Hyposmotic activation of ICl,swell in 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 ICl,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.
Vessey
John P
JP
Department of Physiology and Biophysics, Dalhousie University, Halifax, NS B3H 4H7, Canada.
Shi
Chanjuan
C
Jollimore
Christine Ab
CA
Stevens
Kelly T
KT
Coca-Prados
Miguel
M
Barnes
Steven
S
Kelly
Melanie Em
ME
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000502:physiology; D018118:Chloride Channels / Q000378:metabolism* / Q000502:physiology; D002924:Ciliary Body / Q000166:cytology*; D004553:Electric Conductivity; D004847:Epithelial Cells / Q000378:metabolism / Q000502:physiology; D007038:Hypotonic Solutions; D018408:Patch-Clamp Techniques; D021381:Protein Transport; D011817:Rabbits
D018118:Chloride Channels; C115376:ClC-3 channel; D007038:Hypotonic Solutions