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) (ATPγS) caused transient opening of channels with linear conductances of ~18 and ~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 ~7.4 μM; at 10^-4 M, the peak increase in [Ca²⁺](i) was ATP > UTP > ATPγS > > 2-methyl- thioadenosine 5'-triphosphate, α,β-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 myo-inositol 1,4,5-trisphosphate (50-100 μM) or myo-inositol 1,3,4,5-trisphosphate (20 μM) but opened after increases in Ca²⁺ to greater than ~250 nM, 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.