REGULATION OF CATION CHANNELS IN LIVER-CELLS BY INTRACELLULAR CALCIUM AND PROTEIN-KINASE-C

The regulation of Ca2+-permeant cation channels in HTC hepatoma cells was investigated using patch clamp and fluorescence techniques. In intact cells, exposure to nucleotide analogues ATP, uridine 5'-triphosphate (UTP), and adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) caused transient opening of channels with linear conductances of similar to 18 and similar to 28 pS. Channels were permeable to Na+, K+, and Ca2+ and carried inward (depolarizing) current at the resting potential. Exposure to thapsigargin to increase cytosolic Ca2+ concentration ([Ca2+](i)) opened similar channels, suggesting that opening is stimulated by a rise in [Ca2+](i). In subconfluent monolayers, ATP increased [Ca2+](i) with half-maximal effects at similar to 7.4 mu M; at 10(-4) M, the peak increase in [Ca2+](i) was ATP > UTP > ATP gamma S > > 8-methylthioadenosine 5'-triphosphate, alpha,beta-methyleneadenosine 5'-triphosphate, and adenosine. The relative potency suggests that the effects are mediated by 5'-nucleotide receptors. In excised inside-out patches, channels were not activated by myoinositol 1,4,5-trisphosphate (50-100 mu M) or myo-inositol 1,3,4,5-trisphosphate (20 mu M) but opened after increases in Ca2+ to greater than similar to 250 mu M, consistent with a direct role for Ca2+ in channel opening. In intact cells, channel opening was followed by a prolonged refractory period. Protein kinase C appears to contribute by inhibition of the ATP-stimulated [Ca2+](i) response and by direct inhibitory effects on the channel. These findings indicate that extracellular ATP leads to modulation of liver cell cation channels through activation of 5'-nucleotide receptors and are consistent with a model in which transient opening of channels is stimulated by a rise in [Ca2+](i) and subsequent closure is mediated by protein kinase C-dependent pathways.