Abstract
Volume-sensitive osmolyte and anion channels (VSOACs) are activated upon cell swelling in most vertebrate cells. Native VSOACs are believed to be a major pathway for regulatory volume decrease (RVD) through efflux of chloride and organic osmolytes. ClC-3 has been proposed to encode native VSOACs in Xenopus laevis oocytes and in some mammalian cells, including cardiac and vascular smooth muscle cells. The relationship between the ClC-3 chloride channel, the native volume-sensitive osmolyte and anion channel (VSOAC) currents, and cell volume regulation in HeLa cells and X. laevis oocytes was investigated using ClC-3 antisense. In situ hybridization in HeLa cells, semiquantitative and real-time PCR, and immunoblot studies in HeLa cells and X. laevis oocytes demonstrated the presence of ClC-3 mRNA and protein, respectively. Exposing both cell types to hypotonic solutions induced cell swelling and activated native VSOACs. Transient transfection of HeLa cells with ClC-3 antisense oligonucleotide or X. laevis oocytes injected with antisense cRNA abolished the native ClC-3 mRNA transcript and protein and significantly reduced the density of native VSOACs activated by hypotonically induced cell swelling. In addition, antisense against native ClC-3 significantly impaired the ability of HeLa cells and X. laevis oocytes to regulate their volume. These results suggest that ClC-3 is an important molecular component underlying VSOACs and the RVD process in HeLa cells and X. laevis oocytes.
Highlights
Organic osmolytes accompanied by osmotically obliged water loss
Our results demonstrate that antisense treatment abolished both mRNA and protein expression of ClC-3 in HeLa cells and X. laevis oocytes, significantly reduced native Volume-sensitive osmolyte and anion channels (VSOACs) current density, and significantly diminished the ability of cells to undergo regulatory volume decrease (RVD)
Total RNA from HeLa cell was extracted at 0, 6, 12, 16, 24, and 48 h after antisense oligodeoxynucleotides (ASO)- or MMO-ClC-3 oligonucleotide treatment, and samples were analyzed by semiquantitative PCR
Summary
Organic osmolytes accompanied by osmotically obliged water loss. Approximately 70% of the osmolyte loss during the RVD response is accounted for by loss of KCl via separate conductive pathways [2, 3]. Macroscopic outwardly rectifying VSOAC currents are characterized by activation after cell volume increase [6], a SCN Ͼ I Ͼ Br Ͼ Cl Ͼ F Ͼ gluconate permeability sequence [6], time-dependent inactivation at positive potentials [7], inhibition by tamoxifen, 1,9-dideoxyforskolin, and stilbene derivatives [8], dependence on intracellular ATP [6], and a single channel conductance in the range of 20 – 40 pS [9] These biophysical and pharmacological characteristics are considered to represent classic characteristics of VSOACs. variations in the pharmacological characteristics, voltage dependence, and signaling mechanisms responsible for channel activation have been described in different cell types [5, 10].
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