Generation and Functional Characterization of Epithelial Cells with Stable Expression of SLC26A9 Cl- Channels

Overview

Journal Am J Physiol Lung Cell Mol Physiol

Publisher American Physiological Society

Specialties Cell Biology
Molecular Biology
Physiology
Pulmonary Medicine

Date 2016 Jan 24

PMID 26801567

Citations 24

Authors

Johanna J Salomon

Stephan Spahn

Xiaohui Wang

Joachim Fullekrug

Carol A Bertrand

Marcus A Mall

Affiliations

Soon will be listed here.

Abstract

Recent studies identified the SLC26A9 Cl(-) channel as a modifier and potential therapeutic target in cystic fibrosis (CF). However, understanding of the regulation of SLC26A9 in epithelia remains limited and cellular models with stable expression for biochemical and functional studies are missing. We, therefore, generated Fisher rat thyroid (FRT) epithelial cells with stable expression of HA-tagged SLC26A9 via retroviral transfection and characterized SLC26A9 expression and function using Western blotting, immunolocalization, whole cell patch-clamp, and transepithelial bioelectric studies in Ussing chambers. We demonstrate stable expression of SLC26A9 in transfected FRT (SLC26A9-FRT) cells on the mRNA and protein level. Immunolocalization and Western blotting detected SLC26A9 in different intracellular compartments and to a lesser extent at the cell surface. Whole cell patch-clamp recordings demonstrated significantly increased constitutive Cl(-) currents in SLC26A9-FRT compared with control-transduced FRT (Control-FRT) cells (P < 0.01). Similar, transepithelial measurements showed that the basal short circuit current was significantly increased in SLC26A9-FRT vs. Control-FRT cell monolayers (P < 0.01). SLC26A9-mediated Cl(-) currents were increased by cAMP-dependent stimulation (IBMX and forskolin) and inhibited by GlyH-101, niflumic acid, DIDS, and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), as well as RNAi knockdown of WNK1 implicated in epithelial osmoregulation. Our results support that these novel epithelial cells with stable expression of SLC26A9 will be a useful model for studies of pharmacological regulation including the identification of activators of SLC26A9 Cl(-) channels that may compensate deficient cystic fibrosis transmembrane regulator (CFTR)-mediated Cl(-) secretion and serve as an alternative therapeutic target in patients with CF and potentially other muco-obstructive lung diseases.

Citing Articles

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References

Galietta L, Jayaraman S, Verkman A . Cell-based assay for high-throughput quantitative screening of CFTR chloride transport agonists. Am J Physiol Cell Physiol. 2001; 281(5):C1734-42. DOI: 10.1152/ajpcell.2001.281.5.C1734. View

Ma T, Vetrivel L, Yang H, Pedemonte N, Zegarra-Moran O, Galietta L . High-affinity activators of cystic fibrosis transmembrane conductance regulator (CFTR) chloride conductance identified by high-throughput screening. J Biol Chem. 2002; 277(40):37235-41. DOI: 10.1074/jbc.M205932200. View

Wainwright C, Elborn J, Ramsey B, Marigowda G, Huang X, Cipolli M . Lumacaftor-Ivacaftor in Patients with Cystic Fibrosis Homozygous for Phe508del CFTR. N Engl J Med. 2015; 373(3):220-31. PMC: 4764353. DOI: 10.1056/NEJMoa1409547. View

Liu X, Li T, Riederer B, Lenzen H, Ludolph L, Yeruva S . Loss of Slc26a9 anion transporter alters intestinal electrolyte and HCO3(-) transport and reduces survival in CFTR-deficient mice. Pflugers Arch. 2014; 467(6):1261-75. PMC: 4434866. DOI: 10.1007/s00424-014-1543-x. View

Bakouh N, Bienvenu T, Thomas A, Ehrenfeld J, Liote H, Roussel D . Characterization of SLC26A9 in patients with CF-like lung disease. Hum Mutat. 2013; 34(10):1404-14. DOI: 10.1002/humu.22382. View

Mall M, Galietta L . Targeting ion channels in cystic fibrosis. J Cyst Fibros. 2015; 14(5):561-70. DOI: 10.1016/j.jcf.2015.06.002. View

Schreiber R, Uliyakina I, Kongsuphol P, Warth R, Mirza M, Martins J . Expression and function of epithelial anoctamins. J Biol Chem. 2010; 285(10):7838-45. PMC: 2844227. DOI: 10.1074/jbc.M109.065367. View

Yang C, Liu X, Paliege A, Zhu X, Bachmann S, Dawson D . WNK1 and WNK4 modulate CFTR activity. Biochem Biophys Res Commun. 2006; 353(3):535-40. DOI: 10.1016/j.bbrc.2006.11.151. View

Scott-Ward T, Li H, Schmidt A, Cai Z, Sheppard D . Direct block of the cystic fibrosis transmembrane conductance regulator Cl(-) channel by niflumic acid. Mol Membr Biol. 2003; 21(1):27-38. DOI: 10.1080/09687680310001597758. View

10.

Dorwart M, Shcheynikov N, Wang Y, Stippec S, Muallem S . SLC26A9 is a Cl(-) channel regulated by the WNK kinases. J Physiol. 2007; 584(Pt 1):333-45. PMC: 2277069. DOI: 10.1113/jphysiol.2007.135855. View

11.

Sheppard D, Carson M, Ostedgaard L, Denning G, Welsh M . Expression of cystic fibrosis transmembrane conductance regulator in a model epithelium. Am J Physiol. 1994; 266(4 Pt 1):L405-13. DOI: 10.1152/ajplung.1994.266.4.L405. View

12.

Rich D, Anderson M, Gregory R, Cheng S, Paul S, Jefferson D . Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial cells. Nature. 1990; 347(6291):358-63. DOI: 10.1038/347358a0. View

13.

Li J, Xia F, Reithmeier R . N-glycosylation and topology of the human SLC26 family of anion transport membrane proteins. Am J Physiol Cell Physiol. 2014; 306(10):C943-60. DOI: 10.1152/ajpcell.00030.2014. View

14.

Avella M, Loriol C, Boulukos K, Borgese F, Ehrenfeld J . SLC26A9 stimulates CFTR expression and function in human bronchial cell lines. J Cell Physiol. 2010; 226(1):212-23. DOI: 10.1002/jcp.22328. View

15.

Mall M, Hartl D . CFTR: cystic fibrosis and beyond. Eur Respir J. 2014; 44(4):1042-54. DOI: 10.1183/09031936.00228013. View

16.

Xu J, Song P, Miller M, Borgese F, Barone S, Riederer B . Deletion of the chloride transporter Slc26a9 causes loss of tubulovesicles in parietal cells and impairs acid secretion in the stomach. Proc Natl Acad Sci U S A. 2008; 105(46):17955-60. PMC: 2582584. DOI: 10.1073/pnas.0800616105. View

17.

Richardson C, Sakamoto K, de Los Heros P, Deak M, Campbell D, Prescott A . Regulation of the NKCC2 ion cotransporter by SPAK-OSR1-dependent and -independent pathways. J Cell Sci. 2011; 124(Pt 5):789-800. PMC: 3114804. DOI: 10.1242/jcs.077230. View

18.

Bertrand C, Zhang R, Pilewski J, Frizzell R . SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia. J Gen Physiol. 2009; 133(4):421-38. PMC: 2664976. DOI: 10.1085/jgp.200810097. View

19.

Namkung W, Park J, Seo Y, Verkman A . Novel amino-carbonitrile-pyrazole identified in a small molecule screen activates wild-type and ΔF508 cystic fibrosis transmembrane conductance regulator in the absence of a cAMP agonist. Mol Pharmacol. 2013; 84(3):384-92. PMC: 3876813. DOI: 10.1124/mol.113.086348. View

20.

Sheppard D . CFTR channel pharmacology: novel pore blockers identified by high-throughput screening. J Gen Physiol. 2004; 124(2):109-13. PMC: 2229622. DOI: 10.1085/jgp.200409135. View