title: Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells.
abstract: 1. We have used the patch-clamp technique to study modulation of the inwardly rectifying K+ current (IK(IR)) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole-cell mode, IK(IR) was defined as the Ba(2+)-sensitive current. In single channel recordings, we observed a strongly inwardly rectifying and K(+)-selective channel with a conductance of 31 +/- 3 pS. 2. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis and functional data suggest that the endothelial IRK is most probably Kir2.1. 3. Intracellular ATP is required to prevent run-down of IRK in whole-cell mode. Single channel activity disappeared in inside-out patches exposed to ATP-free solution and in cell-attached patches on cells exposed to metabolic inhibition (KCN, 2-deoxyglucose). 4. The non-hydrolysable ATP analogues, ATP gamma S and adenylyl imidodiphosphate (AMP-PNP), did not prevent run-down. Run-down did not occur in the presence of okadaic acid, a phosphatase inhibitor, but was enhanced in the presence of protamine, an activator of phosphatase 2A (PP2A). 5. GTP gamma S and AlF4- inhibited IRK, also in the presence of ATP. GTP beta S antagonized the GTP gamma S effect. Pretreatment of the cells with PTX did not affect the GTP gamma S-induced inhibition. Okadaic acid, however, slowed this inhibition. 6. Neither activation of protein kinase A (PKA) nor activation of protein kinase C (PKC) affected IRK. Additionally, neither cytochalasin B nor a high concentration of intracellular Ca2+ affected the time course of IRK run-down. 7. We conclude that run-down of IRK is probably due to dephosphorylation by PP2A. Activation of a PTX-insensitive G protein inhibits this current by a mechanism that is neither mediated via the PKA and PKC pathways nor by intracellular Ca2+, but supposedly by a G protein-dependent activation of a phosphatase.
authors.affiliation: Laboratorium voor Fysiologie, KU Leuven, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Kamouchi
authors.forename: K
authors.initials: K
authors.lastname: Van Den Bremt
authors.forename: J
authors.initials: J
authors.lastname: Eggermont
authors.forename: G
authors.initials: G
authors.lastname: Droogmans
authors.forename: B
authors.initials: B
authors.lastname: Nilius
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003571:Cytochalasin B / Q000494:pharmacology; D004594:Electrophysiology; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008564:Membrane Potentials / Q000502:physiology; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D015221:Potassium Channels / Q000187:drug effects / Q000378:metabolism*; D011651:Pulmonary Artery / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*
chemical_list: D015221:Potassium Channels; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D003571:Cytochalasin B; D000255:Adenosine Triphosphate; D019204:GTP-Binding Proteins