The effect of GABAA receptor activation varies from inhibition to excitation depending on the state of the transmembrane anionic concentration gradient (delta anion). delta anion was genetically altered in cultured dorsal root ganglion neurons via adenoviral vector-mediated expression of ClC-2, a Cl- channel postulated to regulate the Cl- concentration in neurons in which GABAA receptor activation is predominantly inhibitory. ClC-2 expression was verified by the presence of the appropriate mRNA, protein, and membrane conductance. CIC-2 expression resulted in a large negative shift in the Cl- equilibrium potential (ECl) that attenuated the GABA-mediated membrane depolarization and prevented GABAA receptor-mediated action potentials. These results establish that gene transfer of transmembrane ion channels to neurons can be used to demonstrate their physiological function, and that delta anion can be genetically manipulated to alter the function of neuronal GABAA receptors in situ.

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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.

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