CAD cells were transiently transfected with 1 µg BKα subunit cDNA plus 1 µg BKβ4 subunit cDNA ( A ) or 1 µg BKβ1 subunit cDNA ( B ), along with either 0.75 µg CSPα or pCMV vector (control). 24 h post-transfection, protein expression was analyzed by Western Blot with primary antibodies recognizing BKα subunit, BKβ4 subunit, BKβ1 subunit, myc epitope tag, and β-actin. C and D, Quantification of BKα subunit expression in CAD cells in the absence or presence of co-transfected CSPα, along with the presence of either BKβ4 (panel C) or BKβ1 (panel D). E CAD cells were transfected with cDNAs encoding pCMV vector alone, BKα subunit or BKα subunit+CSPα and the indicated proteins were evaluated by Western blot analysis. F CAD cells were transfected with TRPC6 channel or syntaxin1A cDNA in either the absence of presence of CSPα cDNA. Detection of β-actin was utilized to verify equal protein sample loading. Data are presented as mean ± SE of 3 similar experiments; statistical significance was determined using one way ANOVA, *p<0.05; **p<0.01. Data shown in E and F are representive of 4 independent experiments.

Physiologically, BKα subunits co-assemble into a tetrameric complex with a single, ion conducting pore structure that is subject to regulation by auxiliary β-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 BKβ-subunits may alter the observed CSPα-mediated regulation of BK channel expression. As shown in Figure 2 , co-expression of the BK channel auxiliary subunits, BKβ4 (panel A) or BKβ1 (panel B), did not influence the CSPα-mediated decrease in BK channel levels in transfected CAD cells. Co-expression of CSPα still reduced BK channel levels in the presence of BKβ1 or BKβ4, compared to vector control. Interestingly, CSPα did not noticeably alter the expression of either BKβ1 or BKβ4 ( Fig. 2A and B , middle panels). In CAD cells transfected with BKα cDNA in either the absence or presence of CSPα, 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 CSPα 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 BKβ1 and BKβ4 do not influence the regulation of BK channel expression by CSPα and that CSPα selectively reduces BKα subunit expression, but not that of either BKβ1 or BKβ4 or a number of other membrane proteins.

We next examined the time dependence of the observed CSPα-mediated regulation of BK channel expression. Figures 1C&D show that the extent of CSPα-mediated decrease in BKα subunit expression was greater at 48 hours post-transfection using either 0.25 or 0.75 µg CSPα cDNA compared with the expression observed at 24 hours. The greater effect of CSPα on BK channel expression at 48 hours was not due to enhanced expression of CSPα at the 48 versus 24 hour time point, as shown by anti-myc detection ( Figure 1C ). To quantify the greater effect of CSPα 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 µg CSPα transfection conditions ( Figure 1D ). With a higher level of CSPα expression (i.e. 0.75 vs 0.25 µg), a more substantial decrease in BK channel expression at the 48 hour time point was noted. No CSPα-mediated changes were detected in cellular level of β-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 CSPα that can be identified by Western blot; a 26 kDa immature form, a 34 kDa mature palmitoylated protein and a 70 kDa CSPα dimer, as described previously [11] , [12] . Taken together, these findings show that the wild-type CSPα is able to lower BKα subunit expression in a dose- and time-dependent manner.

A. Native CAD cells were transiently transfected with 1 µg cDNA encoding a neuronal BKα subunit, along with different amounts of myc-tagged CSPα (0.25 µg, 0.5 µg and 0.75 µg). Empty pCMV expression vector (0.75 µg) was co-transfected with 1 µg BKα subunit cDNA as a transfection control. 24 h post-transfection, the cells were lysed and the expression of BKα subunit and myc-tagged protein was analyzed by Western Blot. β-actin detection is shown to verify comparable sample loading. B. Histogram depicting quantification of BKα subunit levels in CAD cells co-transfected with increasing amounts of CSPα cDNA. Data are presented as mean ± SE of 5 similar experiments; *p<0.05 vs. pCMV vector control. C. Cells were transfected with 1 µg cDNA encoding BKα subunit along with either 0.25 µg or 0.75 µg of myc-tagged CSPα or 1 µg of pCMV. 24 h and 48 h post-transfection, BKα subunit expression was analyzed by Western Blot. D. Histograms depicting quantification of immunoreactive BKα subunit observed in the presence of co-transfected CSPα, as displayed in panel C. BKα subunit immunoreactivity detected at 48 h is expressed relative to the level of BKα subunit observed at 24 h; data are presented as mean ± SE of 4 similar experiments. Statistical significance was determined using one way ANOVA, *p<0.05; **p<0.01.

We have recently reported that interference of CSPα activity, either by genetic disruption (i.e. CSPα −/− mice) or expression of dysfunctional CSPα 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 CSPα function strongly suggest that part of CSPα’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 CSPα truly acts in this capacity, then a strategy involving elevated expression of wild-type CSPα would predictably lead to a decrease in BK channel levels, and provide direct insights into the cellular actions of wild-type CSPα. To test this hypothesis, we utilized a transient transfection strategy in order to express murine brain BK channel α 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 CSPα. As shown in Figure 1A , co-expression of murine brain BKα subunits (Butler et al, 1993) in native CAD cells with increasing amounts of myc-tagged, wild-type CSPα led to a dose-dependent decrease in the cellular level of BKα subunit protein, which correlated with increasing cellular expression of CSPα. Whereas co-transfection of cells with a low amount of CSPα cDNA (i.e. 0.25 µg) had no significant effect on BKα channel expression at 24 hour post-transfection, addition of either 0.5 µg or 0.75 µg CSPα cDNA significantly reduced the level of BKα protein. Figure 1B displays quantification of the CSPα-dependent changes in BKα subunit expression; data are normalized to the level of BKα subunit in the presence of empty pCMV expression vector, which served as the co-transfection control. These experiments revealed that wild-type CSPα is capable of decreasing BK channel density in a dose-dependent manner.

The J Domain is Essential for CSPα-mediated Reduction in BKα Channels

To elucidate the structural elements within CSPα responsible for its regulation of BK channel expression, a series of CSPα deletion mutants were constructed and co-transfected with BKα subunit cDNA in CAD cells. The myc-tagged CSPα 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 CSPα were still capable of decreasing BK channel expression following co-transfection. CSPα 1–90 and CSPα 1–100 reduced BK channel expression to levels of 41.9±10.9% and 48.1±9.5% of control, respectively, and larger reductions in channel expression were observed in the presence of CSPα 1–82 (15.1±8.2% of control) and CSPα 1–112 (16.5±8.6% of control), relative to cells co-transfected with the pCMV vector (Fig. 3C). These findings indicate that the N-terminal region of CSPα, which contains the J domain, is sufficient for the observed regulation of BK channel expression by wild-type CSPα. This conclusion was further confirmed by examining N-terminal truncations of CSPα lacking the J domain. Neither CSPα 113–198 (97.3±30.5%) nor CSPα 137–198 (87.3±7.6%) had a significant effect on BK channel expression (Fig. 3C), emphasizing the importance of the N-terminal region of CSPα for the regulation of BK channel expression. As expected, multiple bands are observed for CSPα 113–198 which includes the cysteine string region, while a single immunoreactive band was detected for CSPα 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 CSPα oligomerization [11] as exemplified in the right hand side of Figure 3B. Interestingly, deletion of the cysteine string region (CSPα ΔC ) did not preclude the ability of CSPα to reduce BK channel expression (18.8±7.0%).

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larger image TIFF original image Download: Figure 3. The J domain of CSPα reduces BK channel expression. A. Schematic of myc-tagged full length CSPα and CSPα deletion constructs. B. Western blot analysis of BK channel expression in CAD cells 24 h post-transfection with 1 µg cDNA encoding BKα subunit along with 0.75 µg myc-tagged full length CSPα cDNA or the indicated deletion constructs. As a transfection control, 0.75 µg empty pCMV was co-transfected with BKα subunit cDNA. 30 µg of cell lysate isolated under each experimental condition was separated by SDS-PAGE, probed with an anti-BKα subunit antibody and an anti-myc antibody. The histograms in panel C quantify changes in BK channel expression in the presence of wild-type CSPα and individual CSPα deletion mutants. Statistically significant differences from the pCMV control (set to 100%) were determined by one-way ANOVA; *p<0.05; **p<0.001. https://doi.org/10.1371/journal.pone.0086586.g003

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 CSPα’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 CSPα 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 CSPα 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 CSPα (rat isoform). Western blot analysis demonstrated that co-transfection with individual CSPα/J-domain chimeras decreased BK channel expression compared to the pCMV vector (Figure 4B). Quantification of these effects revealed that the CSPα chimeras - CSPαJD Hsp40 (17.0±3.7%), CSPαJD Rdj2 (23.8±2.0%) and CSPαJD Rme8 (17.5±9.8%), along with wild-type CSPα (22.6±3.4%) and the truncation mutant CSPα 1–82 (17.4±8.1%), all produced a statistically significant and comparable reduction in BK channel expression. As depicted in Figure 4B, all three CSPα chimeric constructs expressed to a similar level compared with wild-type CSPα, and three distinct molecular species of wild-type and chimeric CSPα 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 CSPα 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, β-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 CSPα 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 CSPα.

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larger image TIFF original image Download: Figure 4. CSPα chimeras with substituted J domains alter BK channel expression. A. Schematic of wild-type CSPα and CSPα chimeras in which the J domain of CSPα is replaced by the J domain of the related J proteins Hsp40, Rdj2 or Rme8. B. Western blot analysis of BK channel expression in CAD cells 24 h following transfection with cDNA encoding BKα subunit and myc-tagged CSPα or the indicated CSPα myc-tagged chimeric constructs. 0.75 µg of pCMV vector was co-transfected with 1 µg cDNA encoding BKα subunit as a transfection control. Expression of myc-tagged proteins is shown by western blot, along with β-actin detection to verify similar sample loading. C. Histogram showing quantification of BK channel expression in the presence of either wild-type or J-domain (JD) substituted CSPα chimeras relative to cells co-transfected with pCMV vector. Mean data were obtained from 3–4 independent experiments, and statistically significant differences were determined by one-way ANOVA; **p<0.001. https://doi.org/10.1371/journal.pone.0086586.g004

Hsc70 is a critical interacting partner of CSPα, and displays a low basal ATPase activity that is activated following interaction with the J-domain of CSPα [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 CSPα. CAD cells were transiently co-transfected with cDNA encoding the BKα 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 (132.9±30.3%) or the active ATPase domain (i.e. Hsc70 1–386 ) of Hsc70 (69.7±18.9%) was co-expressed with the BKα 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 CSPα 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 over-expression of Hsc70 alone had no effect on maturation events [16].