Association of ClC-3 Channel with Cl − Transport by Human Nonpigmented Ciliary Epithelial Cells

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Abstract
Electrophysiologic and volumetric evidence link the swelling-activated Cl− channels [gCl(Vol)] of nonpigmented ciliary epithelial (NPE) cells with the Cl−- channel/Cl−-channel regulator protein pICln. However, inhibitors (verapamil and dideoxyforskolin) of another Cl− channel/regulator (MDR1) have been found to inhibit the volume-activated transport response [the regulatory volume decrease (RVD)] of bovine NPE cells. We have addressed the possible molecular basis for the NPE Cl− channels by volumetric measurements of ODM human NPE cells in hypotonic and isotonic test solutions, and by polymerase chain reaction (PCR) cloning and Northern analyses of the same cells. Verapamil and dideoxyforskolin did inhibit the RVD. However, at a concentration (100 μm) which blocks >90% of the MDR1-associated Cl− currents, forskolin had no effect on the volume-activated Cl− channels or on the inhibition of those channels by protein kinase C. High concentrations of ATP (3.5 and 10 mm) and niflumic acid (IC50∼ 200 μm) also block [gCl(Vol)]. The RVD is inhibited by 9-phenylanthranilic acid (DPC) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), unaffected by anthracene-9-carboxylic acid (9-AC), and stimulated by ionomycin. The Cl−-channel blockers NPPB, niflumic acid, DPC and 9-AC, and the Ca2+-ionophore ionomycin had qualitatively similar effects on the rate of staurosporine-activated isotonic cell shrinkage. These results support the concept that the volume-sensitive protein pICln regulates the Cl− channels, and that the same conduits subserve volume- and staurosporine-activated Cl− release. Of the cloned and sequenced Cl− channels, ClC-3 uniquely conforms to the stationary currents and PKC sensitivity of the NPE Cl− channels. PCR amplifications of human cDNA libraries from ciliary body, NPE cells and retina with primers based on human ClC-3 and ClC-4 cDNA, and Northern analyses using the products generated indicated that ciliary epithelial cells express transcripts for ClC-3 (but not ClC-4). We suggest that ClC-3 provides the same conduit for both volume-activated and isotonically staurosporine-activated Cl− channels of human nonpigmented ciliary epithelial cells.

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