Effects of anti–ClC–3 antibody on swelling–activated Cl– channels in native bovine non–pigmented ciliary epithelial (NPE) cells

Abstract

Abstract: : Purpose:Cl^– channels in NPE cells provide a major conduit for Cl^– efflux to the posterior chamber. Swelling–activated Cl^– current (I_Cl,swell) has been reported in NPE cells, but whether ClC–3 accounts for the I_Cl,swell is unclear. We examined the effects of a specific anti–ClC–3 antibody (Ab) on I_Cl,swell in native bovine NPE cells. Methods:Whole–cell patch clamp measurements were carried out with freshly harvested bovine NPE cells. A_1–14 Ab to the N–terminus of mouse ClC–3 (short form) was generously provided by Dr. Joseph R. Hume and his laboratory. Results:At 25mM cytoplasmic NaCl, the baseline whole–cell currents were small (1.6pA/pF at –80mV) when bathed in a solution contained 122mM NaCl. Hypotonic cell swelling stimulated I_Cl,swell, reflecting an outwardly–rectifying Cl^– current in native bovine NPE cells. Time–dependent current inactivation was observed at +80mV. Reducing the osmolarity of the bath solution, from 310mOsm to 293, 275, 258 and 240mOsm, triggered a stepwise stimulation of I_Cl,swell, which was significantly blocked by NPPB (100µM) and phloretin (30–300µM). Intracellular dialysis of NPE cells with anti–ClC–3 Ab (A_1–14 Ab) had no effects on baseline currents, but significantly delayed and inhibited the I_Cl,swell. The inhibition of I_Cl,swell was about 60–70% for both inward and outward currents in a 240mOsm hypotonic solution. Preabsorption of Ab with corresponding antigen partially reversed the inhibition of I_Cl,swell. Conclusions:Our results suggest that I_Cl,swell triggered by hypotonicity was influenced, at least in part, by endogenous ClC–3 in native bovine NPE cells. The delayed and incomplete inhibition of I_Cl,swell raises the possibility of involvement of other volume–activated Cl^– channels and/or regulators, as well, in mediating I_Cl,swell.

Keywords: ion channels • inflow/ciliary body • intraocular pressure