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KF Webb, R Merriman-Smith, J Kistler, PJ Donaldson; Chloride Conductances In Lens Fiber Cells: A Role For ClC3? . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4648.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The exposure of lenses to variety of Cl- channel inhibitors induces a dilation of the extracellular space between fiber cells localized to a distinct zone some 150µm in from the lens capsule. To directly test whether these dilations are caused by blocking Cl- channels a molecular physiological characterization of the Cl- conductance of fiber cells isolated from this zone was conducted. Methods: Fiber cells of up to 350mm in length were obtained from three-to four-week-old female Wistar rats by mechanical decapsulation and enzymatic dissociation. Isolated cells were seeded into a recording chamber and overlaid with Artificial Aqueous Humour (AAH) that contained the cation channel blocker Gd3+ (3 mM) and the hemi-channel blocker Co2+ (4 mM). In this recording solution cell integrity could be maintained for many hours in AAH that contained Ca2+, thereby allowing the conductance properties of isolated fiber cells to be assayed using the whole cell patch clamp recording configuration. Molecular profiling of Cl- channel transcripts in the rat lens was performed using reverse transcription-polymerase chain reaction (RT-PCR). Levels of transcript expression and protein expression was then verified by Northern and Western analysis, respectively. Finally, immunocytochemistry was used to localize the extent of chloride channel expression. Results: Fiber cells from the zone of extracellular space dilation were typically over 120 µm in length and exhibited an outwardly rectifying conductance. Replacement of external Cl- with the impermeant anion gluconate drastically reduced this current indicating that it is mediated by influx of Cl- ions. This conductance was enhanced by the replacement of Cl- with I- indicating an anion selectivity of I- ≷ Cl- ≷ gluconate. The current was inhibited by the addition of DIDS (100 µM) and ATP (5 mM) to the bath. RT-PCR based screening for members of the ClC channel family indicated that ClC2 and ClC3, but not ClC1, are expressed in lens fiber cells. The expression these two isoforms was confirmed by Northern and Western analysis. Immunocytochemistry showed that ClC2 and ClC3 protein was expressed in both epithelial cells and cortical fiber cells. Conclusion: The electrical fingerprint of fiber cells isolated from the zone of extracellular space dilations suggests that ClC3 channels mediate the Cl- influx observed in this region. This influx is thought to contribute to a circulating flux of Cl- ions that acts to regulate lens volume and therefore maintain lens transparency.
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