ABSTRACT
BACKGROUND & AIMS: Increases in intracellular Ca 2+ are thought to complement cAMP in stimulating Cl - secretion in cholangiocytes, although the site(s) of action and channels involved are unknown. We have identified a Ca 2+ -activated K + channel (SK2) in biliary epithelium that is inhibited by apamin. The purpose of the present studies was to define the role of SK channels in Ca 2+ -dependent cholangiocyte secretion. METHODS: Studies were performed in human Mz-Cha-1 cells and normal rat cholangiocytes (NRC). Currents were measured by whole-cell patch clamp technique and transepithelial secretion by Ussing chamber. RESULTS: Ca 2+ -dependent stimuli, including purinergic receptor stimulation, ionomycin, and increases in cell volume, each activated K + -selective currents with a linear IV relation and time-dependent inactivation. Currents were Ca 2+ dependent and were inhibited by apamin and by Ba 2+. In intact liver, immunoflourescence with an antibody to SK2 showed a prominent signal in cholangiocyte plasma membrane. To evaluate the functional significance, NRC monolayers were mounted in a Ussing chamber, and the short-circuit current ( I sc ) was measured. Exposure to ionomycin caused an increase in I sc 2-fold greater than that induced by cAMP. Both the basal and ionomycin-induced I sc were inhibited by basolateral Ba 2+, and approximately 58% of the basolateral K + current was apamin sensitive. CONCLUSIONS: These studies demonstrate that cholangiocytes exhibit robust Ca 2+ -stimulated secretion significantly greater in magnitude than that stimulated by cAMP. SK2 plays an important role in mediating the increase in transepithelial secretion due to increases in intracellular Ca 2+. SK2 channels, therefore, may represent a target for pharmacologic modulation of bile flow.
Subject(s)
Bile Ducts, Intrahepatic/metabolism , Calcium/metabolism , Potassium Channels, Calcium-Activated , Potassium Channels/metabolism , Animals , Apamin/pharmacology , Bee Venoms/pharmacology , Bile Ducts, Intrahepatic/drug effects , Biological Transport/drug effects , Biological Transport/physiology , Cell Line, Tumor , Cells, Cultured , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Humans , Ionomycin/pharmacology , Ionophores/pharmacology , Potassium/metabolism , Potassium Channels/drug effects , Rats , Small-Conductance Calcium-Activated Potassium ChannelsABSTRACT
In human liver, Ca(2+)-dependent changes in membrane K(+) permeability play a central role in coordinating functional interactions between membrane transport, metabolism, and cell volume. On the basis of the observation that K(+) conductance is partially sensitive to the bee venom toxin apamin, we aimed to assess whether small-conductance Ca(2+)-sensitive K(+) (SK(Ca)) channels are expressed endogenously and contribute to volume-sensitive K(+) efflux and cell volume regulation. We isolated a full-length 2,140-bp cDNA (hSK2) highly homologous to rat brain rSK2 cDNA, including the putative apamin-sensitive pore domain, from a human liver cDNA library. Identical cDNAs were isolated from primary human hepatocytes, human HuH-7 hepatoma cells, and human Mz-ChA-1 cholangiocarcinoma cells. Transduction of Chinese hamster ovary cells with a recombinant adenovirus encoding the hSK2-green fluorescent protein fusion construct resulted in expression of functional apamin-sensitive K(+) channels. In Mz-ChA-1 cells, hypotonic (15% less sodium glutamate) exposure increased K(+) current density (1.9 +/- 0.2 to 37.5 +/- 7.1 pA/pF; P < 0.001). Apamin (10-100 nM) inhibited K(+) current activation and cell volume recovery from swelling. Apamin-sensitive SK(Ca) channels are functionally expressed in liver and biliary epithelia and likely contribute to volume-sensitive changes in membrane K(+) permeability. Accordingly, the hSK2 protein is a potential target for pharmacological modulation of liver transport and metabolism through effects on membrane K(+) permeability.
