Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes

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Abstract
Hepatocellular Cl−flux is integral to maintaining cell volume and electroneutrality in the face of the many transport and metabolic activities that describe the multifaceted functions of these cells. Although a significant volume-regulated Cl−current (VRAC) has been well described in hepatocytes, the Cl−channels underlying the large resting anion conductance have not been identified. We used a combination of electrophysiological and molecular approaches to describe potential candidates for this conductance. Anion currents in rat hepatocytes and WIF-B and HEK293T cells were measured under patch electrode-voltage clamp. With K+-free salts of Cl−comprising the major ions externally and internally, hyperpolarizing steps between −40 and −140 mV activated a time-dependent inward current in hepatocytes. Steady-state activation was half-maximal at −63 mV and 28–38% of maximum at −30 to −45 mV, previously reported hepatocellular resting potentials. Gating was dependent on cytosolic Cl−, shifting close to 58 mV/10-fold change in Cl−concentration. Time-dependent inward Cl−currents and a ClC-2-specific RT-PCR product were also observed in WIF-B cells but not HEK293T cells. All cell types exhibited typical VRAC in response to dialysis with hypertonic solutions. DIDS (0.1 mM) inhibited the hepatocellular VRAC but not the inward time-dependent current. Antibodies against the COOH terminus of ClC-2 reacted with a protein between 90 and 100 kDa in liver plasma membranes. The results demonstrate that rat hepatocytes express a time-dependent inward Cl−channel that could provide a significant depolarizing influence in the hepatocyte.