ClC-3 is a fundamental molecular component of volume-sensitive outwardly rectifying Cl- channels and volume regulation in HeLa cells and Xenopus laevis oocytes.
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.
Hermoso
Marcela
M
Instituto de Ciencias Biomédicas, Facultad de Medicina Universidad de Chile, Santiago 6530499, Chile.
Satterwhite
Christina M
CM
Andrade
Yanire Naty
YN
Hidalgo
Jorge
J
Wilson
Sean M
SM
Horowitz
Burton
B
Hume
Joseph R
JR
D000818:Animals; D000838:Anions; D018118:Chloride Channels / Q000737:chemistry* / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D017931:DNA Primers / Q000737:chemistry; D004594:Electrophysiology; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D017403:In Situ Hybridization; D016297:Mutagenesis, Site-Directed; D016376:Oligonucleotides, Antisense / Q000378:metabolism / Q000494:pharmacology; D009865:Oocytes / Q000378:metabolism; D018408:Patch-Clamp Techniques; D011485:Protein Binding; D018075:RNA, Complementary / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014162:Transfection; D014981:Xenopus; D014982:Xenopus laevis / Q000196:embryology*
D000838:Anions; D018118:Chloride Channels; D002712:Chlorides; C115376:ClC-3 channel; D017931:DNA Primers; D016376:Oligonucleotides, Antisense; D018075:RNA, Complementary; D012333:RNA, Messenger