Regulation of BK(Ca) channels expressed in human embryonic kidney 293 cells by epoxyeicosatrienoic acid.
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.
Fukao
M
M
Department of Physiology & Cell Biology, University of Nevada School of Medicine, Reno, Nevada.
Mason
H S
HS
Kenyon
J L
JL
Horowitz
B
B
Keef
K D
KD
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
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