Hyposmotic activation of I_Cl,swell rabbit nonpigmented ciliary epithelial cells involves increased ClC-3 trafficking to the plasma membrane

In mammalian nonpigmented ciliary epithelial (NPE) cells, hyposmotic stimulation leading to cell swelling activates an outwardly rectifying Cl ^- conductance (I_Cl,swell), which, in turn, results in regulatory volume decrease. The aim of this study was to determine whether increased trafficking of intracellular ClC-3 Cl channels to the plasma membrane could contribute to the I_Cl,SWell following hyposmotic stimulation. Our results demonstrate that hyposmotic stimulation reversibly activates an outwardly rectifying Cl^- current that is inhibited by phorbol-12-dibutyrate and niflumic acid. Transfection with ClC-3 antisense, but not sense, oligonucleotides reduced ClC-3 expression as well as I _Cl,swell. Intracellular dialysis with 2 different ClC-3 antibodies abolished activation of I_Cl,SWell. Immunofluorescence microscopy showed that hyposmotic stimulation increased ClC-3 immunoreactivity at the plasma membrane. To determine whether this increased expression of ClC-3 at the plasma membrane could be due to increased vesicular trafficking, we examined membrane dynamics with the fluorescent membrane dye FM1-43. Hyposmotic stimulation rapidly increased the rate of exocytosis, which, along with I _Cl,swell, was inhibited by the phosphoinositide-3-kinase inhibitor wortmannin and the microtubule disrupting agent, nocodazole. These findings suggest that ClC-3 channels contribute to I_Cl,swell following hyposmotic stimulation through increased trafficking of channels to the plasma membrane.