole-cell BK channel current. Because these studies were carried out in CNS neurons in acute brain slices, these findings may be particularly relevant to channel function in vivo. The dynamic regulation of endogenous BK channel localization in neurons is an exciting avenue for future investigations.