ClC-2 chloride channels contribute to HTC cell volume homeostasis.
Membrane Cl(-) channels play an important role in cell volume homeostasis and regulation of volume-sensitive cell transport and metabolism. Heterologous expression of ClC-2 channel cDNA leads to the appearance of swelling-activated Cl(-) currents, consistent with a role in cell volume regulation. Since channel properties in heterologous models are potentially modified by cellular background, we evaluated whether endogenous ClC-2 proteins are functionally important in cell volume regulation. As shown by whole cell patch clamp techniques in rat HTC hepatoma cells, cell volume increases stimulated inwardly rectifying Cl(-) currents when non-ClC-2 currents were blocked by DIDS (100 microM). A cDNA closely homologous with rat brain ClC-2 was isolated from HTC cells; identical sequence was demonstrated for ClC-2 cDNAs in primary rat hepatocytes and cholangiocytes. ClC-2 mRNA and membrane protein expression was demonstrated by in situ hybridization, immunocytochemistry, and Western blot. Intracellular delivery of antibodies to an essential regulatory domain of ClC-2 decreased ClC-2-dependent currents expressed in HEK-293 cells. In HTC cells, the same antibodies prevented activation of endogenous Cl(-) currents by cell volume increases or exposure to the purinergic receptor agonist ATP and delayed HTC cell volume recovery from swelling. These studies provide further evidence that mammalian ClC-2 channel proteins are functional and suggest that in HTC cells they contribute to physiological changes in membrane Cl(-) permeability and cell volume homeostasis.
Roman
R M
RM
Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Smith
R L
RL
Feranchak
A P
AP
Clayton
G H
GH
Doctor
R B
RB
Fitz
J G
JG
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000818:Animals; D000906:Antibodies / Q000008:administration & dosage; D000090007:CLC-2 Chloride Channels; D006528:Carcinoma, Hepatocellular / Q000378:metabolism* / Q000473:pathology; D002460:Cell Line; D002462:Cell Membrane; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D008845:Microinjections; D018408:Patch-Clamp Techniques; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000906:Antibodies; D000090007:CLC-2 Chloride Channels; D018118:Chloride Channels; D002712:Chlorides; D018076:DNA, Complementary; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid