BK channel was identified as a possible target protein of the CSPα chaperone complex based on our initial observation of robust changes in BK channel density in neuroblastoma cells co-expressing CSPα mutant proteins. Members of the J-protein family have been reported to regulate the trafficking/expression of several ion channel types, including: the hERG (human ether-a-go-go related gene) K+ channel37,38, CFTR (cystic fibrosis transmembrane conductance regulator)39 and the K ATP channel (ATP-sensitive K+ channel)40, leading to the prospect that J-proteins are components of the cellular machinery that make ‘triage’ decisions regarding whether ion channels are trafficked and/or retained at the cell surface or degraded. To study the relationship between CSPα and BK channel expression, we first measured BK channel expression in wild-type and CSPα knockout mice. CSPα null mice are normal at birth, but after a lag period of 2–3 weeks, they stop gaining weight and develop blindness, activity-dependent neuronal degeneration and progressive motor impairments. CSPα null mice typically die prematurely between 40–80 days14. Heterozygous mutant mice contain less CSPα protein than wild-type controls, while homozygous mutant mice lack detectable CSPα14. Figure 1 shows the expression of BK channel, Ca v 2.2, Kv1.1 and K v 1.2 channels in whole brain synaptosomal membrane fractions from CSPα−/−, CSPα−/+ and CSPα+/+ mice, as assessed by western blotting. BK channel expression is significantly elevated in CSPα null mice, whereas heterozygotes show expression levels similar to the wild-types. Moreover, the observed increase in BK channel is selective, as the tissue levels of Ca v 2.2, K v 1.1 and K v 1.2 channels are not significantly altered in the brains of either the CSPα−/− or CSPα−/+ mice compared with the wild-type control. Interestingly, BK channel α subunit mRNA was not elevated in the brains of CSPα−/− mice, as determined by quantitative RT-PCR (Table 1), suggesting that the increased level of BK channel protein in CSPα−/− brain tissue is not due to a transcriptional event. It is also well known that CSPα is primarily expressed in tissues exhibiting regulated secretion, such as the brain and pancreas16,41. In murine aortic smooth muscle, which has prominent BK channel levels, we observed no difference in the average level of BKα subunit expression amongst the three genetic backgrounds (Figure 1C). Given that CSPα is not expressed in the aorta, this finding is not unexpected.

Table 1 BK Channel (KCNMA1) mRNA levels in wild-type and CSPα knockout micea Full size table

Figure 1 Comparison of protein levels for BK, Ca v 2.2, K v 1.1 and K v 1.2 channels in whole brain tissue from CSPα wild-type, heterozygous and null mice. (A) Representative western blot data of the pore-forming α subunits of BK channel, Ca v 2.2 channel, K v 1.1 and K v 1.2 channels detected in synaptosome-enriched membrane fractions prepared from whole brain (age P23–27). The total protein loaded per lane was 40 μg; detection of β-actin on the same blots was used to verify equal loading amongst the various lanes. The data shown in panel A were selected from full-length western blot images, which are displayed in Supplementary Figure 1. (B) Histogram showing average data for BK, Ca v 2.2, K v 1.1 and K v 1.2 channel protein in the wild-type, heterozygous and null brain samples quantified by camera-based detection of emitted chemiluminescence. To perform quantification, the ratio of detected channel protein to β-actin for a given sample was calculated for all three genetic backgrounds. Channel density data for the heterozygote and null tissues were then normalized to the wild-type tissue by dividing all calculated ratios by the wild-type ratio for a given channel species. (C) Histogram showing BK channel expression detected in aorta from CSPα heterozygous and null mice relative to wild-type mice. Quantification of BKα subunit detection was performed as described in panel B. Averaged Western blot data were derived from 5–6 animals (panel B) and 3–4 animals (panel C) of each genetic background (2–3 litters). * indicates a statistically significant difference from the heterozygote and wild-type values, as determined by one-way ANOVA and a Tukey's post-hoc test; p < 0.05. Full size image

In order to gain mechanistic insight into the influence of CSPα on BK channel proteostasis, we generated a murine CNS-derived catecholaminergic (CAD) cell line42 stably expressing a murine neuronal BK channel (43; see Methods Section) that would enable rigorous electrophysiological and immunocytochemical analyses of BK channel levels. The expression of BK channels in these cells was measured in the presence of myc-tagged CSPα or the myc-tagged loss-of-function mutant CSPα HPD-AAA . Functionally, mutation of the HPD motif (residues 43-45) in CSPα disrupts the integrity of the highly conserved J domain, thereby preventing CSPα from activating Hsp70/Hsc70 to carry out conformational protein folding44. Expression of the loss-of-function mutant CSPα HPD-AAA is thus expected to override the activity of endogenous CSPα in CAD cells and act in a dominant-negative fashion45. Figures 2A and B show Western blot analysis and corresponding mean data of BK channel expression in the stable CAD cell line 48 h following transient transfection with myc-tagged CSPα, CSPα HPD-AAA or pCMV plasmid (negative control). As expected, 3 distinct species of CSPα were identified by Western blot in transfected cells; the 26 kDa immature form, the 34 kDa mature palmitoylated protein and the 70 kDa CSPα dimer42,46. In the presence of CSPα HPD-AAA , BK channel expression was elevated ~6-fold (657.5 ± 175.4%) compared with the plasmid control (100%). In contrast, increased levels of wild-type CSPα did not significantly change BK channel expression. As genetic knockout of CSPα is associated with elevated BK channel protein in brain tissue (Figure 1), we speculated that CSPα may interact either directly or indirectly with neuronal BK channels under native conditions. However, we were unable to capture stable CSPα-BK channel complexes from wild-type mouse brain using a classic immunoprecipation strategy (n = 4, data not shown). Such an observation may either reflect a lack of complex formation between native CSPα and BK channels, or that such putative interactions are of a transient, low affinity nature and not detectable using this analytical approach.

Figure 2 CSPα HPD-AAA increases BK channel levels in stably-transfected, BK channel expressing CAD cells. A schematic of wild-type CSPα, CSPα HPD-AAA , wild-type Hsp40 and Hsp40 HPD-AAA is shown at the top of the Figure, in which the J domain and the cysteine string regions are indicated. (A) Western blot analysis of BKα subunit expression in BK stable, CAD cells transiently transfected with either myc-tagged CSPα or CSPα HPD-AAA (0.75 μg cDNA each), or 1 μg cDNA encoding either myc-tagged Hsp40 or Hsp40 HPD-AAA . pCMV plasmid (1 μg cDNA) was used as a transfection control. 48 h post-transfection, transfected cells were harvested and 30 μg of cell lysate were separated by SDS-PAGE and probed with either an anti-BKα subunit antibody (upper) or an anti-myc antibody (middle). Detection of endogenous β-actin (lower) was used to verify similar sample loading. The BKα subunit data shown in the upper image of this panel were selected from a full-length western blot, which is displayed in Supplementary Figure 2A. The histogram (panel B) quantifies the relative changes in cellular BKα subunit expression in the presence of WT and HPD-AAA mutant forms of CSPα and Hsp40. (Panel C) shows a series of representative western blots of proteins reported to undergo palmitoylation. Soluble lysates were prepared from BK stable CAD cells transiently transfected with either wild-type CSPα or the HPD-AAA mutant, as indicated at the top of the panel. eGFP was used as a transfection control. Samples in the left hand column (denoted by the minus sign) are lysates from non-transfected cells. Detection of β-actin was used as a loading control. Results are representative of 4 independent experiments that all provided qualitatively similar data. Statistically significant differences between values were determined by -ANOVA, * p < 0.05. Full size image

As CSPα is a member of the highly conserved J protein family, we reasoned that other J proteins may also influence BK channel expression. Hsp40 (heat shock protein of 40 kDa) is a cytosolic member of the J protein family that is expressed constitutively, as well as in response to cell stress. Like CSPα, Hsp40 is neuroprotective and acts in concert with Hsc70, but the exact mechanism underlying its cell protective actions remains poorly characterized47. In CAD cells stably expressing BK channels, transient expression of either wild-type Hsp40 (99.3 ± 21.7%) or the loss-of-function mutant Hsp40 HPD-AAA (94.7 ± 9.5%) did not alter BKα subunit expression (Figure 2B). Collectively, these results indicate that the loss-of-function mutant CSPα HPD-AAA leads to a selective increase in BK channel levels, whereas an equivalent mutation in Hsp40, a related J protein, has no effect.

It has been reported that BKα subunits can undergo palmitoylation35, prompting us to examine whether other palmitoylated proteins may also be increased in the presence of the CSPα mutant CSPα HPD-AAA . As shown in Figure 2C, CSPα HPD-AAA did not increase expression of the palmitoylated proteins48,49 SNAP25, syntaxin1, GAP43 (growth associated protein of 43 kDa) and flotillin in BK stable CAD cells. In synaptosomes from CSPα null mice, we also did not observe changes in the expression of K v 1.1 (Figure 1A), another protein reported to be palmitoylated50.

In order to evaluate the influence of mutant forms of CSPα on cell surface expression of functional BK channels, we carried out whole cell patch clamp electrophysiology to quantify active BK channels in single CAD cells. To distinguish BK channel current from endogenous voltage-gated K+ channels (i.e. K v 1-type channels), we utilized 4-aminopyridine (4-AP, 5 mM) to block these channels and measured the remaining whole cell currents in the absence and presence of the highly selective BK channel inhibitor, penitrem A (100 nM)51. Figure 3A shows a current-voltage plot of the penitrem A-sensitive whole cell current density recorded from CAD cells stably expressing BK channels that have been transiently transfected with either wild-type or mutant forms of CSPα. BK current density was significantly greater in the presence of the CSPα HPD-AAA mutant compared with eGFP-expressing control cells, demonstrating higher surface expression of functional BK channels. In contrast, transient expression of wild-type CSPα had no effect on single cell BK current density, which is consistent with western blot data demonstrating very little effect of exogenous wild-type CSPα on the level of BKα subunit protein in BK stable CAD cells (Figure 2B). Figure 3B shows representative families of membrane currents recorded from BK stable CAD cells in the absence and presence of penitrem A under the described transfection conditions. The right hand column displays the penitrem A-sensitive current obtained by digital subtraction (i.e. left hand column minus middle column). These data clearly demonstrate that the CSPα loss-of-function mutant HPD-AAA increases cell surface expression of functional BK channels.

Figure 3 CSPα HPD-AAA increases penitrem A-sensitive ionic current in BK channel stably-transfected CAD cells. Domain architecture of CSPα is shown at the top of the Figure. A. Electrophysiological analysis of BK stable CAD cells transiently transfected with either wild-type or mutant forms of CSPα. Single CAD cells were voltage clamped as depicted by the test pulse protocol shown on the left and the selective BK channel inhibitor, penitrem A (100 nM) was used to isolate BK channel current. The current-voltage plot summarizes the penitrem A-sensitive, whole cell current density recorded from transfected cells, as described on the right hand side of the plot. Data are presented as mean ± SE and statistically significant differences were determined using a one-way ANOVA and a Dunnett post-hoc test (vs. GFP alone); * (p<0.05), ** (p<0.01). B. Representative families of whole cell currents recorded from BK stable CAD cells transiently transfected with either wild-type or mutant forms of CSPα in response to voltages steps ranging from −20 to +200 mV. The left hand column displays total whole cell current recorded from cells under the indicated transfection conditions. The middle column shows current remaining in the presence of the BK channel blocker penitrem A and the right hand column displays the calculated difference currents obtained by digitally subtracting the currents evoked in penitrem A from the total whole cell currents. Scale bars are indicated. Full size image

The CSPα Palmitoylation Mutants CSP 116 and CSP L115R Increase BK Channel Expression

In 2011, it was reported that novel mutations in human CSPα (i.e. deletion of residue 116 or replacement of Leu115 by Arg) are directly linked to adult-onset autosomal dominant neuronal ceroid lipofuscinosis (ANCL)52,53,54. ANCL is a rapidly progressive, neurodegenerative disorder in young adults, characterized by psychiatric manifestations, seizures, progressive dementia and motor deficits. Detailed pathogenic mechanisms of these CSPα mutations have yet to be elucidated, but individuals afflicted with ANCL display phenotypes reminiscent of the progressive motor deficits observed in the CSPα KO mouse. Both these mutations interfere with the palmitoylation of CSPα's cysteine string region, which is important for anchoring CSPα to synaptic vesicles. To evaluate the cellular effects of these putative dominant negative mutations, we examined the impact of CSPα Δ116 and CSPα L115R mutations on the level of functional BK channel in our CAD cells stably expressing neuronal BK channels. Analysis of BK channel activity in the presence of either CSPα Δ116 or CSPα L115R is shown in Figure 3. BK channel current density is significantly greater in the presence of CSPα Δ116 and CSPα L115R mutants compared with either wild-type CSPα or eGFP-expressing control cells, but the increases are less than that observed with the CSPα HPD-AAA mutant. Collectively, these data indicate that CSPα dysfunction has a profound effect on BK channel expression in a KO mouse model, as well as a model neuronal cell line.

To determine if the dysfunctional mutant CSPα HPD-AAA could also affect BK channel activity in a non-neuronal cell, we recorded BK currents from a rat aortic smooth muscle cell line (i.e. A7r555) stably transfected with the same BKα subunit cDNA used to create stable CAD cells. The data displayed in Figure 4 demonstrate that transient expression of either wild-type CSPα or CSPα HPD-AAA , as confirmed by immunocytochemistry (Figure 4B), had no effect on BK current density in A7r5 cells stably expressing BKα subunits. These findings are thus consistent with the observed lack of effect of CSPα knockout on native BK channel levels in mouse aorta (Figure 1C). Taken together, our data indicate that CSPα-mediated regulation of BK channels likely involves additional co-factors expressed in secretory cells, such as neurons.

Figure 4 Transient transfection with CSPα or CSPα HPD-AAA has no effect on BK channels in A7r5 cells stably expressing murine brain BK channels. (A) Penitrem A-sensitive current density plot demonstrated no significant difference between the treatments at 100 mV. Evoked current was >5-fold higher than native A7r5 cell current at the same potential (data not shown). (B) Immunocytochemical staining of A7r5 cells with anti-BKα subunit antibody demonstrated BK channel expression. Data shown are representative of at least five different experiments. Full size image

Biochemical confirmation that the CSPα HPD-AAA -mediated increase in BK current density was associated with an increase in BK channel cell surface expression, along with the total cellular pool of channel protein, is shown in Figure 5. For this purpose, CAD cells stably expressing BK channels were first transfected with CSPα variants, followed by labeling of intact cells with biotin. Biotinylated cell surface proteins were then extracted from the total cell lysate by streptavidin pull-down, followed by western blot analysis. The degree of BK channel biotinylation was evaluated in the presence of transfected CSPα, CSPα HPD-AAA or pCMV plasmid as a negative control. Consistent with the data displayed in Figures 2 and 3, the results shown in Figure 5A independently confirm that CSPα HPD-AAA (lane 2) markedly increased the cell surface expression of BK channel compared with wild-type CSPα (lane 1) or the plasmid control (lane 3). These data are complemented by quantifiable changes in the immunofluorescence staining of BKα subunit expression in BK stable CAD cells that were transiently transfected with either wild-type or mutant forms of CSPα (Figures 5B and C). Co-expression of eGFP was used as a marker of transiently transfected CAD cells. The elevated cellular expression of BK channels in the presence of dysfunctional CSPα mutants shown in Figure 5B thus mimics the functional increase in BK current density, as determined by single cell patch clamp recordings (Figure 3A). Whereas transient expression of wild-type CSPα did not significantly alter BK current density, it did reduce BKα subunit staining in BK stable CAD cells (Figure 5B). This difference likely reflects the ability of immunocytochemistry to detect intracellular and cell surface pools of BK channels, the former of which may be more readily affected by increased levels of wild-type CSPα. Note that only very low background fluorescence was detected in BK stable CAD cells stained with only fluorescently-labeled secondary antibody (Figure 5B, 2° Ab alone).