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Enrique J. Rodriguez-Boulan, Ignacio Benedicto, Erwin de la Fuente, Diego Gravotta, Agustin Anastasia, Zelda Salfati, Guillermo Lehmann-Mantaras; ClC-2 chloride channel localizes to the apical surface and primary cilium of RPE cells and regulates ciliogenesis. Invest. Ophthalmol. Vis. Sci. 2017;58(8):358.
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© ARVO (1962-2015); The Authors (2016-present)
Chloride channel 2 (ClC-2) is a ubiquitous polytopic membrane protein localized to the basolateral plasma membrane of many body epithelia that plays important physiological roles in cell volume regulation, ion transport and acid-base regulation. ClC-2 knock-out in mice causes retinal degeneration and blindness. Here, we report an unexpected apical and Primary Cilium (PC) localization of ClC-2 in RPE cells.
We performed confocal immune-fluorescence microscopy to localize ClC-2-HA in polarized ARPE-19 and hfRPE cells transduced with lentiviral vectors. To explore the localization of endogenous ClC-2 we carried out domain-specific cell surface biotinylation assays. To examine the localization of the channel in mouse RPE in vivo, we performed subretinal injection of a plasmid encoding ClC-2-GFP in wild type C57BL/6 mice. Ciliogenesis was induced in cultured ARPE-19 cells by serum starvation for 48h in control and lentivirus-transduced cells with shRNA against CLC-2. To activate the Hedgehog (Hh) pathway, we exposed ARPE-19 cells to 100nM Smoothened agoinst (SAG), a Hh agonist, for 48h.
Surprisingly, in striking contrast with previous physiological studies, our immunofluorescence and surface biotinylation experiments demonstrate that ClC-2 is predominantly localized to the apical surface and PC of cultured RPE cells from different sources. This result was confirmed in vivo by subretinal electroporation of ClC-2-GFP into mice eyes. The apical and PC localization appears to be cell-specific, as there is no detectable signal for ClC-2-HA in the PC of MDCK cells, where ClC-2 localizes basolaterally. Quantification of the percentage of ciliated cells and cilium length showed that ClC-2 depletion significantly impairs cilia formation. Because PC is home for the Hh pathway, we exposed ARPE-19 cells to a Hh agonist (SAG) and found that ClC-2 knockdown attenuates Hh signaling, consistent with a decreased population of ciliated RPE cells.
We show for the first time that ClC-2 is predominantly enriched in the PC of RPE cells, where it seems to play a role in the genesis of primary cilia. We are now exploring additional physiological roles of apical ClC-2 in RPE cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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