Ca(2+)-activated C1- channels in a human biliary cell line: regulation by Ca2+/calmodulin-dependent protein kinase

Abstract

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 Mz-ChA-1 cholangiocarcinoma cell line as a model, and to assess the possible roles of Ca(2+)-dependent kinases in channel regulation. Exposure to ionomycin (1 microM) activated ion channels in the cell-attached configuration in 63 of 74 attempts, increasing open probability (NPo) from 0 to 0.26 +/- 0.15 (n = 17). Multiple channels were present in each patch, and the effects of ionomycin were reversed by subsequent addition of ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'- tetraacetic acid (2 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 microM) decreased NPo in ionomycin-stimulated cells to 0.02 +/- 0.06 (n = 19). The protein kinase C antagonist chelerythrine (50 microM) 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 Ca(2+)-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.

Regulation of Human CLC-3 Channels by Multifunctional Ca2+/Calmodulin-dependent Protein Kinase

“…Regulated (17), and cystic fibrosis-derived pancreatic epithelial cells (18). The molecular identity of the channel or channels mediating the CaMKIIdependent conductance has remained unknown.…”

Regulation of biliary secretion through apical purinergic receptors in cultured rat cholangiocytes

“…In airway cells ATP stimulates Cl Ϫ secretion through activation of multiple signal transduction pathways, including both adenosine 3Ј,5Ј-cyclic monophosphate (cAMP)-dependent and independent mechanisms (44). In preliminary studies of NRC monolayers, the cAMP inhibitor Rp-cAMPs failed to inhibit the response to ATP (data not presented), and in isolated biliary cells, both cytosolic Ca 2ϩ concentration and Ca 2ϩ -calmodulindependent kinases increase membrane Cl Ϫ permeability (30,40). If these findings are relevant to cholangiocytes in vivo, it seems likely that apical ATP modulates Cl Ϫ secretion through one or more cAMP-independent pathways not directly related to CFTR.…”

“…Thus one potential explanation for these clinical differences is that biliary secretion might also be regulated by secretin-and CFTR-independent pathways. Indeed, Cl Ϫ channels that are distinct from CFTR have been identified in biliary cells and cell lines (14,28,39,40). Definition of their physiological role(s) has been limited by the lack of information regarding their cellular localization to the apical (luminal) or basolateral membranes, by the inability to measure transepithelial potentials across polarized cell monolayers, and by the limited information regarding the other receptors and physiological factors involved in regulation of secretory function.…”

Regulation of a Human Chloride Channel

“…channels was known for sure. Other Cl Ca channels that have hitherto been shown to be activated by CaMKII all display substantially higher unitary conductances in the 14 -40-pS range (15,(33)(34)(35).…”