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Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells
Author(s) -
Kamouchi M.,
Bremt K.,
Eggermont J.,
Droogmans G.,
Nilius B.
Publication year - 1997
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1111/j.1469-7793.1997.545bd.x
Subject(s) - okadaic acid , staurosporine , gtp' , chemistry , biophysics , phosphatase , potassium channel , intracellular , patch clamp , microbiology and biotechnology , biochemistry , protein kinase a , biology , enzyme , receptor
1 We have used the patch‐clamp technique to study modulation of the inwardly rectifying K + current ( I K(IR) ) in cultured bovine pulmonary artery endothelial cells (CPAE cells). In whole‐cell mode, I K(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, ATPγS 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 GTPγS and A1F 4 − inhibited IRK, also in the presence of ATP. GTPγS antagonized the GTPγS effect. Pretreatment of the cells with PTX did not affect the GTPγS‐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 Ca 2+ 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 Ca 2+ , but supposedly by a G protein‐dependent activation of a phosphatase.