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Inhibition of Swelling-Activated Cl^– Currents by Functional Anti-ClC-3 Antibody in Native Bovine Non-Pigmented Ciliary Epithelial Cells

¹From the Departments of Physiology, ²Ophthalmology, and ³Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania.

PURPOSE. To determine the potential role of ClC-3, a PKC-inhibitable Cl^– channel, in mediating the swelling-activated Cl^– current (I_Cl,swell) of native bovine nonpigmented ciliary epithelial (NPE) cells.

METHODS. Native bovine NPE cells were freshly harvested by enzymatic digestion. Whole-cell currents were recorded by patch–clamp measurements either in the presence or absence of a functional anti-ClC-3 antibody.

RESULTS. Baseline whole-cell currents were small under isotonic conditions. Hypotonic cell swelling stimulated outwardly rectifying I_Cl,swell, which was reversibly inhibited by the Cl^– channel blockers, phloretin (300 µM) or 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 100 µM). Intracellular dialysis with anti-ClC-3 C_670-687 antibody did not affect baseline currents, but significantly delayed and inhibited hypotonic stimulation of I_Cl,swell. Preabsorption of the antibody with its antigen prevented the inhibition of I_Cl,swell by antibody. In addition, intracellular dialysis with control Ex_133-148 antibody did not affect the I_Cl,swell. Moreover, activation of PKC by pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited the initial stimulation of I_Cl,swell, but had no effects on the steady state currents.

CONCLUSIONS. The results suggest that endogenous ClC-3 is involved in mediating I_Cl,swell of native bovine NPE cells. The delayed stimulation of I_Cl,swell by PDBu may reflect upregulation of swelling-activated Cl^– channels of different subtypes, especially when the function of ClC-3 is blocked. This information will be useful in understanding the mechanisms controlling aqueous humor formation and thereby intraocular pressure.

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