Cysteine String Protein Limits Expression of the Large Conductance, Calcium-Activated K+ (BK) Channel

Large-conductance, calcium-activated K+ (BK) channels are widely distributed throughout the nervous system and play an essential role in regulation of action potential duration and firing frequency, along with neurotransmitter release at the presynaptic terminal. We have previously demonstrated that select mutations in cysteine string protein (CSPα), a presynaptic J-protein and co-chaperone, increase BK channel expression. This observation raised the possibility that wild-type CSPα normally functions to limit neuronal BK channel expression. Here we show by Western blot analysis of transfected neuroblastoma cells that when BK channels are present at elevated levels, CSPα acts to reduce expression. Moreover, we demonstrate that the accessory subunits, BKβ4 and BKβ1 do not alter CSPα-mediated reduction of expressed BKα subunits. Structure-function analysis reveals that the N-terminal J-domain of CSPα is critical for the observed regulation of BK channels levels. Finally, we demonstrate that CSPα limits BK current amplitude, while the loss-of-function homologue CSPαHPD-AAA increases BK current. Our observations indicate that CSPα has a role in regulating synaptic excitability and neurotransmission by limiting expression of BK channels.


Introduction
Cysteine string protein (CSPa) is a presynaptic co-chaperone for Hsc70 that protects neurons from degeneration and synaptic loss [1]. CSPa is a synaptic vesicle-associated protein bearing a characteristic J domain, as well as a cysteine rich 'string' region [2]. Mutations of CSPa in humans are associated with adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL), which is a progressive neurodegenerative disorder characterized by psychiatric manifestations, seizures, progressive dementia and motor impairment [3][4][5]. Disruption of the CSPa gene causes impaired presynaptic neurotransmission in Drosophila melanogaster [6] and fulminant neurodegeneration in mice [1,7]. In CSPa null mice, synapse loss occurs in an age- [1] and activity-dependent manner [8,9]. The cellular mechanisms that underlie CSPa's neuroprotective function remain to be established.
Recent work from our group has shown that the largeconductance, calcium-activated K+ (BK) channel, which plays an important role in neuronal membrane excitability, is markedly increased by disabling mutations of CSPa [10]. Specifically, deletion of CSPa residue 116, replacement of Leu 115 by Arg lead to increased BK channel density in cultured neurons. Moreover, CSPa null mice exhibit 2.5 fold higher BK channel expression compared to wild type mice, whereas the densities of other important cation channels (i.e. Ca v 2.2, K v 1.1 and K v 1.2) do not change. These findings suggest that one particular function of CSPa may be to limit the cellular expression of BK channels.
In the present study, we extend our recent work and characterize the effect of wild type CSPa on BK channel density.
In particular, we have examined the prediction that wild-type CSPa limits the cellular level of BK channels, however, such an effect may only be evident under conditions of elevated BK channel expression. To create such a model system, murine neuroblastoma cells were transfected with murine BK channel cDNA in either the absence or presence of co-transfected wildtype, CSPa, or loss-of-function CSPa HPD-AAA . Using this strategy, we observed that CSPa limits BK channel expression in a timeand dose-dependent manner, and that the J domain of CSPa is essential for this regulatory action, as revealed by structurefunction analysis. Finally, we show that while CSPa reduces BK channel current, loss-of-function CSPa HPD-AAA increases BK current amplitude. These findings thus demonstrate that wildtype CSPa is able to restrict neuronal BK channel expression, and further help to explain why loss of CSPa function, due to genetic mutations, lead to elevated BK density in the CNS.

Robust BK Channel Expression is Reduced by CSPa
We have recently reported that interference of CSPa activity, either by genetic disruption (i.e. CSPa 2/2 mice) or expression of dysfunctional CSPa in a neuronal cell line, is associated with a significant elevation of BK channel density at the cell surface [10]. The data arising from the experimental strategies designed to disrupt CSPa function strongly suggest that part of CSPa's normal function in the CNS may be to regulate neuronal BK expression, which would be expected to influence neuronal excitability. We rationalized that if CSPa truly acts in this capacity, then a strategy involving elevated expression of wild-type CSPa would predictably lead to a decrease in BK channel levels, and provide direct insights into the cellular actions of wild-type CSPa. To test this hypothesis, we utilized a transient transfection strategy in order to express murine brain BK channel a subunits at a high level, thereby providing a robust baseline signal from which one could reliably detect hypothesized decreases in BK channel levels in the presence of increasing amounts of wild-type CSPa. As shown in Figure 1A, co-expression of murine brain BKa subunits (Butler et al, 1993) in native CAD cells with increasing amounts of myc-tagged, wildtype CSPa led to a dose-dependent decrease in the cellular level of BKa subunit protein, which correlated with increasing cellular expression of CSPa. Whereas co-transfection of cells with a low amount of CSPa cDNA (i.e. 0.25 mg) had no significant effect on BKa channel expression at 24 hour post-transfection, addition of either 0.5 mg or 0.75 mg CSPa cDNA significantly reduced the level of BKa protein. Figure 1B displays quantification of the CSPa-dependent changes in BKa subunit expression; data are normalized to the level of BKa subunit in the presence of empty pCMV expression vector, which served as the co-transfection control. These experiments revealed that wild-type CSPa is capable of decreasing BK channel density in a dose-dependent manner.
We next examined the time dependence of the observed CSPamediated regulation of BK channel expression. Figures 1C&D show that the extent of CSPa-mediated decrease in BKa subunit expression was greater at 48 hours post-transfection using either 0.25 or 0.75 mg CSPa cDNA compared with the expression observed at 24 hours. The greater effect of CSPa on BK channel expression at 48 hours was not due to enhanced expression of CSPa at the 48 versus 24 hour time point, as shown by anti-myc detection ( Figure 1C). To quantify the greater effect of CSPa at 48 hours, we compared BK channel expression at this time point with the expression at 24 hours, which was first normalized to 100% for both 0.25 and 0.75 mg CSPa transfection conditions ( Figure 1D). With a higher level of CSPa expression (i.e. 0.75 vs 0.25 mg), a more substantial decrease in BK channel expression at the 48 hour time point was noted. No CSPa-mediated changes were detected in cellular level of b-actin and co-transfection with the pCMV vector alone did not result in any time-dependent alteration of BK channel density. The data displayed in Figure 1 further illustrate the 3 distinct species of wild-type CSPa that can be identified by Western blot; a 26 kDa immature form, a 34 kDa mature palmitoylated protein and a 70 kDa CSPa dimer, as described previously [11,12]. Taken together, these findings show that the wild-type CSPa is able to lower BKa subunit expression in a doseand time-dependent manner.
Physiologically, BKa subunits co-assemble into a tetrameric complex with a single, ion conducting pore structure that is subject to regulation by auxiliary b-subunits. Since chaperones typically regulate the assembly and/or disassembly of protein complexes (e.g. DnaJC6 mediates clathrin disassembly [13]), we investigated the possibility that the presence of auxiliary BKb-subunits may alter the observed CSPa-mediated regulation of BK channel expression. As shown in Figure 2, co-expression of the BK channel auxiliary subunits, BKb4 (panel A) or BKb1 (panel B), did not influence the CSPa-mediated decrease in BK channel levels in transfected CAD cells. Co-expression of CSPa still reduced BK channel levels in the presence of BKb1 or BKb4, compared to vector control. Interestingly, CSPa did not noticeably alter the expression of either BKb1 or BKb4 ( Fig. 2A and B, middle panels). In CAD cells transfected with BKa cDNA in either the absence or presence of CSPa, we further observed that the expression levels of several endogenous membrane proteins (i.e. SNAP25, syntaxin1A and GAP43) were not altered ( Figure 2E). Moreover, we found that co-transfected CSPa does not alter the expression of the membrane proteins syntaxin 1A or the TRPC6 channel isoform following their transient expression in CAD cells ( Figure 2F). Collectively, these results indicate that BKb1 and BKb4 do not influence the regulation of BK channel expression by CSPa and that CSPa selectively reduces BKa subunit expression, but not that of either BKb1 or BKb4 or a number of other membrane proteins.

The J Domain is Essential for CSPa-mediated Reduction in BKa Channels
To elucidate the structural elements within CSPa responsible for its regulation of BK channel expression, a series of CSPa deletion mutants were constructed and co-transfected with BKa subunit cDNA in CAD cells. The myc-tagged CSPa constructs are shown schematically in Figure 3A, and each cDNA construct generated a protein that migrated at the expected molecular weight when analyzed by SDS PAGE and western blotting (Fig. 3B). Experimentally, all C-terminal truncations of CSPa were still capable of decreasing BK channel expression following cotransfection. CSPa 1-90 and CSPa 1-100 reduced BK channel expression to levels of 41.9610.9% and 48.169.5% of control, respectively, and larger reductions in channel expression were observed in the presence of CSPa 1-82 (15.168.2% of control) and CSPa 1-112 (16.568.6% of control), relative to cells co-transfected with the pCMV vector (Fig. 3C). These findings indicate that the N-terminal region of CSPa, which contains the J domain, is sufficient for the observed regulation of BK channel expression by wild-type CSPa. This conclusion was further confirmed by examining N-terminal truncations of CSPa lacking the J domain. Neither CSPa 113-198 (97.3630.5%) nor CSPa 137-198 (87.367.6%) had a significant effect on BK channel expression (Fig. 3C), emphasizing the importance of the N-terminal region of CSPa for the regulation of BK channel expression. As expected, multiple bands are observed for CSPa 113-198 which includes the cysteine string region, while a single immunoreactive band was detected for CSPa 137-198 which does not include the cysteine string region. We have previously demonstrated that residues 83-136, encoding the linker region and cysteine string region are required for CSPa oligomerization [11] as exemplified in the right hand side of Figure 3B. Interestingly, deletion of the cysteine string region (CSPa DC ) did not preclude the ability of CSPa to reduce BK channel expression (18.867.0%).
Several neuronal proteins contain a J-domain [14], which represents the signature motif of all members of the J protein family. Given the functional importance of the J-domain for CSPa's observed regulation of BK channel levels, we asked whether J-domains from related chaperones were sufficiently conserved to substitute in this process. To address this question, we generated chimeras of CSPa in which the native J-domain was replaced by the J-domain from another J protein chaperone. The resulting chimeric constructs, shown schematically in Figure 4A, consist of a CSPa background and a substituted J-domain obtained from Hsp40 (DnaJB1), Rdj2 (DnaJA2) and Rme8 (DnaJC13), which display 52%, 52% and 44% amino acid identity, respectively, with the J-domain of CSPa (rat isoform). Western blot analysis demonstrated that co-transfection with individual CSPa/J-domain chimeras decreased BK channel expression compared to the pCMV vector ( Figure 4B). Quantification of these effects revealed that the CSPa chimeras -CSPaJD Hsp40 (17.063.7%), CSPaJD Rdj2 (23.862.0%) and CSPaJD Rme8 (17.569.8%), along with wild-type CSPa (22.663.4%) and the truncation mutant CSPa 1-82 (17.468.1%), all produced a statistically significant and comparable reduction in BK channel expression. As depicted in Figure 4B, all three CSPa chimeric constructs expressed to a similar level compared with wild-type CSPa, and three distinct molecular species of wild-type and chimeric CSPa isoforms (i.e. 26 kDa, 34 kDa and 70 kDa) were readily identified by Western blot, regardless of the substituted J-domain. (Note that the CSPa 1-82 construct was not detected on this blot, due to its rapid migration during SDS-PAGE. However, it is evident from Figure 3B that this construct expresses well under our experimental conditions). As displayed in preceding figures, b-actin staining was utilized to ensure similar protein loading for the various cell lysates. Based on these data, it appears that the J-domain of CSPa is necessary for the regulation of BK channel expression and that individual J-domains from related J protein family members can functionally substitute for the native J-domain in CSPa.  Hsc70 is a critical interacting partner of CSPa, and displays a low basal ATPase activity that is activated following interaction with the J-domain of CSPa [15]. We examined the possibility that over-expression of Hsc70 alone may be able to independently evoke a reduction in BK channel levels, similar to that observed for CSPa. CAD cells were transiently co-transfected with cDNA encoding the BKa subunit, along with either HA-tagged, wild-type Hsc70, the HA-tagged ATPase domain of Hsc70, which displays constitutive activity [15] or pCMV vector (negative control). Figure 5 demonstrates that no significant changes in BK channel expression were observed when either full length Hsc70 (132.9630.3%) or the active ATPase domain (i.e. Hsc70 1-386 ) of Hsc70 (69.7618.9%) was co-expressed with the BKa subunit, compared with pCMV vector alone. These data indicate that increased cellular levels of Hsc70 are not sufficient to regulate BK channel expression and that activation of Hsc70 by CSPa is likely required. Our observations are thus similar to those described by Walker and colleagues, who reported that DnaJA1, DnaJA2 and DnaJA4 reduced hERG channel maturation, whereas overexpression of Hsc70 alone had no effect on maturation events [16].

BK Channel Current Density is Decreased by CSPa
To determine whether the observed CSPa-mediated decrease in the cellular BKa protein level also reflected a reduction in functional BK channels at the cell surface, we carried out single cell patch clamp recordings of CAD cells transiently transfected with BKa subunit cDNA in the absence or presence of CSPa cDNA. Co-transfection of GFP under all conditions was utilized as a marker to identify transfected cells. Figure 6A shows representative current families recorded from transfected CAD cells expressing BKa subunit alone, or BKa along with either wild-type CSPa or the dysfunctional CSPa mutant CSPa HPD-AAA . Following control recordings, cells were treated with the highly selective BK channel blocker penitrem A to isolate BK channel currents. The bottom row of traces in Figure 6A display the magnitude of BK channel-mediated, penitrem A-sensitive current observed under the three different transfection conditions. The currentvoltage plot displayed in Figure 6B quantifies the effect of CSPa co-expression on BK channel current density. In the presence of wild-type CSPa, BK current density was significantly decreased compared with BK channel alone, whereas co-transfection with the loss-of-function CSPa HPD-AAA led to a higher current density at very positive test pulse voltages. In parallel experiments, these observed CSPa-mediated changes in cell surface BK channel density and were confirmed by cell surface biotin labeling, followed by streptavidin pull-down and Western analysis (refer to inset). We have previously reported that CSPa HPD-AAA did not alter the expression of other membrane-associated proteins (e.g. GAP43 and flotillin) [10]. Taken together, these data demonstrate that CSPa limits the expression of functional BK channels at the cell surface.

Discussion
We have found that CSPa, a presynaptic neuroprotective chaperone, acts to significantly limit BK channel current and BK channel expression. The CSPa-mediated reduction in BK channels is dose-and time-dependent and the N terminal J-domain of CSPa is essential for the noted decrease ( Figures 1&3).
Our earlier results reporting that loss/disruption of CSPa activity in either a knockout mouse or a neuronal cell line markedly increase BK channel expression [10] indirectly suggested that in the CNS, wild-type CSPa may function to limit cellular BK channel expression. In order to directly examine such an effect of wild-type CSPa, we utilized an expression strategy to force an imbalance between BK channels and endogenous CSPa, thereby allowing BK channel protein to be present at elevated levels so that decreases in BK channel density induced by CSPa could be precisely monitored. Physiologically, BK channel activity is regulated by multiple mechanisms, including modulatory accessory subunits [17], alternative splicing [18], phosphorylation [19] and palmitoylation [20]. Data presented here suggest that the CSPa-mediated reduction in BK channel density occurs in a graded fashion that is dependent upon the relative expression between BK channels and CSPa (i.e. when BK channel expression is high, the cellular level of CSPa must also be elevated in order for a reduction to occur). While BK channel expression was reduced by CSPa, at low expression levels of BK channels, the CSPamediated reduction was minimal. These data thus provide evidence that wild-type CSPa normally acts to restrict neuronal BK channel expression.
It is possible that the CSPa-induced regulation of BK channel current represents a key event contributing to the CSPa-mediated synapse protection. By influencing membrane excitability and action potential firing, abnormal BK channel current density could be the trigger for the cascade of events leading to neurodegeneration in ANCL patients and the fulminant neurodegeneration observed in CSPa-KO mice. Nonetheless, association studies have identified several other synaptic proteins that are also likely clients for CSPa and may contribute to the neuropathology associated with the loss/dysfunction of CSPa [21][22][23][24][25][26][27][28]. Trafficking proteins in CSPa controlled pathways include t-SNARE protein SNAP25 (synaptosomal associated protein of 25 kDa), which is required for exocytosis, the GTPase dynamin1, which is essential for endocytosis, and a-synuclein, which is implicated in synaptic vesicle function [7,[29][30][31][32]. It will therefore be important to determine whether the increased BK channel expression observed in our studies reflects a membrane trafficking defect involving one or more these putative pathways associated with CSPa activity and degeneration. While the importance of CSPa in synapse protection is well established, the series of events underlying protection and degeneration cascades remains to be determined.
In conclusion, we provide evidence that the presynaptic chaperone, CSPa, limits BK channel density. We further speculate that elevated BK channel expression may be involved in the severe age-dependent [1] and activity-dependent degeneration [8,9] reported in the CNS and motor neurons of animal models displaying loss/dysfunction of CSPa [1,8,32,33].

Immunoblotting
Proteins (30 mg) were electro-transferred from SDS-polyacrylamide gels to nitrocellulose membrane (0.2 mm pore size) in 20 mM Tris, 150 mM glycine and 12% methanol. Membranes were blocked in phosphate-buffered saline (PBS) containing 0.1% Tween 20, 4% skim milk powder and then incubated with primary antibody overnight at 4uC. The membranes were washed and incubated with horseradish peroxidase-coupled secondary antibody. The signal was developed using West Pico reagent (Pierce Biotechnology Inc.) and exposed to Kodak x-ray film. Bound antisera were quantified using a Biorad Fluor-S MultiImager Max and QuantityOne 4.2.1 software.

Whole Cell Patch Clamp Recordings
Voltage-clamp measurements were performed using conventional, ruptured membrane patch clamp methodology in combination with an Axopatch 200B amplifier, Digidata 1440 series analogue/digital interface and pClamp v10 software. Whole cell electrical signals were typically filtered at 1-2 kHz and sampled at 5 kHz. Glass micropipettes (2-4 MV tip resistance) were pulled from thin-walled borosilicate capillaries and were filled with a solution containing 100 mM KOH, 30 mM KCl, 1 mM MgCl 2 , 0.005 mM CaCl 2 , 10 mM HEPES, pH 7.3 with methanesulfonic acid. The bath chamber was placed on the stage of Nikon TE2000 inverted microscope equipped with epifluorescence illumination and perfused with a modified Ringer's saline solution containing 135 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 2.5 mM CaCl 2 , 5 mM 4-aminopyridine and 10 mM HEPES, pH 7.3 with 1N NaOH. Cells in the bath chamber were constantly superfused at ,2 ml/ min and solution changes were performed by gravity flow from a series of elevated solution reservoirs using manually controlled solenoid valves. All electrophysiological recordings were performed at 35-37uC. CAD cells seeded on 35 mm sterile plastic dishes were transiently transfected with separate pcDNA3.1-based constructs encoding BKa subunit, wild-type or mutant CSPa. A separate cDNA construct encoding enhanced GFP was included in all conditions as a fluorescent marker of transfection. Cells were transfected for 5-6 hours using Lipofectamine 2000 as the transfection reagent, and the total amount of cDNA added per dish was typically 1.8-2 mg. Transfected cells were identified in the recording chamber by their green fluorescence using 488 nm excitation and 510 nm emission filters.

Biotinylation of Cell Surface BK Channels
CAD cells transiently co-expressing murine brain BKa subunit [35] and CSPa variants were washed three times with PBS and incubated with EZ-Link Sulfo-NHS-SS Biotin (Thermo Scientific) (1 mg/ml) in PBS for 30 min at 4uC. As a negative control, cells were incubated only with PBS. The reaction was neutralized by addition of 1% (w/v) BSA in PBS for 10 min at 4uC. After neutralization, cells were washed with ice-cold PBS to remove non-reacted biotin, and were harvested in 1ml of PBS containing 1% v/v Triton X-100 and protease inhibitor (complete, EDTAfree, Sigma) by an incubation for 2-5 minutes on ice. The lysates were centrifuged at 15,0006g for 15 min at 4uC and the soluble protein concentration was determined using the Bradford assay (Bio Rad).
For streptavidin pull-down, 1 mg of the soluble protein lysate was incubated with 100 ml streptavidin agarose beads (50% slurry) (Thermo Scientific) overnight at 4uC on a rotator. Beads were centrifuged at 3.0006g and washed with 1% Triton X-100 in PBS. Following centrifugation, biotinylation proteins were eluted from the beads by adding 26 Laemmli sample buffer (62.2 mM Tris HCl pH 6.8, 7.5% v/v Glycerol, 2% w/v SDS, 0.015 mM Bromophenol Blue, 1.2% v/v b-Mercaptoethanol and 100 mM DTT) and incubated at 37uC for 1 h. Following elution, proteins were separated by SDS-PAGE.