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B.R. Pattnaik, B.A. Hughes; Characterization of the resting chloride (Cl–) conductance in freshly dissociated mouse retinal pigment epithelial (RPE) cells. . Invest. Ophthalmol. Vis. Sci. 2004;45(13):420.
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Purpose: Cl– channels are key elements in the pathways mediating Cl– and fluid transport across the RPE. The purpose of the present study was to characterize Cl– channels in freshly dissociated mouse RPE cells. Methods: RPE cells were enzymatically dissociated from eyecups of C57/BL6 mice. Whole–cell currents were recorded at room temperature using either the whole–cell or perforated–patch configuration of the patch–clamp technique while continuously perfusing the recording chamber with experimental solutions. Cl– currents were isolated by ion substitution or ion channel blockers. Results: Freshly dissociated mouse RPE cells had a characteristic hour–glass shape. When recorded with physiological pipette solution, both inwardly rectifying and delayed outwardly rectifying K+ currents were observed. When K+ currents were blocked with 5 mM external Ba2+, a residual inwardly rectifying current that reversed at ECl was revealed. The inwardly rectifying Cl– current (IClir) was further characterized under the condition of 0 Na+, 0 K+, and elevated internal Cl–. Substitution of external Cl– with foreign anions resulted in reversal potential shifts that specified an anion selectivity sequence of NO3– > I– >> Br– > Cl– >> Gluc– > MeSO4–. The Cl– channel antagonists niflumic acid, DIDS and NPPB reversibly blocked IClir, while 9–AC, Zn2+ and Cd2+ had no effect. Exposure to ionomycin did not affect Cl– current. Conclusions: Isolated mouse RPE cells exhibit a robust inwardly rectifying Cl– current. The anion selectivity and pharmacology profile of this current indicate that it is not mediated by ClC channels. We conclude that the underlying channel may provide an important pathway for Cl– efflux across the RPE basolateral membrane. Support: NIH grants EY08850 and EY07003, Foundation Fighting Blindness, and RPB Lew R. Wasserman Award (BAH)
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