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C.W. Do, M.M. Civan; Effects of anti–ClC–3 antibody on swelling–activated Cl– channels in native bovine non–pigmented ciliary epithelial (NPE) cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):418.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:Cl– channels in NPE cells provide a major conduit for Cl– efflux to the posterior chamber. Swelling–activated Cl– current (ICl,swell) has been reported in NPE cells, but whether ClC–3 accounts for the ICl,swell is unclear. We examined the effects of a specific anti–ClC–3 antibody (Ab) on ICl,swell in native bovine NPE cells. Methods:Whole–cell patch clamp measurements were carried out with freshly harvested bovine NPE cells. A1–14 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 ICl,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 ICl,swell, which was significantly blocked by NPPB (100µM) and phloretin (30–300µM). Intracellular dialysis of NPE cells with anti–ClC–3 Ab (A1–14 Ab) had no effects on baseline currents, but significantly delayed and inhibited the ICl,swell. The inhibition of ICl,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 ICl,swell. Conclusions:Our results suggest that ICl,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 ICl,swell raises the possibility of involvement of other volume–activated Cl– channels and/or regulators, as well, in mediating ICl,swell.
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