Regulation of CLC-1 chloride channel biosynthesis by FKBP8 and Hsp90β

Mutations in human CLC-1 chloride channel are associated with the skeletal muscle disorder myotonia congenita. The disease-causing mutant A531V manifests enhanced proteasomal degradation of CLC-1. We recently found that CLC-1 degradation is mediated by cullin 4 ubiquitin ligase complex. It is currently unclear how quality control and protein degradation systems coordinate with each other to process the biosynthesis of CLC-1. Herein we aim to ascertain the molecular nature of the protein quality control system for CLC-1. We identified three CLC-1-interacting proteins that are well-known heat shock protein 90 (Hsp90)-associated co-chaperones: FK506-binding protein 8 (FKBP8), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). These co-chaperones promote both the protein level and the functional expression of CLC-1 wild-type and A531V mutant. CLC-1 biosynthesis is also facilitated by the molecular chaperones Hsc70 and Hsp90β. The protein stability of CLC-1 is notably increased by FKBP8 and the Hsp90β inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) that substantially suppresses cullin 4 expression. We further confirmed that cullin 4 may interact with Hsp90β and FKBP8. Our data are consistent with the idea that FKBP8 and Hsp90β play an essential role in the late phase of CLC-1 quality control by dynamically coordinating protein folding and degradation.

A cardinal process during protein biogenesis involves conformation surveillance of nascent polypeptide by a network of molecular chaperones and cofactors (co-chaperones) that efficiently assist protein folding, thereby minimizing degradation/aggregation of proteins in nonnative states [19][20][21] . In this study, we aim to investigate the molecular nature of the chaperone/co-chaperone network monitoring the biosynthesis of human CLC-1. We demonstrate that CLC-1 channels are associated with the molecular chaperones heat shock cognate protein 70 (Hsc70) and heat shock protein 90β (Hsp90β ), as well as their co-chaperones FK506-binding protein 8 (FKBP8; also known as FKBP38), activator of Hsp90 ATPase homolog 1 (Aha1), and Hsp70/Hsp90 organizing protein (HOP). Biochemical and electrophysiological characterizations reveal that these co-chaperones and chaperones enhance both the protein level and the functional expression of CLC-1 WT and A531V mutant. Importantly, we present additional evidence suggesting that, in addition to promoting CLC-1 protein folding, FKBP8 and Hsp90β may also play a critical role in regulating CLC-1 degradation by the CUL4A/B-DDB1-CRBN complex.

Promotion of CLC-1 protein level by co-chaperones.
To study the molecular nature of CLC-1 protein quality control system, we began by searching for potential CLC-1-binding partners that are previously demonstrated to play a role in the chaperone/co-chaperone network. By performing yeast two-hybrid screening of a mouse skeletal muscle cDNA library with a bait sequence corresponding to an intracellular carboxyl-terminal region of the human CLC-1 channel (see Supplementary Methods), we identified the co-chaperones FKBP8 and Aha1. FKBP8 is an Hsp90-associated membrane-anchored immunophilin with potential peptidyl-prolyl cistrans isomerase function, whereas Aha1 is a cytosolic protein regulating the ATPase activity of Hsp90 19,[22][23][24] . Furthermore, both FKBP8 and Aha1 have been demonstrated to play critical roles in ER quality control of cystic fibrosis transmembrane conductance regulator (CFTR) Cl − channels [25][26][27][28] . The interaction of CLC-1 with FKBP8/ Aha1 was further confirmed by GST pull-down assay (see Supplementary Methods) with GST fusion proteins comprising C-terminal regions of CLC-1 (GST-CLC-1-C1, -C2, and -C3) (Suppl. Fig. 1A), and by immunoprecipitation experiment with full-length CLC-1 channel (Suppl. Fig. 1B). Over-expression of FKBP8/Aha1 substantially increases the protein level of CLC-1 WT and A531V mutant heterologously expressed in HEK293T cells ( Fig. 1A) (Suppl . Table S1). CLC-1 surface expression, as determined by the surface biotinylation assay, is also significantly promoted by both FKBP8 and Aha1 (Fig. 1B) (Suppl . Table S1). Moreover, the membrane trafficking efficiency of CLC-1, which was quantified by the ratio of surface expression to total protein level, appears to be enhanced by FKBP8, but not by Aha1 (Fig. 1B) (Suppl . Table S1). Interestingly, FKBP8, but not Aha1, seems to display a more prominent effect on the A531V mutant than on its WT counterpart. For CLC-1 WT, FKBP8 co-expression leads to about 2.5-fold and 3.8-fold increase in the total protein and surface expression, respectively. For the A531V mutant, however, FKBP8 co-expression results in about 3.7-fold and 9.9-fold increase in the total protein and surface expression, respectively. On the other hand, upon down-regulating endogenous FKBP8/Aha1 level in HEK293T cells with the RNA interference technique, we found that CLC-1 protein level is effectively reduced by lentiviral infection with the shRNA for Aha1, but not by that for FKBP8 (Fig. 1C) (Suppl . Table S1).
CLC-2 is a ubiquitously expressed Cl − channel that, along with CLC-1, belongs to the CLC Cl − channel family 29 . In a different set of experiments in which we performed yeast two-hybrid screening of a rat brain cDNA library with a carboxyl-terminal region of mouse CLC-2, we identified the co-chaperone HOP. HOP is a soluble protein mediating the interaction of Hsp70 and Hsp90, as well as regulating Hsp90 ATPase activity 19,22 . In addition, HOP has been implicated in ER-associated folding of CFTR as well 25 . To test the idea that HOP may also contribute to CLC-1 protein quality control, we first employed GST pull-down and immunoprecipitation assays to verify the interaction of CLC-1 with HOP (Suppl. Fig. 2A). Heterologous expression studies in HEK293T cells further confirm that HOP promotes both the total protein level and the surface expression of CLC-1 WT and A531V mutant ( Fig. 2A,B). Similar to the result observed for Aha1, HOP fails to discernibly affect the membrane trafficking of CLC-1 (Fig. 2B). Furthermore, also like Aha1, shRNA knock-down of endogenous HOP in HEK293T cells leads to a notable reduction of CLC-1 protein level (Fig. 2C).
Together, these observations suggest that the co-chaperones FKBP8, Aha1, and HOP promote CLC-1 biosynthesis. In addition, our data seem to imply that the role of FKBP8 in CLC-1 protein quality control may be considerably different from that of Aha1 and HOP. employed for immunoblotting analyses (total) or subject to streptavidin pull-down prior to immunoblotting analyses (surface). Expressions of GAPDH are displayed as the loading control. (Bottom) Quantification of surface protein level (Surface) and membrane trafficking efficiency (Surface/Total). The surface protein density was standardized as the ratio of surface signal to cognate total GAPDH signal, followed by normalization to that of the corresponding vector control. The total protein density was standardized as the ratio of input signal to GAPDH signal. The membrane trafficking efficiency was expressed as surface protein density divided by the corresponding standardized total protein density. The mean ratios in the presence of FKBP8/Aha1 were normalized to those of corresponding vector controls. (C) shRNA knock-down of endogenous FKBP8/Aha1 in HEK293T cells. The shRNA for GFP was used as the lentiviral infection control. Expressions of tubulin are displayed as the loading control. The protein density was standardized as the ratio of Flag-CLC-1 signal to cognate total tubulin signal, followed by normalization to that of the corresponding GFP control. Asterisks denote significant difference from the control (*, t-test: p < 0.05). See Supplementary Table S1 for more details on quantification values. The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in Supplementary Fig. S4.
Scientific RepoRts | 6:32444 | DOI: 10.1038/srep32444 Facilitation of CLC-1 biosynthesis by chaperones. FKBP8, Aha1, and HOP serve as co-chaperones for the interconnected Hsp70 and Hsp90 molecular chaperone systems 19,22,30 . Moreover, a recent report employing mass spectrometry combined with luminescence-based mammalian interactome assays indicates that the  . Asterisks denote significant difference from the control (*, t-test: p < 0.05). The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in Supplementary Fig. S4.
co-chaperone FKBP8 may interact more strongly with Hsp90β (the constitutive Hsp90 isoform) than with Hsp90α (the inducible Hsp90 isoform) 31 . We therefore went on to investigate the potential role of the constitutively expressed chaperone isoforms Hsc70 and Hsp90β in CLC-1 biogenesis. Representative GST pull-down and immunoprecipitation data illustrated in Supplementary Fig. 2B indicate that, in HEK293T cells, CLC-1 may be physically associated or at least stably co-exist in the same protein complex with Hsc70. Over-expression of Hsc70 significantly increases both the protein level and the surface expression of CLC-1 WT and A531V mutant, but does not appreciably alter the membrane trafficking efficiency of the Cl − channel (Fig. 3A,B). Conversely, suppressing endogenous Hsc70 expression in HEK293T cells with shRNA prominently reduces CLC-1 protein level (Fig. 3C).
The interaction between CLC-1 and Hsp90β was only demonstrated by GST pull-down (Suppl. Fig. 2C), but not by immunoprecipitation, suggesting that the two protein molecules probably form transient association with each other. Similar to the effect of Hsc70, Hsp90β promotes CLC-1 protein level and surface expression, but not membrane trafficking (Fig. 3A,B), suggesting that both Hsc70 and Hsp90β facilitate CLC-1 biosynthesis. Since over-expressing the inducible chaperone isoforms Hsp70/Hsp90α fails to discernibly increase CLC-1 protein level (data not shown), the foregoing observations do not appear to result from non-specific CLC-1 responses to Hsc70/Hsp90β over-expression in HEK293T cells. Surprisingly, shRNA knock-down of endogenous Hsp90β in HEK293T cells results in a remarkable enrichment of the protein level of CLC-1 WT and A531V mutant (Fig. 3C), which may be caused by a unknown compensatory response to Hsp90β suppression in HEK293T cells. Alternatively, this result may imply a differential role between Hsc70 and Hsp90β in CLC-1 quality control system.

Regulation of CLC-1 degradation by FKBP8 and Hsp90β.
Given the regulatory role of ER quality control system in protein homeostasis (proteostasis), an increase in the net expression level can be attributed to an enhanced chaperone-assisted folding of native protein and/or a reduced chaperone-directed degradation of misfolded protein [19][20][21]32 . To address whether the abovementioned co-chaperones and chaperones may control protein degradation of CLC-1, next we examined their role in CLC-1 protein stability by performing the cycloheximide chase experiment. Figure 4A and Supplementary Table S2 exemplify the effect of FKBP8 over-expression on the protein degradation time course of CLC-1 WT and A531V mutant in HEK293T cells. FKBP8 effectively raises the protein half-life of CLC-1 WT from about 6.6 to 10.1 hours. Furthermore, for the A531V mutant that is associated with enhanced proteasomal degradation 14 , FKBP8 considerably increases its protein half-life from about 3.6 to 8.0 hours. We then investigated the effect of over-expressing Aha1, HOP, Hsc70, or Hsp90β . Figure 4B and   Table S2 show that only FKBP8 is capable of significantly improving protein stability of the A531V mutant, suggesting that FKBP8 is remarkably effective in correcting the protein folding defect of the CLC-1 mutant.
In addition to its common role in stabilizing client proteins, Hsp90β may also promote the degradation of misfolded client proteins by directly interacting with E3 ubiquitin ligases [33][34][35] . In other words, depending on the mechanistic role of Hsp90β in the ER quality control system of its client proteins, pharmacological inhibition of Hsp90β function may lead to either enhanced or reduced protein degradation [36][37][38] . To gain further insight into the role of Hsp90β in the proteostatic mechanism of CLC-1, we assessed the effect of the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG), which suppresses the ATPase activity by blocking ATP binding to Hsp90 39,40 . Figure 5A and Supplementary Table S3 demonstrate that, in HEK293T cells, treatment with 17-AAG for 24 hours dramatically increases the protein level of both CLC-1 WT and A531V mutant in a concentration-dependent manner. A simple interpretation of this result appears to be that, rather than assisting the formation of mature CLC-1 conformation, Hsp90β promotes the degradation of the Cl − channel. Nevertheless, given that Hsp90β may interact with multiple client proteins in HEK293T cells, an alternative possibility is that the observed CLC-1 up-regulation arises from 17-AAG-induced disruption of Hsp90β interaction with endogenous client protein(s) in HEK293T cells, which in turn alters CLC-1 proteostasis. To test the latter hypothesis, we examined the effect of 17-AAG treatment on endogenous FKBP8, Aha1, HOP, Hsc70, and Hsp90β levels in HEK293T cells. Figure 5B and Supplementary Table S3 clearly show that 17-AAG fails to noticeably affect the protein expression of these co-chaperones and chaperones. Pharmacological inhibition of Hsp90 with geldanamycin or 17-AAG is known to induce prominent up-regulation of Hsp70 (Fig. 5B) (Suppl . Table S3) 41,42 ; however, as mentioned above, over-expressing Hsp70 did not appreciably increase CLC-1 protein level, suggesting that, at least in HEK293T cells, up-regulation of Hsp70 does not seem to contribute to the enhancement of CLC-1 expression by 17-AAG. Since suppression of endogenous CUL4A/B with either dominant-negative CUL4 mutants or shRNA for CUL4 effectively enhances CLC-1 expression in HEK293T cells 18 , we then examined the effect of 17-AAG treatment on endogenous CUL4A/B expression in HEK293T cells. Figure 5C and Supplementary Table S3 depict that 17-AAG indeed significantly decreases endogenous CUL4A/B expression in HEK293T cells, with CUL4B showing a more prominent concentration-dependent reduction pattern. Moreover, similar to the effect of FKBP8 over-expression, 17-AAG treatment markedly raises the protein half-life of the A531V mutant from about 3.8 to 8.4 hours, which can be attributed to reduced polyubiquitination of CLC-1 channels (Fig. 5D) (Suppl . Table S2). However, unlike FKBP8, 17-AAG treatment fails to discernibly alter the membrane trafficking efficiency of CLC-1 (Fig. 5E).
The foregoing data strongly suggest that Hsp90 may be essential for stabilizing CUL4A/B. A previous study involving luminescence-based interactome assays suggests that Hsp90 may directly interact with CUL4B 43 . To confirm this potential interaction between CUL4 and Hsp90β in HEK293T cells, we went on to perform immunoprecipitation experiments. Figure 6A shows that CUL4B, but not CUL4A, can be effectively co-immunoprecipitated with Hsp90β in HEK293T cells. Importantly, shRNA knock-down of endogenous Hsp90β in HEK293T cells significantly enhances CLC-1 protein level (see Fig. 3C). Consistent with this notion, shRNA knock-down of endogenous Hsp90β in HEK293T cells results in a noticeable interruption of the polyubiquitination of CLC-1 (Fig. 6B). Together, these observations imply that suppression of Hsp90β function may vigorously down-regulate CUL4 expression in HEK293T cells, consequently reducing CLC-1 degradation. Furthermore, results from immunoprecipitation experiments support the idea that FKBP8 may also co-exist in the same protein complex with CUL4A/B (Fig. 6C). Therefore, when we consider the overall impact of shRNA knock-down or pharmacological inhibition of Hsp90β /FKBP8 on CLC-1 proteostasis, the effect of disrupted CLC-1 folding may be virtually diminished or even out-balanced by that of reduced CLC-1 degradation.
Enhancement of CLC-1 current level by co-chaperones and chaperones. One critical question that remains unanswered is whether the increased protein biosynthesis induced by the abovementioned co-chaperones and chaperones really corresponds to enhanced amount of surface CLC-1 protein capable of forming functional Cl − channels. To address this question, we studied the effect of the co-chaperones and chaperones on CLC-1 functional expression in HEK293T cells. Due to their drastically different protein level, CLC-1 WT and A531V mutant were subject to cell-attached and whole-cell patch clamp analyses, respectively 14,18 . Figure 7A illustrates that FKBP8 over-expression results in remarkable augmentation of cell-attached current amplitude and whole-cell current density of CLC-1 WT and A531V mutant, respectively. Consistent with the aforementioned biochemical observations (Fig. 1) (Suppl. Table S1), the functional enhancement effect of FKBP8 is slightly more prominent for the A531V mutant (~2.5-fold increase) than for CLC-1 WT (~1.9-fold increase). Moreover, FKBP8 The effect of 1 μ M 17-AAG on protein turn-over kinetics of Flag-CLC-1 A531V mutant. DMSO treatment was used as the control experiment. See Supplementary Table S2 for

polyubiquitination [CLC-1-(Ub)n] by endogenous ubiquitin was identified by immunoblotting the immunoprecipitates with the anti-ubiquitin (Ub) antibody. (E)
The effect of 1 μ M 17-AAG on surface protein level (Surface) and membrane trafficking efficiency (Surface/Total) of CLC-1. The mean ratios were normalized to those of the corresponding DMSO controls. The mean relative surface expression ratios (n = 4) are about 3.6 (WT) and 1.7 (A531V). The mean relative membrane trafficking ratios are about 0.9 (WT) and 1.1 (A531V). Asterisks denote significant difference from the control (*, t-test: p < 0.05). The gels were run under the same experimental conditions. Uncropped images of immunoblots are shown in Supplementary Fig. S4.
Scientific RepoRts | 6:32444 | DOI: 10.1038/srep32444 over-expression does not appreciably alter the steady-state voltage-dependence (P o -V curve) of CLC-1 currents, suggesting that FKBP8 probably induces up-regulation of CLC-1 channels endowed with mature and correct protein conformation. Similar current enhancement effects were also observed when we co-expressed CLC-1 with Aha1, HOP, Hsc70, or Hsp90β (Fig. 7B-E). Furthermore, treatment with the Hsp90 inhibitor 17-AAG also effectively increases the functional expression of CLC-1 WT and A531V mutant (Fig. 7F).

Discussion
Anomalous protein maturation arising from disrupted ER quality control, excessive ERAD, or defective membrane trafficking has been implicated in the molecular pathogenesis of many ion channel diseases (channelopathies) 21,44 . Given that the myotonia congenita-related A531V mutant is associated with enhanced protein degradation and defective membrane trafficking properties 14,15 , it is imperative to decipher the molecular machinery essential for protein biosynthesis of CLC-1. The discovery of the CUL4A/B-DDB1-CRBN complex as the E3 ubiquitin ligase catalyzing CLC-1 polyubiquitination 18 prompted us to further identify the conformation surveillance mechanism regulating CLC-1 proteostasis. In this study, we show that the co-chaperones FKBP8, Aha1, and HOP, as well as the constitutively expressed chaperones Hsc70 and Hsp90β , may directly interact with CLC-1 Cl − channels. Over-expression of these co-chaperones and chaperones substantially promotes the biosynthesis and functional expression of CLC-1 WT and A531V mutant. Conversely, shRNA knock-down of endogenous Aha1, HOP, or Hsc70 expression in HEK293T cells results in notable down-regulation of CLC-1 protein level, consistent with idea that Aha1, HOP, and Hsc70 are responsible for facilitating CLC-1 protein folding in the quality control system. By contrast, shRNA knock-down of endogenous FKBP8 level in HEK293T cells does not appreciably change CLC-1 protein level. Moreover, infection with shRNA for Hsp90β prominently enhances CLC-1 biosynthesis. These apparently paradoxical results appear to imply that FKBP8 and Hsp90β may play additional roles in the CLC-1 quality control system. Consistent with this notion, suppression of Hsp90β function with 17-AAG dramatically down-regulates endogenous CUL4 expression in HEK293T cells, thereby reducing polyubiquitination and degradation of CLC-1. In addition, like Hsp90β , FKBP8 co-exists in the same protein complex with CUL4, raising the possibility that FKBP8 may also contribute to the stabilization of CUL4 by Hsp90β . Importantly, FKBP8 displays three unique features: 1) a more prominent biosynthesis-enhancement effect on the A531V mutant than on its WT counterpart (see Fig. 1A, Suppl. Table S1, and Fig. 7A); 2) effective improvement of CLC-1 protein stability (see Fig. 4 and Suppl. Table S2), and 3) significant promotion of CLC-1 membrane trafficking efficiency (see Fig. 1B and Suppl. Table S1). Therefore, our results appear to suggest that FKBP8 plays a decisive role in correcting protein folding defect of CLC-1.
Taken together, we suggest a model of CLC-1 proteostasis mechanism that is schematically represented in Fig. 8. The inferred protein folding pathway for CLC-1 resembles the ER quality control model previously proposed for another Cl − channel, CFTR 25,27,28,45,46 . We propose that Hsc70 and HOP may assist the early stage of CLC-1 folding before passing the channel protein to the Hsp90β cycle, wherein Aha1, Hsp90β , and FKBP8 work in concert to further promote CLC-1 folding. We hypothesize that FKBP8 is responsible for the last stage of protein folding and is essential for determining whether CLC-1 protein can be properly exported for membrane trafficking. The major difference between our scheme and the CFTR model concerns the coupling between molecular chaperones and the protein degradation pathway. One of the best characterized E3 ubiquitin ligases for  CFTR is carboxyl terminus of Hsc70-interacting protein (CHIP); misfolded CFTR is thought to be recognized through Hsc70-CHIP interaction and thereafter subject to CHIP-mediated polyubiquitination 45,47 . By contrast, the present report suggests that Hsp90β and FKBP8 may play an additional role in regulating CLC-1 degradation by interacting with CUL4. We therefore suggest that misfolded CLC-1 may be primarily processed by Hsp90β / FKBP8-CUL4 interaction. Nevertheless, we cannot rule out the possibility that Hsc70 may also be essential for CLC-1 ubiquitination mediated by as yet unknown E3 ligases.
The majority of our conclusions are based on biochemical experiments using heterologous expression system. One potential limitation with immunoblotting analyses involving protein over-expression is that the sensitivity of the protein detection system from time to time needs to be reduced to avoid signal saturation. Consequently, it is likely that we may occasionally overlook the contribution of chaperones/co-chaperones imparting minute effects on CLC-1 biosynthesis. This potential bias against weaker protein signals may additionally contribute to the quantitative discrepancy between immunoblotting and electrophysiological analyses reported in this study. Due to the voltage clamp capacity of the signal amplifier for the patch clamp system, there is a stringent limitation on the size of the CLC-1 current amplitudes that can be properly recorded; in other words, cells with higher CLC-1 protein expression levels are more likely to be excluded from our functional data analyses. Nevertheless, the results derived from immunoblotting and electrophysiological analyses do agree nicely on a qualitative basis. Another related issue on electrophysiology concerns the fact that CLC-1 WT currents were recorded using the cell-attached configuration. As we reported previously, under the whole-cell mode, CLC-1 currents from WT channels were recorded 4-7 hours post-transfection; by contrast, no significant Cl − currents were observed for A531V until 8-11 hours post-transfection 14 . The current amplitudes of A531V reach a steady-state level at about 24 hours post-transfection, at which whole-cell WT current amplitudes invariably exceed the range of optimal voltage clamp efficiency for the patch amplifier system. This problem can only be overcome by a dramatic reduction of the amount of CLC-1 cDNA used for transfection, which, however, would render it virtually impossible to effectively co-express any chaperone or co-chaperone protein. On the other hand, a likely caveat associated with the cell-attached configuration is that CLC-1 proteins may not be evenly distributed over the cell surface. To minimize the impact of this potential problem, we incorporated a large number of WT membrane patches (ranging from 10 to 31; see Fig. 7) for statistical analyses. Moreover, the validity of the WT data collected with the cell-attached configuration was confirmed by repeating the identical co-expression experiments with the A531V mutant using the whole-cell mode.
One of the surprising findings of our research is the promotion of CLC-1 biosynthesis by the Hsp90 inhibitor 17-AAG. The suppressive effect of 17-AAG treatment on CUL4A/B protein level does not seem to be a special case for HEK293T cells, as a similar result was also observed in HeLa cells treated with a chemical analogue of 17-AAG 37 . Together these data are consistent with the idea that, in human cells, Hsp90β is essential for the stabilization of CUL4. Further investigations are required to elucidate the detailed mechanisms underlying how 17-AAG treatment leads to down-regulation of CUL4. Since Hsp90β is a binding partner of CUL4B, but not CUL4A (see Fig. 6A) 43 , we speculate that Hsp90β may indirectly stabilize CUL4A through other client protein(s). Interestingly, FKBP8 has been shown to interact with the S2 subunit of the 19S proteasome in HEK293T cells and may contribute to anchoring a subset of the proteasome complexes to the organellar membrane of mitochondria and ER in various cell lines 48 . Therefore, it remains to be determined whether FKBP8 may facilitate protein folding/stability of CUL4 via certain Hsp90-dependent and/or -independent processes. Overall, our findings seem to raise an intriguing possibility that Hsp90β may serve as a molecular hub that facilitates the association of CLC-1 channels with Aha1, FKBP8, and CUL4, thereby dynamically coupling the protein folding and degradation pathways of CLC-1 biosynthesis.
Another fascinating discovery arising from the present study is that only FKBP8 can pronouncedly enhance the membrane trafficking efficiency of CLC-1 channels. Emerging evidence indicates that membrane-bound protein is also susceptible to stringent conformation surveillance and substantial degradation by the endosomal-lysosomal pathway, a mechanism known as peripheral quality control 49,50 . FKBP8 is usually considered as an internal membrane protein resident at mitochondria and ER 23,24 . In line with this notion, FKBP8 was previously suggested to contribute to the ER biogenesis, but not the peripheral quality control, of CFTR 51 . However, an alternative interpretation of the unique membrane trafficking effect of FKBP8 on CLC-1 is that the co-chaperone may additionally contribute to the peripheral quality control of CLC-1 localized at the plasma membrane. Future experiments will be required to address whether FKBP8, as well as the other CLC-1-related to the corresponding vector control. The mean relative current levels for WT and A531V are about 1.9 and 2.5, respectively. (Lower right panels) Steady-state voltage-dependence of the open probability (P o -V curve) of CLC-1 channels. Compared to CLC-1 WT, the A531V mutant displays an apparently left-shifted P o -V curve, which primarily arises from the different cytoplasmic ATP concentrations between cell-attached (WT) and whole-cell (A531V) configurations (refer to Suppl. Fig. S3 for experimental evidence). See Supplementary Methods for more details on the analysis of P o -V curves. (B) The effect of Aha1 co-expression. The mean relative current levels for WT and A531V are about 1.4 and 2.5, respectively. (C) The effect of HOP coexpression. The mean relative current levels for WT and A531V are about 1.5 and 1.9, respectively. (D) The effect of Hsc70 co-expression. The mean relative current levels for WT and A531V are about 2.2 and 2.3, respectively. (E) The effect of Hsp90β co-expression. The mean relative current levels for WT and A531V are about 2.7 and 2.7, respectively. (F) The effect of treatment with 1 μ M 17-AAG. The mean relative current levels for WT and A531V are about 3.5 and 2.0, respectively. Two types of drug-free incubation (control and 0.1% DMSO) were used to verify the effect of 17-AAG. Asterisks denote a significant difference from the control condition (*, t-test: p < 0.05).
co-chaperones, chaperones, or even E3 ubiquitin ligase complex proteins, may also be involved in the peripheral quality control of CLC-1 WT and A531V mutant. In addition, as defective biosynthesis appears to contribute to other myotonia congenita mutations 16,17 , it is critical to investigate whether FKBP8 may also improve the protein expression and membrane trafficking of the other disease-related CLC-1 mutant channels.
Recent advances in the molecular elucidation of defective protein biosynthesis mechanisms underlying numerous human diseases drive the developments of novel therapeutic strategies aiming at adapting proteostasis networks to restore normal physiology 32,52 . For example, a substantial amount of different chemical compounds have been developed to correct the biosynthetic anomaly of disease-associated mutant CFTR proteins 53,54 . Current treatment for myotonia primarily focuses on the symptomatic relief of enhanced muscle tone with action potential-reducing agents such as Mexiletine, which exerts use-dependent block of surface voltage-gated Na + channels (Na V1.4 ) in skeletal muscles [55][56][57] . No CLC-1-targeting drug is available at present. Previously, we provided the first evidence showing that CLC-1 ubiquitination is suppressed by MLN4924, which blocks the neddylation of cullin E3 ligases and has emerged as an anti-cancer agent 58,59 . In the current study, we presented the novel discovery that CLC-1 degradation is prevented by the Hsp90 inhibitor 17-AAG, which is also being tested in various clinical trials as an anti-cancer agent 39,40,60 . Together, these data highlight the therapeutic potential of CLC-1 proteostasis modification in treating myotonia patients. In order to further address the clinical significance of our findings, more work is required to verify the specificity, as well as the effectiveness, of MLN4924 and 17-AAG in skeletal muscles. For example, since the biosynthetic mechanism of Na V1.4 remains unclear 56 , it will be imperative to determine whether (and perhaps how) these drugs may affect the protein expression and membrane trafficking of the Na + channels in skeletal muscles. Methods cDNA constructs. Epitope-tagged CLC-1 constructs were generated by subcloning human CLC-1 cDNA into either the pcDNA3 vector (Invitrogen) (for Myc and HA tags) 14 or the pFlag-CMV2 vector (Sigma) (for Flag tag) 18 . Other cDNA constructs employed in this study include pcDNA3.1-Myc mouse Aha1, pcDNA3-Myc human cullin 4A/4B (Addgene 19951/19922), pcDNA3.1-Myc mouse FKBP8, pcDNA3-HA rat HOP, pcD-NA5-V5 human Hsc70 (Addgene 19514), pcDNA5-V5 human Hsp70 (Addgene 19510), pcDNA3-HA human Hsp90α (modified from an original clone kindly provided by Dr. Didier Picard, University of Geneva), and pcD-NA3-HA human Hsp90β (Addgene 22847).

Cell culture and DNA transfection. Human embryonic kidney (HEK) 293T cells were grown in
Dulbecco's modified Eagle's medium (DMEM) supplemented with 2 mM glutamine, 10% heat-inactivated fetal bovine serum (Hyclone), 100 units/ml penicillin, and 50 μ g/ml streptomycin, and were maintained at 37 °C in a humidified incubator with 95% air and 5% CO 2 . Transient transfection was performed by using the Lipofectamine 2000 (LF2000) reagent (Invitrogen). Briefly, cells were plated onto 6-or 12-well plates (for biochemical experiments) or poly-D-lysine-coated coverslips in 24-well plates (for electrophysiological recordings) 24 hrs before transfection. The amount of CLC-1 cDNA used in each well was about 300 (for biotinylation) to 700 (for shRNA knock-down) ng/mL, and the molar ratio for co-transfection (relative to CLC-1 cDNA) ranged from 1 to 3. Various expression constructs were incubated with LF2000 reagent for 20 min at room temperature, Figure 8. A model of the ER quality control system for CLC-1 channel. In this schematic diagram of CLC-1 biogenesis process, the ER protein quality control system is hypothesized to comprise the co-chaperones FKBP8, Aha1, and HOP, as well as the constitutively expressed chaperones Hsc70 and Hsp90β . Hsc70 and HOP may assist the early stage of CLC-1 folding, whereas Aha1, Hsp90β , and FKBP8 may promote the late stage of CLC-1 folding. FKBP8 is further proposed to be essential for determining whether CLC-1 protein can be properly folded for subunit assembly and thereafter exported for membrane trafficking. In addition, Hsp90β and FKBP8 may regulate ER-associated degradation (ERAD) of CLC-1 by interacting with the cullin 4 E3 ligase complex that catalyzes the covalent linkage of ubiquitin (Ub) to CLC-1. The degradation of CLC-1 can be effectively attenuated by the cullin E3 ligase blocker MLN4924, as well as the Hsp90 inhibitor 17-AAG. and DNA-lipofectamine diluted in Opti-MEM (Invitrogen) was added to culture wells. After 6-hr incubation at 37 °C, the medium was changed and the culture cells were maintained in the 37 °C incubator for 24-48 hrs before being used for biochemical or electrophysiological experiments. Where indicated, drugs [cycloheximide (Sigma) or 17-AAG (Sigma)] were applied to the culture medium.