Ca²⁺-activated Cl^- channels in a human biliary cell line: Regulation by Ca²⁺/calmodulin-dependent protein kinase

Biliary epithelial cells contribute to bile formation through absorption and secretion of fluid and electrolytes. Recent studies indicate that membrane Cl^- permeability is regulated in part by increases in intracellular Ca²⁺ concentration. The purpose of these studies was to evaluate the effects of intracellular Ca²⁺ on channel activity, using the human M(z)- ChA-1 cholangiocarcinoma cell line as a model, and to assess the possible roles of Ca²⁺-dependent kinases in channel regulation. Exposure to ionomycin (1 μM) activated ion channels in the cell-attached configuration in 63 of 74 attempts, increasing open probability (NP(o)) from 0 to 0.26 ± 0.15 (n -17). Multiple channels were present in eacb patch, and the effects of ionomycin were reversed by subsequent addition of ethylene glycol-bis(β- aminoethyl ether)-N,N,N',N'-tetraacetic acid 12 mM) to the bath. With Cl^-- containing solutions, channels had a slope conductance of 14 ± 4 pS (n 11), and the mean open time was estimated to be 5.3 ± 1.8 ms. These channels were anion selective, and currents were carried by efflux of Cl^- at the resting potential. Exposure to the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) antagonist calmidazolium (100 μM) decreased NP(o) in ionomycin- stimulated cells to 0.02 ± 0.06 (n = 19). The protein kinase C antagonist chelerythrine (50 μM) was without effect. In parallel studies in subconfluent cell monolayers, CaMKII antagonists were also potent inhibitors of ionomycin-stimulated ¹²⁵I efflux. These findings indicate that Ca²⁺- dependent increases in membrane Cl^- permeability are related in part to opening of 14-pS anion channels through a mechanism that depends on both Ca²⁺ and CaMKII. These channels represent a potential target for pharmacological modulation of biliary cell transport and function.