Inhibition of swelling-activated Cl- currents by functional anti-ClC-3 antibody in native bovine non-pigmented ciliary epithelial cells

PURPOSE. To determine the potential role of ClC-3, a PKC-inhibitable Cl- channel, in mediating the swelling-activated Cl- current (I-Cl,I-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,I-swell, which was reversibly inhibited by the Cl- channel blockers, phloretin (300 muM) or 5-nitro-2-(phenylpropylamino)-benzoate (NPPB, 100 muM). Intracellular dialysis with anti-ClC-3 C670-687 antibody did not affect baseline currents, but significantly delayed and inhibited hypotonic stimulation of I-Cl,I-swell. Preabsorption of the antibody with its antigen prevented the inhibition of I-Cl,I-swell by antibody. In addition, intracellular dialysis with control Ex(133-148) antibody did not affect the I-Cl,I-swell, Moreover, activation of PKC by pretreatment with 100 nM phorbol 12,13-dibutyrate (PDBu) significantly inhibited the initial stimulation of I-Cl,I-swell, but had no effects on the steady state currents. CONCLUSIONS. The results suggest that endogenous ClC-3 is involved in mediating I-Cl,I-swell of native bovine NPE cells. The delayed stimulation of I-Cl,I-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.