Polarized MDCK Cells Research Articles

Entry of PIP3-containing polyplexes into MDCK epithelial cells by local apical-basal polarity reversal.

The polarized architecture of epithelium presents a barrier to therapeutic drug/gene carriers, which is mainly due to a limited (apical) internalization of the carrier systems. The bacterium Pseudomonas aeruginosa invades epithelial cells by inducing production of apical phosphatidylinositol-3, 4, 5-triphosphate (PIP3), which results in the recruitment of basolateral receptors to the apical membrane. Since basolateral receptors are known receptors for gene delivery vectors, apical PIP3 may improve the internalization of such vectors into epithelial cells. PIP3 and nucleic acids were complexed by the cationic polymer polyethylenimine (PEI), forming PEI/PIP3 polyplexes. PEI/PIP3 polyplexes showed enhanced internalization compared to PEI polyplexes in polarized MDCK cells, while basolateral receptors were found to redistribute and colocalize with PEI/PIP3 polyplexes at the apical membrane. Following their uptake via endocytosis, PEI/PIP3 polyplexes showed efficient endosomal escape. The effectiveness of the PIP3-containing delivery system to generate a physiological effect was demonstrated by an essentially complete knock down of GFP expression in 30% of GFP-expressing MDCK cells following anti-GFP siRNA delivery. Here, we demonstrate that polyplexes can be successfully modified to mimic epithelial entry mechanisms used by Pseudomonas aeruginosa. These findings encourage the development of pathogen-inspired drug delivery systems to improve drug/gene delivery into and across tissue barriers.

Abstract 2143: A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation

Abstract The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell surface of polarized MDCK cells. Previously, we showed that EREG basolateral trafficking is regulated by a conserved tyrosine residue within a YXXΦ motif (Y156ERV) in its cytoplasmic domain. Interestingly, a Y156A substitution led to apical mistrafficking of EREG and transformation of polarized MDCK cells (PNAS 110: 8960-5, 2013). We have identified an EREG mutation (R147stop) in human tumors that we now report mistrafficks to the apical surface. To test the hypothesis that apical mistrafficking of EREG can be a driver event, we have generated MDCK cells stably expressing inducible (Tet-ON) wild-type and Y156A mutant EREG. In 3D Matrigel cultures, we show that uninduced clones form normal-appearing cysts, regardless of EREG status. Induction of WT or mutant EREG leads to increased cyst diameter. However, only (Y156A)EREG-expressing cells form abnormal cysts with ectopic lumens and inward growth, features of in vivo transformation. Furthermore, we show that ligand processing and EGFR activity is required for EREG mistrafficking-induced ectopic lumen formation as preincubation with a broad-spectrum metalloprotease inhibitor, BB-94, abrogates ectopic lumen formation. We have extended these studies to polarizing human colon cancer cells, HCA-7, which preserve the trafficking fidelity of WT and mutant EREG. Furthermore, using EREG cytoplasmic domain as a bait in a yeast-2-hybrid screen, we have identified APOBEC3C as an interacting protein. APOBECs are a family of cytidine deaminases that have been identified as a major contributor to human cancer mutations. We have subsequently confirmed EREG-APOBEC3C interaction by co-immunoprecipitation. Results from MDCK cells stably expressing differentially inducible EREG (Tet-ON) and APOBEC3C (Cumate-ON) will be presented. Note: This abstract was not presented at the meeting. Citation Format: Bhuminder Singh, Galina Bogatcheva, Robert J. Coffey. A 3D system to elucidate a direct role for epiregulin mistrafficking in epithelial transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2143. doi:10.1158/1538-7445.AM2015-2143

Recycling of galectin-3 in epithelial cells

Galectins, a family of β-galactoside binding proteins, do not possess a signalling sequence to enter the endoplasmic reticulum as a starting point for the classical secretory pathway. They use a so-called unconventional secretion mechanism for translocation across the plasma membrane and/or into the lumen of transport vesicles. The β-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium. The lectin re-enters the cell by non-clathrin mediated endocytosis and passages through endosomal organelles. This internalized galectin-3 plays an important role in apical protein trafficking by directing the subcellular targeting of apical glycoproteins via oligomerization into high molecular weight clusters, a process that can be fine-tuned by changes in the environmental pH. Following release at the apical plasma membrane, the lectin can reenter the cell for another round of recycling and apical protein sorting. This review will briefly address galectin-3-functions in epithelia and focus on distinct phases in apical recycling of the lectin.

Stable knockdown of Kif5b in MDCK cells leads to epithelial–mesenchymal transition

Polarization of epithelial cells requires vectorial sorting and transport of polarity proteins to apical or basolateral domains. Kif5b is the mouse homologue of the human ubiquitous Kinesin Heavy Chain (uKHC). To investigate the function of Kif5b in epithelial cells, we examined the phenotypes of Kif5b-deficient MDCK cells. Stable knockdown of Kif5b in MDCK cells resulted in reduced cell proliferation rate, profound changes in cell morphology, loss of epithelial cell marker, and gain of mesenchymal marker, as well as increased cell migration, invasion, and tumorigenesis abilities. E-cadherin and NMMIIA could interact with Kif5b in polarized MDCK cells, and their expression levels were decreased in Kif5b-deficient MDCK cells. Overexpression of E-cadherin and NMMIIA in Kif5b depleted MDCK cells could decrease mesenchymal marker expression and cell migration ability. These results indicate that stable knockdown of Kif5b in MDCK cells can lead to epithelial–mesenchymal transition, which is mediated by defective E-cadherin and NMMIIA expression.

A Renal Olfactory Receptor Aids in Glucose Handling in the Proximal Tubule

Olfactory receptors (ORs) are seven transmembrane‐domain G protein‐coupled chemosensors that detect odorants in the nose. We previously reported that ORs play roles in the kidney, and identified 9 renal ORs including Olfr1393. In reverse‐transcribed RNA from microdissected renal segments, Olfr1393 is exclusively expressed in the proximal tubule (PT; S1, S2 and S3). When stably expressed in polarized MDCK cells, Olfr1393 localizes to the apical plasma membrane (PM), but not to cilia. Because Olfr1393 is an “orphan receptor” with no known ligand, we screened over 1400 chemicals and found that Olfr1393 detects preconstrained cyclic molecules containing carbonyl or alcohol groups. To determine the physiological role of Olfr1393, we generated knockout (KO) mice. At baseline, KO mice are similar to wild‐type (WT) littermates with regard to plasma electrolytes, blood pressure and GFR (measured via iStat, tail cuff, and elimination kinetics of plasma sinistrin). However, despite being euglycemic, KO mice exhibit glycosuria (1.4x increase in glucose/creatinine vs WT) and can better handle a glucose challenge during a glucose tolerance test as indicated by plasma glucose values (area under curve: WT 22.4 ± 1.9 vs KO 15.7 ± 1.1; P = 0.001). These data suggest that Olfr1393 helps to regulate glucose reabsorption on the apical PM of the PT. In support of this, preliminary data suggests that heterologously expressed Olfr1393 can co‐immunoprecipitate with Sglt1 and Sglt2, the two Na+/glucose co‐transporters responsible for apical glucose reabsorption. We are currently examining the possibility that Olfr1393 influences the function of these transporters to regulate PT glucose handling.Funding: ASN, NIDDK

Measuring receptor recycling in polarized MDCK cells.

Recycling of proteins such as channels, pumps, and receptors is critical for epithelial cell function. In this chapter we present a method to measure receptor recycling in polarized Madin-Darby canine kidney cells using an iodinated ligand. We describe a technique to iodinate transferrin (Tf), we discuss how (125)I-Tf can be used to label a cohort of endocytosed Tf receptor, and then we provide methods to measure the rate of recycling of the (125)I-Tf-receptor complex. We also show how this approach, which is easily adaptable to other proteins, can be used to simultaneously measure the normally small amount of (125)I-Tf transcytosis and degradation.

Induction of lateral lumens through disruption of a monoleucine-based basolateral-sorting motif in betacellulin.

Directed delivery of EGF receptor (EGFR) ligands to the apical or basolateral surface is a crucial regulatory step in the initiation of EGFR signaling in polarized epithelial cells. Herein, we show that the EGFR ligand betacellulin (BTC) is preferentially sorted to the basolateral surface of polarized MDCK cells. By using sequential truncations and site-directed mutagenesis within the BTC cytoplasmic domain, combined with selective cell-surface biotinylation and immunofluorescence, we have uncovered a monoleucine-based basolateral-sorting motif (EExxxL, specifically (156)EEMETL(161)). Disruption of this sorting motif led to equivalent apical and basolateral localization of BTC. Unlike other EGFR ligands, BTC mistrafficking induced formation of lateral lumens in polarized MDCK cells, and this process was significantly attenuated by inhibition of EGFR. Additionally, expression of a cancer-associated somatic BTC mutation (E156K) led to BTC mistrafficking and induced lateral lumens in MDCK cells. Overexpression of BTC, especially mistrafficking forms, increased the growth of MDCK cells. These results uncover a unique role for BTC mistrafficking in promoting epithelial reorganization.

Abstract 3322: Spatio-temporal regulation of epithelial transformation by mistrafficking of EGFR ligand, epiregulin

Abstract The EGF receptor (EGFR) ligand epiregulin (EREG) is delivered preferentially to the basolateral cell surface of polarized MDCK cells. Recently, we showed that EREG basolateral trafficking is regulated by a conserved tyrosine residue within a YXXΦ motif (Y156ERV) in its cytoplasmic domain. Interestingly, a Y156A substitution led to apical mistrafficking of EREG and transformation of polarized MDCK cells (PNAS 110: 8960-5, 2013). We have identified EREG mutations (R147stop) in human tumors that would disrupt the basolateral sorting motif of EREG and now report that EREG harboring these patient mutations mistrafficks to the apical surface. We propose that ligand mistrafficking is a driver of transformation rather than being a mere passenger. To test our proposal wild-type and mutant EREG will be inducibly (Tet-ON) expressed at various stages of MDCK cyst formation and maintenance in 3D Matrigel cultures where we have now recapitulated the transformation phenotype induced by EREG-mistrafficking. Results from these experiments will be presented at the meeting. Citation Format: Bhuminder Singh, Galina Bogatcheva, Mary K. Washington, Robert J. Coffey. Spatio-temporal regulation of epithelial transformation by mistrafficking of EGFR ligand, epiregulin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3322. doi:10.1158/1538-7445.AM2014-3322

TBC1D9B functions as a GTPase-activating protein for Rab11a in polarized MDCK cells.

Rab11a is a key modulator of vesicular trafficking processes, but there is limited information about the guanine nucleotide-exchange factors and GTPase-activating proteins (GAPs) that regulate its GTP-GDP cycle. We observed that in the presence of Mg(2+) (2.5 mM), TBC1D9B interacted via its Tre2-Bub2-Cdc16 (TBC) domain with Rab11a, Rab11b, and Rab4a in a nucleotide-dependent manner. However, only Rab11a was a substrate for TBC1D9B-stimulated GTP hydrolysis. At limiting Mg(2+) concentrations (<0.5 mM), Rab8a was an additional substrate for this GAP. In polarized Madin-Darby canine kidney cells, endogenous TBC1D9B colocalized with Rab11a-positive recycling endosomes but less so with EEA1-positive early endosomes, transferrin-positive recycling endosomes, or late endosomes. Overexpression of TBC1D9B, but not an inactive mutant, decreased the rate of basolateral-to-apical IgA transcytosis--a Rab11a-dependent pathway--and shRNA-mediated depletion of TBC1D9B increased the rate of this process. In contrast, TBC1D9B had no effect on two Rab11a-independent pathways--basolateral recycling of the transferrin receptor or degradation of the epidermal growth factor receptor. Finally, expression of TBC1D9B decreased the amount of active Rab11a in the cell and concomitantly disrupted the interaction between Rab11a and its effector, Sec15A. We conclude that TBC1D9B is a Rab11a GAP that regulates basolateral-to-apical transcytosis in polarized MDCK cells.

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.

Clathrin and its adaptor AP1 are required for apical sorting of GPI‐anchored proteins in biosynthetic and recycling routes in MDCK cells (598.4)

To fulfill their functions, epithelial cells establish asymmetric protein distributions in apical and basolateral plasma membranes. To date, the role of clathrin and its adaptor, the AP1 complex, has been documented exclusively for basolateral protein trafficking in mammalian epithelial cells. We found that the apical localization of endogenous glycosylphosphatidylinositol (GPI)‐anchored proteins is severely affected by gene silencing of the clathrin heavy chain and components of the AP1 adaptor complex in MDCK cells. In these cells, apical targeting is affected not only for GPI‐ anchored proteins but also for endogenous secretory proteins and the apical‐destined t‐SNARE, syntaxin 3. Analysis of apical secretion of lysoGPI‐APs reveals that the biosynthetic delivery of GPI‐APs toward apical plasma membrane is impaired in these cells. Furthermore, apical recycling and transcytosis from basolateral to apical membranes of GPI‐APs are also impaired in these cells. Our results formally demonstrate the importance of biosynthetic and endocytic routes for establishment and maintenance of apical localization of GPI‐APs in polarized MDCK cells.Grant Funding Source: Swiss National Sciences Foundation Assistant Professorship

Elucidating the role of a renal proximal tubule‐specific olfactory receptor (892.42)

Olfactory receptors (ORs) are seven transmembrane domain G protein‐coupled chemosensors that detect odorants in the nose. We previously reported that OR signaling plays a role in the kidney (PNAS 2009), and to date we have identified 9 renal ORs. One of these is Olfr1393, which is widely expressed in a number of tissues, including kidney (by RT‐PCR screen). Using reverse‐transcribed RNA from microdissected renal segments, we found that Olfr1393 is exclusively expressed in the proximal tubule (PT; S1, S2 and S3). Like the majority of ORs, Olfr1393 is an orphan receptor with no known ligand. To screen for ligands, we cloned this receptor from the kidney and expressed it in HEK293T cells after optimizing conditions to allow for surface expression. We comprehensively screened over 1400 chemicals, and found that Olfr1393 detects preconstrained cyclic molecules containing either a carbonyl or alcohol group. We also found that when stably expressed in polarized MDCK cells, Olfr1393 localizes to the apical PM yet is excluded from the primary cilia. To determine the function of this receptor, we generated knockout (KO) mice. At baseline, KO mice appear to be in homeostatic balance based on plasma electrolytes, BUN, creatinine, urinalysis, and blood pressure measurements ‐ in future studies, we will challenge WT and KO mice with Olfr1393 ligands to ascertain how PT and renal function are regulated by Olfr1393.Grant Funding Source: Supported by: NIDDK and ASN

Real time monitoring of NKCC2 endocytosis by total internal reflection fluorescence microscopy (892.19)

The apical Na‐K‐2Cl cotransporter NKCC2 mediates NaCl reabsorption by the thick ascending limb (TAL). The amount of NKCC2 at the apical membrane of TALs is determined by dynamic endocytosis from the apical membrane mediated by clathrin and lipid rafts. However, the time resolution of previous methods used to measure endocytosis was low (5 min), and internalization of NKCC2 containing vesicles not measurable. We hypothesize that TIRF microscopy imaging of fluorescently labeled apical NKCC2 would allow us to monitor its endocytosis in real‐time. We transduced polarized MDCK cells (in Transwell filters) with an N‐terminus GFP‐tagged NKCC2 using adenoviruses. Similar to native TALs, GFP‐NKCC2 was located in the apical membrane, at and above tight junctions. Surface biotinylation confirmed targeting of mature GFP‐NKCC2 (≍190 kDa) to the apical membrane. For TIRF imaging, cells were placed in a chamber with the apical membrane facing the TIRF illumination field. To selectively label NKCC2 at the apical surface we generated NKCC2 containing a biotin acceptor domain of 16 aa between one of the extracellular loops. Once expressed in MDCK cells, we specifically biotinylated surface NKCC2 with recombinant biotin ligase. Labeling apical NKCC2 with streptavidin‐Qdot, or streptavidin‐AlexaFluor allowed us to monitor single endocytic events and apical surface NKCC2 distribution in real‐time. In the absence of biotin ligase we did not observe labeling, indicating specificity for NKCC2 labeling. The overall rate of constitutive NKCC2 endocytosis was 1.2±0.1 % per minute (n=6). In MDCK and TAL cells most apical NKCC2 was located within small discrete domains that exhibited little lateral movement. We conclude that TIRF microscopy of labeled NKCC2 allows the imaging of individual endocytic events at the apical membrane.Grant Funding Source: NIH‐AHA

Golgi sorting regulates organization and activity of GPI proteins at apical membranes.

Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the apical surface.

An image analysis method to quantify CFTR subcellular localization

Aberrant protein subcellular localization caused by mutation is a prominent feature of many human diseases. In Cystic Fibrosis (CF), a recessive lethal disorder that results from dysfunction of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the most common mutation is a deletion of phenylalanine-508 (pF508del). Such mutation produces a misfolded protein that fails to reach the cell surface. To date, over 1900 mutations have been identified in CFTR gene, but only a minority has been analyzed at the protein level. To establish if a particular CFTR variant alters its subcellular distribution, it is necessary to quantitatively determine protein localization in the appropriate cellular context. To date, most quantitative studies on CFTR localization have been based on immunoprecipitation and western blot. In this work, we developed and validated a confocal microscopy-image analysis method to quantitatively examine CFTR at the apical membrane of epithelial cells. Polarized MDCK cells transiently transfected with EGFP-CFTR constructs and stained for an apical marker were used. EGFP-CFTR fluorescence intensity in a region defined by the apical marker was normalized to EGFP-CFTR whole cell fluorescence intensity, rendering “apical CFTR ratio”. We obtained an apical CFTR ratio of 0.67 ± 0.05 for wtCFTR and 0.11 ± 0.02 for pF508del. In addition, this image analysis method was able to discriminate intermediate phenotypes: partial rescue of the pF508del by incubation at 27 °C rendered an apical CFTR ratio value of 0.23 ± 0.01. We concluded the method has a good sensitivity and accurately detects milder phenotypes. Improving axial resolution through deconvolution further increased the sensitivity of the system as rendered an apical CFTR ratio of 0.76 ± 0.03 for wild type and 0.05 ± 0.02 for pF508del. The presented procedure is faster and simpler when compared with other available methods and it is therefore suitable as a screening method to identify mutations that completely or mildly affect CFTR processing. Moreover, it could be extended to other studies on the biology underlying protein subcellular localization in health and disease.

Polarize MDCK Hücrelerinde Osteopontin Ekspresyonu

Polarize MDCK Hücrelerinde Osteopontin Ekspresyonu

The nephronophthisis gene product NPHP2/Inversin interacts with Aurora A and interferes with HDAC6-mediated cilia disassembly

Nephronophthisis (NPH) is a rare recessive disease caused by several different gene mutations. Most gene products localize to the cilium, and thus, the various NPH manifestations including kidney cysts and situs inversus have been linked to ciliary defects. Here, we describe that targeted knockdown of NPHP2 significantly reduced the number of cilia on polarized MDCK cells. As one of the underlying molecular mechanisms, we identified a direct interaction between NPHP2 and Aurora A, a cell cycle kinase that promotes ciliary disassembly after activation by Hef1. NPHP2 inhibited the phosphorylation and activation of Aurora A, and reduced its kinase activity in vitro. Aurora A and histone deacetylase inhibitors ameliorated the ciliogenesis defect in NPHP2-deficient MDCK cells, supporting our hypothesis that NPHP2 is involved in the control of ciliary disassembly. Furthermore, we observed that nephrocystin (NPHP1), an interaction partner of NPHP2, also binds Aurora A, exerting very similar inhibitory effects on Hef1-mediated Aurora A activation. Taken together, these findings suggest that NPHP gene products can interfere with ciliary disassembly through interaction with the Hef1/Aurora A module, thereby modulating cell cycle control and cell proliferation.

PH‐Dependent Recycling of Galectin‐3 at the Apical Membrane of Epithelial Cells

The β-galactoside binding protein galectin-3 is highly expressed in a variety of epithelial cell lines. Polarized MDCK cells secrete this lectin predominantly into the apical medium by non-classical secretion. Once within the apical extracellular milieu, galectin-3 can re-enter the cell followed by passage through endosomal organelles and modulate apical protein sorting. Here, we could show that galectin-3 is internalized by non-clathrin mediated endocytosis. Within endosomal organelles this pool associates with newly synthesized neurotrophin receptor in the biosynthetic pathway and assists in its membrane targeting. This recycling process is accompanied by transient interaction of galectin-3 with detergent insoluble membrane microdomains in a lactose- and pH-dependent manner. Moreover, in the presence of lactose, apical sorting of the neurotrophin receptor is affected following endosomal deacidification. Taken together, our results suggest that internalized galectin-3 directs the subcellular targeting of apical glycoproteins by membrane recycling.

P38 MAPK mediates calcium oxalate crystal-induced tight junction disruption in distal renal tubular epithelial cells

We examined whether p38 MAPK plays role in calcium oxalate monohydrate (COM) crystal-induced tight junction disruption. Polarized MDCK cells were pretreated with or without 20 μM SB239063 (p38 MAPK inhibitor) for 2-h, and then incubated with 100 μg/ml COM crystals for up to 48-h. Western blotting showed increased level of phospho-p38, not total p38, in COM-treated cells, whereas SB239063 pretreatment successfully maintained phospho-p38 at its basal level. COM crystals also caused decreased levels of two tight junction proteins, zonula occludens-1 (ZO-1) and occludin. Immunofluorescence study revealed disruption of tight junction, redistribution, and dissociation of ZO-1 and occludin. Moreover, transepithelial resistance (TER) showed defective barrier function, whereas Western blotting for Na+/K+-ATPase-α1 revealed defective fence function of tight junction in COM-treated cells. All these expression and functional defects were successfully prevented by SB239063 pretreatment. These findings indicate that COM crystals cause tight junction disruption in distal renal tubular epithelial cells through p38 MAPK activation.

Translational Read-Through of a Nonsense Mutation Causing Bartter Syndrome

Bartter syndrome (BS) is classified into 5 genotypes according to underlying mutant genes and BS III is caused by loss-of-function mutations in the CLCNKB gene encoding for basolateral ClC-Kb. BS III is the most common genotype in Korean patients with BS and W610X is the most common CLCNKB mutation in Korean BS III. In this study, we tested the hypothesis that the CLCNKB W610X mutation can be rescued in vitro using aminoglycoside antibiotics, which are known to induce translational read-through of a nonsense mutation. The CLCNKB cDNA was cloned into a eukaryotic expression vector and the W610X nonsense mutation was generated by site-directed mutagenesis. Cultured polarized MDCK cells were transfected with the vectors, and the read-through was induced using an aminoglycoside derivative, G418. Cellular expression of the target protein was monitored via immunohistochemistry. While cells transfected with the mutant CLCNKB failed to express ClC-Kb, G418 treatment of the cells induced the full-length protein expression, which was localized to the basolateral plasma membranes. It is demonstrated that the W610X mutation in CLCNKB can be a good candidate for trial of translational read-through induction as a therapeutic modality.