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Departments of 1 Medicine, 2 Neurology, 3 Pediatrics, and 4 Cell Biology, University of Colorado Health Sciences Center, Denver, Colorado 80262
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 µM). 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.
hepatocyte; purinergic receptors; liver
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