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Guillermo Lehmann-Mantaras, Ignacio Benedicto, Erwin de la Fuente, Diego Gravotta, Enrique Rodriguez-Boulan; Apical localization of the CLC-2 chloride channel in polarized RPE cells. Invest. Ophthalmol. Vis. Sci. 2014;55(13):2977.
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The chloride channel 2 (ClC-2) is a polytopic membrane protein ubiquitously expressed in the human body that plays important physiological roles in cell volume regulation, ion transport and acid-base balance. Knock-out of this channel in mice causes retinal degeneration, suggesting that ClC-2 is involved in retinal homeostasis. It is known that ClC-2 localizes to the basolateral surface of most epithelia such as duodenum, colon and MDCK cells. There is functional evidence based on pharmacologic manipulation of Cl- conductance that suggests that ClC-2 is basolaterally localized in RPE. However, the localization of ClC-2 in RPE has not been addressed yet. Here we present a morphological analysis of the subcellular localization of hClC-2 in RPE cells.
We performed confocal fluorescence microscopy in highly polarized ARPE-19 and hfRPE cells grown on Transwell filters. To explore the localization of endogenous hClC-2 we used an affinity-purified anti-ClC-2 antibody. We also studied the localization of exogenous ClC-2 by transduction of hClC-2-GFP and hClC-2-HA via liposomes, electroporation and lentivirus vectors.. To examine the localization of the channel in RPE in situ, we performed in vivo subretinal injection of a plasmid encoding hClC-2-GFP in wild type C57BL/6 mice.
To our surprise, endogenous hClC-2 was predominantly localized to the apical membrane of polarized hfRPE cells. Independently of the gene delivery method used, both hClC-2-GFP and hClC-2-HA were found highly enriched at the apical domain in ARPE-19 and hfRPE cells. The apical localization was confirmed by colocalization of hClC-2-HA with the apical marker MCT1 in non-permeabilized hfRPE monolayers. Finally, we analyzed the ClC-2 localization in situ on mouse RPE transfected in vivo. Confocal analysis of whole mount RPE sheets and transverse retinal vibratome sections showed that hClC-2 was highly enriched at apical RPE microvilli.
We provide for the first time a morphological analysis of ClC-2 localization in RPE cells. To our surprise we found by different methods that that hClC-2 is predominantly localized to the apical plasma membrane in RPE cells.
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