title: Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.
abstract: Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+ activated K+ channels (BK(Ca)) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BK(Ca) channels via ADP-ribosylation of the G protein Galphas with a subsequent membrane-delimited action on the channel [Circ Res 78:415-423, 1996; 80:877-884, 1997; 85:349-356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BK(Ca) alpha-subunit (cslo-alpha) is expressed without the beta-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-alpha channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPgammaS alone also activated cslo-alpha channels. Dialysis of cells with anti-Galphas antibody completely blocked the activation of cslo-alpha channels by 11,12-EET, whereas anti-Galphai/o and anti-Gbetagamma antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-alpha channels in cell-attached patches. These data suggest that EET leads to Galphas-dependent activation of the cslo-alpha subunits expressed in HEK293 cells and that the cslo-beta subunit is not required.
authors: Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
authors: M
authors: M
authors: Fukao
authors: H S
authors: HS
authors: Mason
authors: J L
authors: JL
authors: Kenyon
authors: B
authors: B
authors: Horowitz
authors: K D
authors: KD
authors: Keef
mesh_terms: D015126:8,11,14-Eicosatrienoic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology*; D036002:ADP Ribose Transferases / Q000037:antagonists & inhibitors / Q000378:metabolism; D000225:Adenine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000906:Antibodies / Q000494:pharmacology; D002227:Carbazoles; D002478:Cells, Cultured; D004347:Drug Interactions; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000276:immunology; D005786:Gene Expression Regulation / Q000187:drug effects*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007668:Kidney / Q000166:cytology / Q000196:embryology; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels / Q000096:biosynthesis / Q000235:genetics*; D024681:Potassium Channels, Calcium-Activated; D011758:Pyrroles / Q000494:pharmacology
chemical_list: D000906:Antibodies; D002227:Carbazoles; D004791:Enzyme Inhibitors; D007211:Indoles; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D051038:Large-Conductance Calcium-Activated Potassium Channel beta Subunits; D051036:Large-Conductance Calcium-Activated Potassium Channels; D015221:Potassium Channels; D024681:Potassium Channels, Calcium-Activated; D011758:Pyrroles; C017759:9-(tetrahydro-2-furyl)-adenine; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C057416:KT 5720; C046783:11,12-epoxy-5,8,14-eicosatrienoic acid; D006160:Guanosine Triphosphate; D036002:ADP Ribose Transferases; D019204:GTP-Binding Proteins; D015126:8,11,14-Eicosatrienoic Acid; D000225:Adenine