Contribution of a time-dependent and hyperpolarization-activated chloride conductance to currents of resting and hypotonically shocked rat hepatocytes.
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.
Lan
Wen-Zhi
WZ
Gastrointestinal Diseases Research Unit, Hotel Dieu Hospital, Queen's University, Kingston, Ontario, Canada.
Abbas
Houria
H
Lam
Hung D
HD
Lemay
Anne-Marie
AM
Hill
Ceredwyn E
CE
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D018118:Chloride Channels / Q000502:physiology*; D002712:Chlorides / Q000502:physiology; D005260:Female; D015870:Gene Expression; D022781:Hepatocytes / Q000502:physiology*; D015640:Ion Channel Gating; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D008969:Molecular Sequence Data; D009997:Osmotic Pressure; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D018118:Chloride Channels; D002712:Chlorides; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid