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• W2079132038 abstract "Article10 August 2010free access α-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells Bojun Chen Bojun Chen Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Ping Liu Ping Liu Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Sijie J Wang Sijie J Wang Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Qian Ge Qian Ge Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Haiying Zhan Haiying Zhan Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author William A Mohler William A Mohler Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Zhao-Wen Wang Corresponding Author Zhao-Wen Wang Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Bojun Chen Bojun Chen Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Ping Liu Ping Liu Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Sijie J Wang Sijie J Wang Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Qian Ge Qian Ge Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Haiying Zhan Haiying Zhan Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author William A Mohler William A Mohler Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Zhao-Wen Wang Corresponding Author Zhao-Wen Wang Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA Search for more papers by this author Author Information Bojun Chen1, Ping Liu1, Sijie J Wang1, Qian Ge1, Haiying Zhan1, William A Mohler2 and Zhao-Wen Wang 1 1Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA 2Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA *Corresponding author. Department of Neuroscience, University of Connecticut Health Center, 263 Farmington Avenue, Farmington, CT 06030-3401, USA. Tel.: +1 860 679 7659; Fax: +1 860 679 8766; E-mail: [email protected] The EMBO Journal (2010)29:3184-3195https://doi.org/10.1038/emboj.2010.194 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The BK channel, a voltage- and Ca2+-gated large-conductance potassium channel with many important functions, is often localized at specific subcellular domains. Although proper subcellular localization is likely a prerequisite for the channel to perform its physiological functions, little is known about the molecular basis of localization. Here, we show that CTN-1, a homologue of mammalian α-catulin, is required for subcellular localization of SLO-1, the Caenorhabditis elegans BK channel α-subunit, in body-wall muscle cells. CTN-1 was identified in a genetic screen for mutants that suppressed a lethargic phenotype caused by expressing a gain-of-function (gf) isoform of SLO-1. In body-wall muscle cells, CTN-1 coclusters with SLO-1 at regions of dense bodies, which are Z-disk analogs of mammalian skeletal muscle. In ctn-1 loss-of-function (lf) mutants, SLO-1 was mislocalized in body-wall muscle but its transcription and protein level were unchanged. Targeted rescue of ctn-1(lf) in muscle was sufficient to reinstate the lethargic phenotype in slo-1(gf);ctn-1(lf). These results suggest that CTN-1 plays an important role in BK channel function by mediating channel subcellular localization. Introduction The BK channel (also known as Slo1, KCa or MaxiK channel) is almost ubiquitously expressed and performs many important physiological functions. Malfunction of the channel may cause a variety of disorders, including epilepsy (Brenner et al, 2005; Du et al, 2005), hypertension (Brenner et al, 2000b), progressive hearing loss (Ruttiger et al, 2004), cerebellar ataxia (Sausbier et al, 2004), overactive bladder (Meredith et al, 2004), penile erectile dysfunction (Werner et al, 2005), impaired renal glomerular filtration and potassium excretion (Pluznick et al, 2003) and paroxysmal dyskinesia (Du et al, 2005). The central components of a BK channel are four α-subunits, which form the channel pore and contain voltage and calcium sensing domains (Adelman et al, 1992; Butler et al, 1993; Pallanck and Ganetzky, 1994). In addition, the α-subunits may interact with other proteins that modulate channel functional properties or expression, such as β-subunits and a MinK-related peptide in mammals (Knaus et al, 1994; Wallner et al, 1999; Xia et al, 1999, 2000; Uebele et al, 2000; Weiger et al, 2000; Brenner et al, 2000a; Levy et al, 2008), and Slob and dSLIP1 in Drosophila (Xia et al, 1998; Zhou et al, 1999). BK channels are often localized/enriched at specific subcellular domains. For example, in neurons, BK channels are enriched at the presynaptic nerve terminal (Robitaille et al, 1993; Knaus et al, 1996; Zhou et al, 1999; Hu et al, 2001; Misonou et al, 2006), where they colocalize with voltage-gated Ca2+ channels (VGCCs) (Roberts et al, 1990; Robitaille et al, 1993; Issa and Hudspeth, 1994; Yazejian et al, 2000) to regulate neurotransmitter release (Robitaille et al, 1993; Hu et al, 2001; Wang et al, 2001; Raffaelli et al, 2004; Liu et al, 2007; Wang, 2008). In epithelial cells, BK channels are localized to the apical membrane, where they may regulate potassium secretion and cell volume (Segal and Reuss, 1990; Pacha et al, 1991; Takeuchi et al, 1992; Hirsch et al, 1993; James and Okada, 1994; Huang et al, 1999). In mouse inner hair cells of the cochlea, BK channels are localized to the apical membrane (Pyott et al, 2004) but their role in mammalian auditory function is unclear (Pyott et al, 2007). Proper subcellular localization of the channel is likely important to its physiological functions. However, this has not been experimentally demonstrated. The molecular basis of BK channel subcellular localization is also poorly understood. Although a recent study reported that ISLO-1, a protein with two putative membrane-spanning domains, contributes to SLO-1 subcellular localization in Caenorhabditis elegans (Kim et al, 2009), no homologues could be identified in mammals. The C. elegans BK channel α-subunit SLO-1 is enriched at synaptic regions in the nervous system and clusters in the vicinity of dense bodies in body-wall muscle cells (Wang et al, 2001). In a genetic screen for suppressors of a lethargic phenotype caused by expressing a slo-1(gf) transgene, we identified CTN-1 as a protein indispensable for BK channel function in C. elegans body-wall muscle cells, presumably because of its function in channel subcellular localization. This finding may serve as a starting point for elucidating the molecular basis of BK channel subcellular localization in mammals. Results ctn-1 Mutants were isolated as suppressors of a lethargic phenotype caused by slo-1(gf) To identify novel molecules related to BK channel function in vivo, we screened for mutants that suppressed the lethargic phenotype of a worm strain expressing SLO-1(gf) under the control of slo-1 promoter (Pslo-1). SLO-1(gf) was created by mutating SLO-1 glutamate 350 to glutamine (E350Q). E350 is the same residue that was changed to lysine in a previously described slo-1(gf) mutant (Davies et al, 2003), and the equivalent of mouse Slo1 E321, which contributes to one of the two negative rings at the entrance to the intracellular vestibule of the channel (Brelidze et al, 2003). Worms expressing Pslo-1::SLO-1(E350Q) exhibited distorted locomotion waveform and greatly decreased locomotion speed (Supplementary Movies 1 and 2). From a screening of 24 000 haploid genomes, we isolated 25 mutants. Twelve of the isolated mutants belong to one gene, which was mapped to a 107-kb interval on chromosome I (2562–2669 kb) through single-nucleotide polymorphism (SNP)-based mapping (Davis et al, 2005). We then tested whether cosmids or PCR-amplified genomic DNA fragments of predicted genes within this interval could reinstate the lethargic phenotype of slo-1(gf) when they were expressed in one of the mutants that harboured the slo-1(gf) transgene. We found that two PCR-amplified overlapping genomic DNA fragments (total ∼15 kb) corresponding to the predicted ctn-1 gene (locus Y23H5A.5, http://www.wormbase.org) and 3 kb sequence upstream of its initiation site completely restored the lethargic phenotype of slo-1(gf) (not shown). The ctn-1 encodes a homologue of mammalian α-catulin (Janssens et al, 1999). Although several splice variants of ctn-1 have been identified (http://www.wormbase.org), we found that ctn-1d (Y23H5A.5d) is the predominant isoform based on reverse transcription PCR (RT–PCR). The predicted translational product of this isoform shares 41% identity with mammalian α-catulin (Janssens et al, 1999) in amino-acid sequence (Figure 1). Sequencing of five randomly picked ctn-1 mutants revealed molecular lesions of this gene in all of them (Figure 1). The ctn-1 (zw1), which is a putative null resulting from a premature stop codon, was used for all subsequent analyses. Figure 1.The ctn-1 encodes a homologue of mammalian α-catulin. Shown is the alignment between predicted amino-acid sequences of CTN-1 and human α-catulin (41% identity). The molecular lesions of five ctn-1 alleles were determined. Four alleles have mutations leading to premature stop codon (marked with ‘*’) and one allele (marked with an arrow) disrupts a splice donor site leading to a frame shift after amino-acid Y92 and then a stop codon (CFNGQPIMCM STOP). Download figure Download PowerPoint ctn-1 was expressed in muscle cells and neurons To understand how CTN-1 contributes to SLO-1 function in vivo, we first analysed the expression pattern of ctn-1 and compared it with that of slo-1. Two independent transgenic strains were created that expressed GFP under the control of the ctn-1 promoter (Pctn-1) and slo-1 promoter (Pslo-1), respectively. The expression pattern of ctn-1 largely overlapped with that of slo-1. Specifically, both ctn-1 and slo-1 were expressed in many neurons and several types of muscles, including body-wall muscle, vulval muscle and stomatointestinal muscle. However, slo-1 appeared to be expressed in more neurons in the head than ctn-1, whereas ctn-1 was expressed in pharyngeal muscle cells and some other unidentified cells that did not express slo-1 (Figure 2). Figure 2.The expression pattern of ctn-1 was similar to that of slo-1. The expression patterns were analysed by expressing GFP under the independent controls of slo-1 promoter (Pslo-1, 5.2 kb) and ctn-1 promoter (Pctn-1, 4.3 kb). Strong expression was observed in body-wall muscle (BM) (including head muscle, HM), vulval muscle (VM), stomatointestinal muscle (SIM), nerve ring (NR), and many neurons in the head (not labelled), ventral cord (VC) and tail (not labelled) with both transcriptional fusions. In addition, Pslo-1::GFP was expressed in more neurons in the head, and anal depressor muscle (ADM) in the tail, whereas Pctn-1::GFP was expressed in some pharyngeal muscles (PM) in the corpus and terminal bulb. Scale bar=20 μm. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint The identified ctn-1 splice variants (http://www.wormbase.org) include 9 to 13 exons. All of the splice variants share the first 8 exons but each of them has a unique exon afterward. To determine where these splice variants might be expressed, we created transgenic strains expressing ctn-1 genomic DNA with GFP-coding sequence inserted into each of the unique exons separately (Figure 3A). As GFP was fused to full-length CTN-1, GFP epifluorescence could reflect both the expression and subcellular localization patterns of the CTN-1 isoforms. We detected strong ctn-1d expression in both neurons and muscle cells, obvious ctn-1c expression in muscle cells but weak ctn-1c expression in neurons, and no ctn-1a or ctn-1b expression (Figure 3B). As CTN-1d::GFP appeared as puncta in neuronal processes but not in the soma, it is difficult to tell whether it is expressed in all or a subset of motoneurons in the ventral nerve cord. We then tested whether the expression of wild-type ctn-1c in a ctn-1;slo-1(gf) double mutant could reinstate the lethargic phenotype as ctn-1d did. However, such an effect of ctn-1c was not observed, suggesting CTN-1c is either unrelated or unimportant to SLO-1 function in vivo. Therefore, CTN-1d appeared to be the most important isoform with respect to SLO-1 function, and was used in subsequent experiments. CTN-1d is referred to as CTN-1 hereafter. Figure 3.Expression patterns of ctn-1 splicing variants. (A) Intron–exon organization of ctn-1 splicing variants. GFP was inserted into the unique exon of each splicing variant. The arrows indicate the locations of GFP insertion. (B) No expression was observed for CTN-1a and CTN-1b. Both CTN-1c and CTN-1d were strongly expressed in body-wall muscle cells. CTN-1d was also strongly expressed in neurons, as indicated by the bright fluorescence signal in the nerve ring (arrow), whereas CTN-1c appeared to be weakly expressed only in some neurons (arrow). A corresponding DIC image was shown below each fluorescent image. Scale bar=20 μm. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint CTN-1 was required for SLO-1 function in body-wall muscle cells As the expression patterns of ctn-1 and slo-1 appeared to overlap in body-wall muscle cells and some neurons, the suppression of slo-1(gf) lethargic phenotype by ctn-1(lf) could be due to CTN-1 deficiency in muscle cells, neurons or both. To answer this question, we tested whether targeted expression of wild-type CTN-1 in body-wall muscle cells or neurons could reinstate the lethargic phenotype in the slo-1(gf);ctn-1(lf) double mutant. Quantitative analyses of locomotion speed showed that ctn-1(lf) partially but significantly suppressed the inhibitory effect of slo-1(gf) on locomotion, which could be reversed by expressing wild-type CTN-1 under the control of the muscle-specific myo-3 promoter (Pmyo-3) (Okkema et al, 1993) but not the pan-neuronal rab-3 promoter (Prab-3) (Nonet et al, 1997) (Figure 4A; Supplementary Movies 3, 4, 5, 6 and 7). These observations suggest that the suppression of slo-1(gf) phenotype by ctn-1(lf) resulted mainly from CTN-1 deficiency in muscle cells. Figure 4.CTN-1 was required for SLO-1 function in body-wall muscle cells but not neurons. (A) The ctn-1(lf) mutant counteracted the inhibitory effect of slo-1(gf) on locomotion speed, and this effect could be reversed by expressing wild-type CTN-1 in body-wall muscle cells under the control of the myo-3 promoter (Pmyo-3) but not in neurons under the control of the pan-neuronal rab-3 promoter (Prab-3). (B, C) Quantification of the maximal head-bending angle (B) and root mean square (RMS) of head-bending angle (C). The ctn-1(lf) and slo-(lf) mutants showed increased head-bending angle, and the severity of this phenotype was nonadditive in the slo-1(lf);ctn-1(lf) double mutant. The abnormal bending phenotype of the ctn-1(lf) mutant could be rescued by expressing wild-type CTN-1 in muscle cells using Pmyo-3 but not in neurons using Prab-3. Data are shown as mean±s.e. The asterisk (*) indicates a statistically significant difference (P<0.01) for comparisons either between the specified groups (A), or between the wild type and a mutant strain (B, C). The number of worms in each group analysed was 20–30 for the data shown in (A) and 10 for the data shown in (B, C). Download figure Download PowerPoint Both slo-1(lf) and ctn-1(lf) mutants appeared to be grossly distinct from the wild type in head movement behaviours. To further examine functional relationship between CTN-1 and SLO-1, we quantified the head-bending angle of the wild type and mutant animals using an automated tracking and analysis system. Consistent with a previous report (Kim et al, 2009), we observed a significant increase in head-bending angle in slo-1(lf). This phenotype was shared by ctn-1(lf), and that its severity was not additive in the ctn-1(lf);slo-1(lf) double mutant (Figure 4B and C), suggesting that CTN-1 and SLO-1 likely function together. The head-bending phenotype of ctn-1(lf) could be rescued by expressing wild-type CTN-1 in muscle cells but not in neurons (Figure 4B and C), suggesting that the mutant phenotype was mainly caused by CTN-1 dysfunction in muscle cells. SLO-1 is an important negative regulator of neurotransmitter release at the C. elegans neuromuscular junction (NMJ) (Wang et al, 2001; Liu et al, 2007). The identification of ctn-1 expression in ventral cord motoneurons (Figure 2) raised the possibility that CTN-1 might be needed for SLO-1 function in regulating neurotransmitter release. To investigate this possibility, we analysed the effect of ctn-1(lf) on miniature and evoked postsynaptic currents (mPSCs and ePSCs) recorded from body-wall muscle cells at two different extracellular Ca2+ concentrations (5 and 0.25 mM). The higher [Ca2+] is more suitable for determining whether slo-1(gf) reduces ePSC amplitude and whether this effect may be reversed by ctn-1(lf), whereas the lower [Ca2+] is more suitable for testing whether ctn-1(lf) could increase ePSC amplitude as slo-1(lf) does (Liu et al, 2007). ePSCs were evoked by photoactivation of motoneurons expressing channelrhodopsin-2 under the control of the unc-17 promoter (Liewald et al, 2008; Liu et al, 2009). At 5 mM [Ca2+]o, slo-1(gf) significantly decreased ePSC amplitude and mPSC frequency without affecting mPSC amplitude compared with the wild type, and these effects of slo-1(gf) were not suppressed by ctn-1(lf) (Figure 5). At 0.25 mM [Ca2+]o, slo-1(lf) significantly increased ePSC amplitude and mPSCs frequency without affecting mPSC amplitude, and these effects of slo-1(lf) were not shared by ctn-1(lf) (Figure 5). These observations suggest that CTN-1 is not required for the function of SLO-1 in regulating neurotransmitter release at the NMJ. Figure 5.The ctn-1(lf) mutant did not affect neurotransmitter release at the NMJs. (A) Representative traces of miniature postsynaptic currents (mPSCs) (top) and photo-evoked postsynaptic currents (ePSCs) (bottom) from the wild type, slo-1(gf), and slo-1(gf);ctn-1(lf), slo-1(lf), and ctn-1(lf) recorded at either 5 or 0.25 mM [Ca2+]o. (B) Comparisons of mPSC frequency, mPSC amplitude and ePSC amplitude between the different groups. Data are shown as mean±s.e. The asterisk (*) indicates P<0.01 compared with the wild type. The number of samples analysed is indicated inside each column. One-way ANOVA (with Bonferroni post hoc test) was used for statistical comparisons of the means. Download figure Download PowerPoint CTN-1 was required for SLO-1 subcellular localization in body-wall muscle cells CTN-1 could contribute to SLO-1 function in body-wall muscle cells through several potential mechanisms. We first tested whether CTN-1 has a function in SLO-1 subcellular localization. We previously showed that SLO-1 is enriched at dense body areas in body-wall muscle cells and in the synapse-rich nerve ring in the nervous system, and that these subcellular localization patterns may be recapitulated by an SLO-1::GFP fusion protein (Wang et al, 2001). We created two transgenic strains expressing integrated Pslo-1::SLO-1::GFP and Pmyo-3::SLO-1::GFP for analysing SLO-1 subcellular localization in the nerve ring and body-wall muscle cells, respectively. These two transgenes were then separately crossed into ctn-1(lf) mutant. Although SLO-1::GFP appeared as puncta at locations matching dense bodies in body-wall muscle cells of the wild type, the fluorescent puncta were almost absent in ctn-1(lf) (Figure 6A and B). In contrast, SLO-1::GFP localization in the nerve ring appeared indistinguishable between the wild-type and ctn-1(lf) mutant (Figure 6A). We then asked whether ctn-1(lf) would affect the subcellular localization of two other proteins in body-wall muscle cells, including INX-11 and vinculin. INX-11 is an innexin that may form gap junctions or hemichannels, whereas vinculin is a membrane-cytoskeletal protein. Both proteins are expressed in body-wall muscle cells and localized to dense body regions (Francis and Waterston, 1985; Barstead and Waterston, 1989; Altun et al, 2009). We found that both GFP-tagged INX-11 and native vinculin were normally localized in the ctn-1(lf) mutant (Supplementary Figure S2). These observations suggest that CTN-1 may be specifically required for SLO-1 subcellular localization in body-wall muscle cells. Figure 6.CTN-1 mediates SLO-1 subcellular localization in body-wall muscle cells. (A) SLO-1::GFP was mislocalized in body-wall muscle cells but not neurons of ctn-1(lf), as indicated by the absence of fluorescent puncta (upper panel) in muscle cells but apparently normal epifluorescence in the nerve ring (indicated with arrow) (lower panel). (B) Quantification of the intensity of SLO-1::GFP puncta in body-wall muscle cells. The intensity in ctn-1(lf) was normalized to that in the wild type. Data are shown as mean±s.e. Compared with the wild type, the intensity was significantly reduced in the ctn-1(lf) mutant (P<0.01, unpaired t-test). The numbers of cells analysed were 24 and 22 for the wild-type and ctn-1 mutant, respectively. (C) slo-1 Transcription was unaltered in ctn-1(lf). A slo-1 promoter and GFP transcriptional fusion was expressed in wild-type animals and ctn-1(lf). GFP epifluorescence in body-wall muscle cells, imaged under identical exposure conditions, was indistinguishable between the two groups. The body muscle (BM) and ventral cord (VC) are indicated (arrows). (D) Western blot shows that the level of total SLO-1::GFP protein was comparable between wild-type and ctn-1(lf) mutant worms. α-Tubulin was blotted to show equal loading of the protein samples. (E) SLO-1 and CTN-1 colocalized in dense body areas of body-wall muscle cells. GFP-tagged SLO-1 (green) and mStrawberry-tagged CTN-1 (red) were coexpressed in body-wall muscle cells under the control of the myo-3 promoter. The merged picture shows colocalization of the two fusion proteins. Scale bar=20 μm. Download figure Download PowerPoint The disappearance of SLO-1::GFP puncta in body-wall muscle cells of ctn-1(lf) mutants could be due to decreased gene transcription or decreased protein synthesis/stability. To determine whether CTN-1 controls slo-1 transcription in muscle cells, we compared the expression of a Pslo-1::GFP transcriptional fusion between the wild-type and ctn-1(lf) mutant. GFP expression in body-wall muscle cells was similar between the two groups (Figure 6C). To determine whether CTN-1 has an effect on SLO-1 protein level, we compared the total SLO-1::GFP protein level between the wild-type and ctn-1(lf) mutant by western blot using a GFP antibody but found no difference (Figure 6D). These observations suggest that the apparent SLO-1 mislocalization observed in ctn-1(lf) mutant did not result from a deficiency in slo-1 transcription or SLO-1 protein synthesis/stability. We also asked whether CTN-1 regulates SLO-1 surface protein level by performing biotinylation assays with transfected HEK293 cells, and found that both the total and surface SLO-1 protein levels were comparable between cells transfected with SLO-1 alone and SLO-1 plus CTN-1 (Supplementary Figure S3), suggesting that CTN-1 probably does not regulate SLO-1 trafficking to the plasma membrane. However, given that HEK293 cells are different from C. elegans body-wall muscle cells in many ways, we cannot exclude a function of CTN-1 in SLO-1 membrane trafficking in vivo solely based on this observation. CTN-1 physically interacted with SLO-1 both in vivo and in vitro To determine whether CTN-1 mediates SLO-1 subcellular localization through a local effect, we analysed subcellular localization pattern of a CTN-1::EGFP fusion protein in body-wall muscle cells. The fusion protein was expected to recapitulate the subcellular localization pattern of wild-type CTN-1 because it reinstated the lethargic phenotype when expressed in the slo-1(gf);ctn-1(lf) double mutant (not shown). We found that CTN-1::EGFP was enriched at dense body regions (Supplementary Figure S4A), which was independent of SLO-1 (Supplementary Figure S4B). Furthermore, we found that CTN-1::mStrawberry, which was also fully functional, colocalized with SLO-1::GFP at dense body regions (Figure 6E). These observations suggest that CTN-1 likely mediates SLO-1 subcellular localization through a local effect. The colocalization data shown in Figure 6E did not have enough resolution to suggest whether CTN-1 physically interacts with SLO-1. To address this question, we performed bimolecular fluorescence complementation (BiFC) assays (Chen et al, 2007; Shyu et al, 2008), which tells not only whether but also where two proteins interact in vivo. In this assay, the nonfluorescent amino- and carboxyl-terminal portions of yellow fluorescent protein (YFPa and YFPc) are fused separately to a pair of proteins of interest. The fluorophore of YFP may be reconstituted if these two proteins are physically very close (Shyu et al, 2008). We inserted YFPa into the linker region between the two RCK domains of SLO-1 and fused YFPc to the carboxyl terminus of CTN-1, and coexpressed these two fusion proteins in body-wall muscle cells under the control of Pmyo-3. Fluorescent puncta were observed in body-wall muscle cells coexpressing SLO-1::YFPa and CTN-1::YFPc (Figure 7A), suggesting that SLO-1 and CTN-1 are physically very close in the muscle cells. SLO-1 may be divided into two major structural components, including the amino-terminal portion (1–352 aa) that contains seven membrane-spanning domains and the channel pore domain, and the cytoplasmic carboxyl terminal portion (353–1140) that contains two RCK domains (Wang et al, 2001; Jiang et al, 2002; Salkoff et al, 2006; Yusifov et al, 2008; Yuan et al, 2010). To determine which part of SLO-1 is important for the interaction with CTN-1, we tested whether the amino- or carboxyl-terminal portion of SLO-1 is required for BiFC with CTN-1. In these experiments, YFPa was either fused to the carboxyl terminus of SLO-1(1–370) or inserted into the linker region between the two RCK domains of SLO-1(371–1140). We found that SLO-1(371–1140) but not SLO-1(1–370) allowed BiFC with CTN-1 (Figure 7A). These observations suggest that CTN-1 is physically very close to SLO-1, and likely interacts with the carboxyl terminal portion of SLO-1 to mediate SLO-1 subcellular localization. Figure 7.CTN-1 physically interacts with SLO-1. (A) CTN-1 and SLO-1 were physically very close. The nonfluorescent amino- and carboxyl-terminal portions of YFP (YFPa and YFPc) were fused to SLO-1 (or its variants) and CTN-1, respectively. Bimolecular fluorescence complementation (BiFC) assays were performed by coexpressing CTN-1::YFPc with each of the SLO-1 fusions (full-length SLO-1::YFPa, SLO-1(1–370)::YFPa and SLO-1(371–1140)::YFPa) in body-wall muscle cells under the control of the myo-3 promoter. Fluorescent puncta were observed in dense body areas when CTN-1::YFPc was coexpressed with either full-length SLO-1::YFPa (upper panel) or SLO-1(371–1140)::YFPa (lower panel) but not with SLO-1(1–370)::YFPa (middle panel). The negative result with SLO-1(1–370)::YFPa was not due to poor expression of the fusion protein because SLO-1(1–370)::YFPa was able to reconstitute YFP epifluorescence when coexpressed with another protein (not shown). Scale bar=20 μm. (B) CTN-1 and SLO-1 coimmunoprecipitated from transfected HEK293 cells. CTN-1 coimmunoprecipitated with either full-length SLO-1 or SLO-1(371–1140) but not with SLO-1(1–370). CTN-1 was tagged with HA, whereas SLO-1 and its variants were tagged with Myc. Immunoprecipitation (IP) and immunoblot (IB) were performed with Myc and HA antibodies. All specific bands are indicated by their relative molecular sizes. The antibody heavy chain dimmer is indicated with an arrow. A full-colour version of this figure is available at The EMBO Journal Online. Download figure Download PowerPoint To obtain independent evidence showing the interaction between CTN-1 and SLO-1, we performed coimmunoprecipitation experiments with HA-tagged CTN-1, and Myc-tagged full-length SLO-1 or SLO-1 amino- and carboxyl-terminal portions. We found that either full-length SLO-1 or SLO-1(371–1140) coimmunoprecipitated with CTN-1 whereas SLO-1(1–370) did not (Figure 7B). These observations reinforced the notion that the cytoplasmic carboxyl terminal portion of SLO-1 i" @default.
• W2079132038 created "2016-06-24" @default.
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• W2079132038 creator A5010260063 @default.
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• W2079132038 date "2010-08-10" @default.
• W2079132038 modified "2023-09-26" @default.
• W2079132038 title "α-Catulin CTN-1 is required for BK channel subcellular localization in C. elegans body-wall muscle cells" @default.
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