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. 1997 Nov 1;504(Pt 3):545–556. doi: 10.1111/j.1469-7793.1997.545bd.x

Modulation of inwardly rectifying potassium channels in cultured bovine pulmonary artery endothelial cells.

M Kamouchi 1, K Van Den Bremt 1, J Eggermont 1, G Droogmans 1, B Nilius 1
PMCID: PMC1159959  PMID: 9401963

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.

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Selected References

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