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

Abstract

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. 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. 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 microM) or 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 100 microM). 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. 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.