Protein kina8e c modulates a swelling-activated cl^- channel in htc rat hepatoma cells

Hepatocytes exhibit facilitated uptake of organic and inorganic solutes which impose osmotic demands resulting in cell swelling. Cell volume recovery depends upon solute efflux mediated in part by activation of Cl^- channels (I_cl-swell) but the mechanisms coupling cell volume to channel opening are unknown. These studies used whole cell patch clamp techniques to characterize the properties and regulation of I_cl-swell in HTC cells. Intracellular perfusion with hypertonic sucrose-containing buffer activated anion-selective currents with outward rectification, time-dependent inactivation, and reversal potentials near E_cl. Incremental increases in intracellular osmolality through addition of 20-100 mM sucrose caused a concentration-dependent increase in I_cl-swell ranging from -3.4±0.9 to -69.9±11.2 pA/pF at -80 mV. Perfusion with Ca⁺⁺-free intracellular buffer (2 mM EGTA) markedly reduced I_cl-swelln (-3.7+0.6 pA/pF, p<0.01) from control values (-35.6+8.8 pA/pF). Inhibition of protein kinase C (PKC) by 25 MM chelerythrine decreased I_cl-swell (-3.5+0.6 pA/pF, p<0.01), while preactivation of PKC with 2 /iM 4 beta-phorbol 12-myristate, 13-acetate (PMA) modestly increased currents (-47.3±21.8 pA/pF, p=0.69). These studies demonstrate that changes in cell volume are closely coupled to membrane Cl^- permeability and suggest that activation of Cl^- channels contributing to I_cl-swell depends on both Ca⁺⁺ and PKC-dependent pathways.