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

Abstract

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.

Chloride channel ClC-3 in gills of the euryhaline teleost,Tetraodon nigroviridis: expression, localization and the possible role of chloride absorption

“…ClC-3 expression in mammalian cells is strongly inhibited by hyperosmotic cell shrinkage whereas it is activated by hyposmotic cell swelling (Duan et al, 1997). Furthermore, several studies demonstrated that ClC-3 represents a major molecular entity responsible for native volume sensing of outwardly rectifying anion channels in hyposmotic medium in cardiac cells (Duan et al, 1997;Duan et al, 2001;Wang et al, 2003), smooth muscle cells (Duan et al, 2001;Wang et al, 2003), bovine epithelial cells (Wang et al, 2000), HeLa cells and ClC-3-expressed Xenopus oocytes (Kawasaki et al, 1994;Hermoso et al, 2002) as well as non-pigmented ciliary epithelial cells (Vessey et al, 2004). Because the gill is in direct contact with the external environment and is crucial to osmoregulation in fish (Evans et al, 2005), environmental salinity changes would cause alteration of the levels of related genes or proteins to compensate for osmotic homeostasis.…”

“…Weylandt et al provided the evidence to show that a proportion of the ClC-3 protein is present on the plasma membrane (Weylandt et al, 2001). In rabbit non-pigmented ciliary epithelial cells, an enhanced shift in ClC-3 immunofluoresence from intracellular locations to the plasma membrane is clearly evident in cells exposed to hyposmotic solutions as compared with isosmotic solutions (Vessey et al, 2004). Moreover, Huang et al demonstrated that the Cl -current was significantly increased in the ClC-3 gene-transfected mammalian epithelial cell line, tsA, followed by calcium/calmodulin-dependent protein kinase II (CaMKII) activation (Huang et al, 2001).…”

Molecular Interaction and Functional Regulation of ClC-3 by Ca2+/Calmodulin-dependent Protein Kinase II (CaMKII) in Human Malignant Glioma

“…To determine whether ClC-3 and CaMKII are expressed by glioma cells, we immunocytochemically labeled cultured D54 human glioma cells. The cellular location of ClC-3 has been controversial, with some groups finding expression on the cell membrane (16,28) and others seeing predominantly intracellular expression on endosomes and vesicles (12,29). We hypothesized that glioma cells express ClC-3 on the cell membrane, providing a resting chloride conductance that plays a role in malignant characteristics (4).…”

Physiology of Cell Volume Regulation in Vertebrates

“…322). Third, cell shrinkage inhibits, and cell swelling stimulates, exocytosis (and vesicle recycling in general), in a manner which is, not surprisingly, dependent on the cytoskeleton (747,852,1024,1034). Changes in vesicle recycling may regulate the transporters/channels both through signaling events, exemplified by the vesicle recycling-dependent, swellinginduced release of ATP from intestine 407 cells (1024) and through plasma membrane insertion/retrieval of the transport proteins.…”