Investigation of the role of TASK-2 channels in rat pulmonary arteries; pharmacological and functional studies following RNA interference procedures.
In the present study, we investigated the ability of RNA interference technology to suppress TASK-2 potassium channel expression in human embryonic kidney (HEK293) cells stably transfected with TASK-2 cDNA and in rat isolated intact pulmonary arteries. Lipofectamine-induced transfection of a specific siRNA sequence targeted against TASK-2 resulted in a dose- and time-dependent decrease in TASK-2 channel protein expression. In siRNA-transfected cells the TASK-2 peak currents were significantly smaller than in control cells at every investigated pH, while the pH sensitivity was not altered. Using scrambled siRNA as a negative control, there were no significant changes in TASK-2 protein expression or current compared to mock-transfected cells. In TASK-2 siRNA-transfected small pulmonary arteries, but not in scrambled siRNA-treated vessels, myocyte resting membrane potential at pH 7.4 was significantly less negative and the hyperpolarisations in response to increasing pH from 6.4 to 8.4 were significantly smaller compared with control. The application of levcromakalim (10 microM), NS1619 (33 microM) and a potassium channel inhibitor cocktail (5 mM 4-aminopyridine, 10 mM tetraethylammonium chloride, 30 microM Ba2+ and 10 microM glibenclamide) had similar effects in control and in siRNA-transfected vessels. The TASK-1 (anandamide-sensitive) contribution to resting membrane potential was comparable in each group. Clofilium (100 microM) generated significantly smaller responses in transfected artery segments. These results suggest that RNA interference techniques are effective at inhibiting TASK-2 channel expression in cultured cells and in intact vessels and that TASK-2 channels have a functional role in setting the membrane potential of pulmonary artery myocytes.
Gönczi
Mónika
M
Faculty of Life Sciences, University of Manchester, Manchester, M13 PT, UK.
Szentandrássy
Norbert
N
Johnson
Ian T
IT
Heagerty
Anthony M
AM
Weston
Arthur H
AH
D000818:Animals; D002460:Cell Line; D006863:Hydrogen-Ion Concentration; D008297:Male; D008564:Membrane Potentials; D009131:Muscle, Smooth, Vascular / Q000502:physiology; D015221:Potassium Channels / Q000502:physiology; D024683:Potassium Channels, Tandem Pore Domain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011651:Pulmonary Artery / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley
C498740:Kcnk5 protein, rat; D015221:Potassium Channels; D024683:Potassium Channels, Tandem Pore Domain; D034741:RNA, Small Interfering