A forward genetic screen identifies chaperone CNX-1 as a conserved biogenesis regulator of ERG K+ channels

The mechanism underlying the biogenesis of hERG channels is not fully understood. Bai et al. identify CNX-1 as a novel regulator of ERG K+ channel biogenesis that is conserved from Caenorhabditis elegans to humans.


Introduction
The human ether-a-go-go-related gene (hERG) channel is a delayed rectifier voltage-gated K + channel that controls repolarization of action potentials in cardiomyocytes (Vandenberg et al., 2012). Dysfunction of hERG is associated with multiple human diseases, including long QT syndrome (LQTS; Curran et al., 1995;Sanguinetti et al., 1995;Trudeau et al., 1995), schizophrenia (Huffaker et al., 2009), epilepsy (Johnson et al., 2009), and multiple types of cancers (Pardo and Stühmer, 2014). Although most LQTS-related hERG mutations result in decreased surface expression of the channel (Harkcom and Abbott, 2010;Anderson et al., 2014), the molecular mechanism underlying maturation of hERG K + channel is not fully understood.
The sole Caenorhabditis elegans ERG-type K + channel, UNC-103, is homologous to hERG, with 70% amino acid identity in functionally important transmembrane domains and cyclic nucleotide-binding domain (Garcia and Sternberg, 2003;Li et al., 2017). Previous studies have demonstrated that worm homologues of hERG modifiers, KCR1, Hyperkinetic, and some J-proteins also affect the function of UNC-103 channels (Petersen et al., 2004;Li et al., 2017), suggesting the conserved regulatory mechanisms of ERG K + channels between C. elegans and humans. Given the convenience of genetic manipulation, C. elegans thus can be used as an animal model to identify novel cellular factors important for regulating ERG channel biogenesis.
In the previous work, through a genetic screen, we found DNJ-1, an ER-located chaperone, is critical for the biogenesis of the UNC-103 K + channel in C. elegans (Li et al., 2017). Although the expression level of UNC-103 was markedly decreased because of a loss-of-function (LOF) mutation in dnj-1, there were still remaining UNC-103 proteins in the dnj-1 mutant worms, suggesting that other cell factors act independently of DNJ-1 to facilitate the biogenesis of ERG channels. In this study, by forward genetic screening, we identified CNX-1, the C. elegans homologue of mammalian chaperone Calnexin, as a critical factor for the biogenesis of ERG K + channels.
Calnexin, also known as CNX, IP90, or P88, is an ER-located chaperone with carbohydrate-binding activity (Ware et al., 1995;Schrag et al., 2001). A previous study has demonstrated that a trafficking defect in a LQTS-related mutant, hERG N470D , is caused by a prolonged association with Calnexin . This result suggests that Calnexin may be involved in the biogenesis of hERG channels. However, the effect of Calnexin (or its worm homologue, CNX-1) on the biogenesis of ERG K + channels in vivo remains unclear. In this study, we demonstrated that Calnexin/CNX-1 interacted with the ERG channel and facilitated ERG channel biogenesis in the ER. The effect of CNX-1 on the biogenesis of the ERG channel involved its ability to promote tetrameric assembly of the channel subunits. Further experiments showed that the action of CNX-1 was independent of DNJ-1, suggesting that these two different types of chaperones facilitate the maturation of ERG channels via parallel pathways. Thus, we reveal a conserved role of CNX-1 in promoting ERG channel biogenesis.

Materials and methods
Worm strains and culture OP50 bacteria-seeded nematode growth medium was used to cultivate worms at 20°C. N2, unc-103(e1597), cnx-1(nr2010), and CB4856 strains were obtained from the Caenorhabditis Genetics Center. The dnj-1(yfh0001) mutant was generated in our laboratory previously (Li et al., 2017). For detailed information of all the strains used in this study, please refer to Table 1.
Genetic screens and whole-genome sequencing Synchronized L3-L4 stage unc-103(e1597) worms were used for mutagenesis. After treatment with 50 mM ethyl methanesulfonate for 4 h at room temperature, the F2 progenies of revertants (mutated worms that can move better and lay eggs) were selected. Hawaii CB4856 was crossed with the revertants to perform genetic mapping of the mutated genes (Davis et al., 2005). Genomic DNA of mutant worms was then prepared for whole-genome sequencing (Doitsidou et al., 2010). Genome sequencing was performed on an Illumina platform (HiSeq 2000/2500 or Hiseq X Ten), and all data were analyzed by CloudMap online software (https:// usegalaxy .org). Rescue experiments of LOF alleles of cnx-1 were performed by reexpressing wild-type CNX-1 proteins in cnx-1 mutants.

Cell-free expression
Cell-free syntheses were performed using the WEP RO7240H Expression kit (Cell Free Sciences) according to protocol described previously (Li et al., 2017), with slight modifications. In brief, 2 µg purified plasmid DNA was transcribed at 37°C for 6 h. Translation reactions were then performed in the presence of 4 mg/ml soybean liposomes (Avanti Polar Lipids) at 25°C for 20 h. The proteoliposomes were solubilized by 1% m/v dodecylmaltoside (Sigma) and subjected to native PAGE electrophoresis.
C. elegans neuron culture Primary cultures of C. elegans embryos were performed according to protocol described previously (Strange et al., 2007). In brief, worm embryos were isolated from adult hermaphrodites. After removal of the worm eggshells by enzymatic digestion, cells were dissociated and plated onto the dishes precoated with peanut lectin (Sigma). Whole-cell patch clamp was performed 4-6 d after seeding.

Immunocytochemistry
HEK293T cells were fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature, followed by permeabilization with 0.25% Triton X-100. After washing three times with PBS buffer, cells were blocked with 1% BSA for 1 h at room temperature. The primary antibodies used in this experiment were anti-HA (1:1,000, Roche) and anti-Calnexin (1:1,000; Abcam). The secondary antibodies were Alexa Fluor 488 (green) or Alexa Fluor 594 (red)-conjugated antibodies.
Western blot and immunoprecipitation HEK293T and SH-SY5Y cells were cultured in Dulbecco's modified Eagle's medium (Gibco) with 10% FBS (Gibco), and 1% penicillin/streptomycin (Gibco) at 37°C in a 5% CO 2 incubator. Lipofectamine 2000 (Invitrogen) was used for plasmid transfection. The components of cell lysis buffer were 1% NP-40, 150 mM NaCl, 1 mM NaF, 50 mM Tris, pH 7.6, and protease inhibitor mixture (Roche). Cells were lysed in lysis buffer at 4°C for 1 h. After centrifugation at 4°C at 12,000 g for 10 min, the supernatants were collected and incubated with the primary antibody at 4°C for 3.5 h. The immune complexes were associated with protein A/G agarose beads (Santa Cruz) at 4°C for 2.5 h. The beads were then washed six times and boiled for 5 min. The Western blot analyses of UNC-103 protein levels were not conducted blind to genotypes, but two researchers independently did the experiments and obtained similar results. The following antibodies were used in these experiments: rabbit anti-hERG (Millipore), rabbit anti-c-Myc (Sigma), rat anti-HA (Roche), mouse anti-tubulin (Sigma), mouse anti-GFP (Abmart), and rabbit anti-actin (Abmart).

Statistical analyses
The band intensities in immunoblots were determined by ImageJ software. The amplitudes of K + currents and the membrane capacitances of the recorded cells were measured using Clampfit software (version 10.4; Molecular Devices). Peak currents and steady-state currents at each voltage step were used for statistical analyses. Graphing and statistical comparisons were performed with GraphPad 7.0 software using either ANO VA or t test as specified in the figure legends.

LOF mutations of cnx-1 suppress the behavioral defects in unc-103(GOF) worms
The C. elegans ERG K + channel UNC-103 showed homology with the hERG channel ( Fig. 1 A). unc-103(e1597) mutant worms exhibit severe defects in locomotion and egg-laying caused by a gainof-function (GOF) mutation (A334T) in the S6 transmembrane domain of UNC-103 (Garcia and Sternberg, 2003;Reiner et al., 2006). The mutation is a GOF allele that alters the channel activation potential to a more negative value (Petersen et al., 2004) and inhibits the excitability of the cell (Collins and Koelle, 2013). We thus hypothesized that inhibition of the function of UNC-103 will alleviate the behavioral defects in unc-103(e1597) mutant worms. To identify novel regulators of ERG K + channels, we performed a forward genetic screen for suppressors of the behavioral defects of unc-103(e1597) worms. The screening strategy is depicted in Fig. 1 B. From a screen of 14,000 haploid genomes, we isolated eight mutants with reverted phenotypes. Four of the isolated mutants were mapped to one gene, cnx-1 (ZK632.6), which encodes a chaperone protein showing homology with mammalian Calnexin, through single-nucleotide polymorphism-based mapping (Fig. 1,  C and D). Molecular lesions were identified in all the four cnx-1 alleles through DNA sequencing (Fig. 1 D and Table 2). As shown in Fig. 2 (A-C), the behavioral defects in locomotion (quantified by thrashing) and egg laying (quantified by egg numbers) found in unc-103(e1597) worms were partially prevented by a LOF mutation or a deletion allele (nr2010) of the cnx-1 gene. We then performed rescue experiments by reexpressing the wild-type CNX-1 with the driver of its own promoter in unc-103(e1597);cnx-1(nr2010) worms and found that reintroduction of the wild-type CNX-1 could reinstate the behavioral defects of unc-103(e1597) (Fig. 2, B and C), demonstrating that the lack of the CNX-1 protein, but not other mutations in the genetic background, is responsible for the observed phenotypes. Together, these results suggest that CNX-1 is essential for the function of the UNC-103 K + channel in C. elegans.

CNX-1 is required for UNC-103 biogenesis
Our previous study generated a transgenic strain (named unc-103 A334T ) expressing the GFP fused unc-103 with the same GOF mutation found in unc-103(e1597) worms (Li et al., 2017). Like unc-103(e1597), the transgenic worm exhibited severe defects in locomotion and egg laying, indicating that expression of UNC-103 A334T ::GFP was sufficient to induce functional defects caused by the UNC-103 mutation, presumably by causing membrane hyperpolarization after its delivery to the plasma membrane.
Formation of functional K + channels requires channel subunits assembly, an important biogenic step occurring in the ER (Doyle et al., 1998;Isacoff et al., 2013). We then explored whether CNX-1 plays a role in the tetrameric assembly of UNC-103 channel subunits using a wheat germ cell-free system to synthesize proteins in vitro (Fig. 4 A). UNC-103 and CNX-1 were expressed in the cell-free extracts with liposomes that provide a hydrophobic  Whole-cell patch clamp was performed 4-6 d after seeding C. elegans embryonic cells. (C) Representative whole-cell K + currents recorded in C. elegans ALA neurons with or without the environment to help the folding and assembly of these membrane proteins (Goren et al., 2009). We found that in vitro-synthesized UNC-103 existed as monomers, whereas part of the UNC-103 proteins appeared as tetramers upon coexpression with CNX-1 (Fig. 4, B and C), indicating that CNX-1 promotes tetrameric assembly of UNC-103 channel subunits in vitro.
The effects of CNX-1 and DNJ-1 on UNC-103 protein biogenesis are additive Previous studies have demonstrated that DNJ-1, another ER-located chaperone protein, is critical for the biogenesis of the UNC-103 K + channel (Li et al., 2017). We then asked whether CNX-1 and DNJ-1 regulate the biogenesis of UNC-103 via the same pathway. We found that the expression level of UNC-103 A334T ::GFP was markedly reduced by introducing either null mutations in cnx-1 or dnj-1 and almost abolished by introducing both the cnx-1 and dnj-1 mutations (Fig. 3, A and B). The locomotion and egg-laying defects found in unc-103 A334T worms could be partially prevented by null mutations in either cnx-1 or dnj-1 and further prevented by a combination of cnx-1 and dnj-1 null mutations (Fig. 3, C and D, left). Notably, locomotion and egg production in cnx-1(nr2010);dnj-1(yfh0001) worms were not significantly different from those in unc-103 A334T ;cnx-1(n-r2010);dnj-1(yfh0001) worms, suggesting that the combination of cnx-1 and dnj-1 null mutations fully suppressed the locomotion and egg-laying defects caused by the GOF mutation in UNC-103. Thus, the effects of CNX-1 and DNJ-1 on UNC-103 channel biogenesis are additive.

CNX-1 regulates the function of endogenous UNC-103 K + channels
To determine the subcellular localization of CNX-1 proteins, we fused GFP and mCherry to the C termini of CNX-1 and the ER marker protein SP12, respectively. We found that CNX-1 was colocalized with SP12 in C. elegans neurons (Fig. 5 A), indicating that CNX-1 is an ER-located protein. Further study showed that cnx-1 promoter-driven GFP was ubiquitously expressed in large numbers of neurons in the tail, head, and vulva (Fig. 5 B) and colocalized with unc-103 promoter-driven mCherry in many neurons (Fig. 5 B), suggesting that CNX-1 and UNC-103 may interact with each other in vivo.
Calnexin regulates the biogenesis of hERG K + channel in mammalian cells C. elegans CNX-1 is homologous with mammalian protein Calnexin. We thus investigated whether Calnexin plays an evolutionarily conserved role in regulating the biogenesis of hERG channels. Immunostaining in HEK293T cells showed that hERG proteins distributed in both the cell surface and the ER (Fig.7 A). Consistent with the previous studies Anderson et al., 2006), we found two forms of hERG proteins corresponding to ER (∼135 kD) and post-ER (∼155 kD) forms with different extents of glycosylation (Fig. 7 B). Coimmunoprecipitation assays showed that Calnexin only interacted with the ER-residing form of hERG proteins (Fig. 7 B), suggesting that Calnexin interacts with hERG proteins in the ER.
We next investigated whether hERG K + channels could be regulated by Calnexin. In HEK293T cells expressing hERG proteins, down-regulation of calnexin by siRNA markedly decreased the protein level (Fig. 7 C) and current density (Fig. 7 D) of hERG K + channels. In human neuroblastoma SH-SY5Y cells that endogenously express hERG K + channels, deletion of calnexin by CRI SPR-Cas9-mediated genome editing significantly decreased the endogenous hERG protein level (Fig. 7 E). Furthermore, the deactivating inward currents, which are mainly conducted by hERG channels (Meyer and Heinemann, 1998;Taglialatela et al., 1998;Li et al., 2017), were significantly reduced in calnexin −/− SH-SY5Y cells compared with wild-type SH-SY5Y cells (Fig. 7 F). Collectively, these findings suggest that Calnexin is essential for the protein biogenesis of native hERG channels in human cells.

Discussion
In this study, through forward genetic screen in C. elegans, we found that CNX-1 is a critical regulator of the UNC-103 K + channel. LOF mutations of cnx-1 resulted in a reduced protein level ERG channel-specific blocker E-4031 (1 µM) in the bath solution. The voltage protocol is shown on the right. Traces shown are the mean of all recordings. UNC-103-elicited K + currents in ALA neurons are shown by mean outward K + currents (i) subtracting mean retained currents (ii) after treatment with the ERG channel-specific blocker E-4031. The magnified currents of the corresponding dotted boxes are shown. (D) Peak current density of voltage-gated outward K + currents at different membrane potentials in ALA neurons in the absence (black line) and presence (red line) of 1 µM E-4031 in the pipette solution. (E) Steadystate current density of voltage-gated outward K + currents at different membrane potentials in ALA neurons in the absence (black line) and presence (red line) of 1 µM E-4031 in the pipette solution. For D and E, the cell numbers used for analyses are indicated. Data shown are mean ± SEM. N.S., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 (t test). and function of UNC-103 K + channels in C. elegans. Down-regulation of its human homologue, Calnexin, significantly decreased the protein level and current density of the hERG K + channel in human SH-SY5Y cells. Thus, our findings reveal an evolutionally conserved role of CNX-1/Calnexin in regulating ERG-type K + channels across species. Calnexin has been shown to play an important role in the protein folding of many secreted proteins, including ion channels Li et al., 2010), G protein-coupled receptors (Free et al., 2007;Markkanen and Petäjä-Repo, 2008;Noorwez et al., 2009), MHC class I molecules (Ireland et al., 2007), and the nicotinic acetylcholine receptor (Chang et al., 1997). It has been also suggested that Calnexin facilitates the assembly of channel subunits into a stable complex (Chang et al., 1997;Green, 1999), but so far, there is no direct experimental evidence to support this idea. In this study, our findings show that CNX-1 facilitates biogenesis of ERG K + channels in C. elegans and promotes tetrameric assembly of ERG K + channels in a cell-free translation system, providing direct evidence to support this notion. However, the detailed mechanism underlying how Calnexin facilitates the biogenesis of ERG K + channels remains to be further studied.
Two major subtypes of chaperones exist in the ER: classical HSP family chaperones and carbohydrate-binding chaperones (Braakman and Hebert, 2013). It is generally thought that these two different types of chaperones cooperate during protein biogenesis to ensure adequate protein flux through the ER (Braakman and Hebert, 2013), but how they work together to control protein biogenesis remains largely unknown. Here, we demonstrate that two types of ER-located chaperones, Calnexin and J-proteins, facilitate the biogenesis of ion channels via distinct mechanisms, providing new insight into the role of ER-located chaperones in protein homeostasis.
The biogenesis of ion channels in the ER involves several steps, including glycosylation, folding, assembly, quality control, and ER exit. Besides Calnexin and DNA JB12/DNA JB14, some other chaperone proteins, including HSP70/HSC70 (Li et al., 2011), HSP90 (Ficker et al., 2003), DNA JA1/DNA JA2 (Walker et al., 2010), sig1R (Crottès et al., 2011), and FKBP38 (Walker et al., 2007) have been reported to be involved in the quality control, ER exit, or degradation of hERG. These chaperones may act at different stages of ERG channel biogenesis with distinct molecular mechanisms. This notion was supported by the findings that DNJ-1 and CNX-1 act in parallel to regulate the biogenesis of ERG K + channels, and previous studies have reported that different trafficking-defective hERG mutants show abnormal binding with different chaperones (Delisle et al., 2004;Curran and Mohler, 2015). Further dissection of the role of chaperones in ion channel biogenesis will facilitate our understanding of the channelopathies caused by defective biogenesis of ion channels.