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

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.

TMEM16 Proteins Produce Volume-regulated Chloride Currents That Are Reduced in Mice Lacking TMEM16A

“…7C). In hepatocytes I Cl,swell was of similar magnitude in both cells from ϩ/ϩ and Ϫ/Ϫ animals, however, currents activated in Ϫ/Ϫ cells showed a different time dependence, suggesting some contribution of TMEM16A to I Cl,swell also in hepatocytes (30).…”

Modulation of hepatocellular swelling-activated K+ currents by phosphoinositide pathway-dependent protein kinase C

“…Whole cell currents were digitized (Digidata 1200B) at 5 kHz, and sampled data were analyzed using Origin 6.0 software. To minimize swellingactivated anion currents, we carried out whole cell recordings using low Cl Ϫ -containing pipette and bath solutions, and 0.1 mM 4,4Ј-diisothiocyanostilbene-2,2Ј-disulfonic acid (Toronto Biochemicals, Toronto, ON), a blocker of volume-activated Cl Ϫ channels in rat hepatocytes (26,31), was included in the bath solution. Cells were bathed in a solution containing (in mM) 140 Na-gluconate, 5 Kgluconate, 5 glucose, 5 HEPES, 1 CaCl2, and 1 MgCl2 (pH adjusted to 7.4 with NaOH).…”

Regulatory volume decrease and P receptor signaling in fish cells: mechanisms, physiology, and modeling approaches

“…The broader relevance of these findings has been confirmed in hepatocytes from several mammalian species (Lan et al, 2005), but also in hepatocytes from goldfish, trout (Pafundo et al, 2004), and the marine flatfish turbot (Ollivier et al, 2006b), where the removal of extracellular ATP (using nucleotide scavengers) or blockage of P2 receptors (using antagonists) inhibited osmolyte efflux and RVD. Moreover, a similar pattern was found in Necturus erythrocytes (Light et al, '99, 2001), extending the relevance to nonhepatic cells.…”