Carbon Monoxide Stimulates the Ca2+–activated Big Conductance K (BK) Channels in Cultured Human Endothelial Cells.
De-Li Dong1&2, Yan Zhang2, Dao-Hong Lin2, Jun Chen3, Susann Patschan3, Michael S. Goligorsky3, Alberto Nasjletti2, Bao-Feng Yang1, Wen-Hui Wang2
1Department of Pharmacology, Harbin Medical University, Harbin 150086, China, 2Department of Pharmacology and 3Department of Medicine, New York Medical College, Valhalla, NY 10595
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Methods
Western blot
Proteins were separated by electrophoresis on 8-10% SDS- polyacrylamide gels and transferred to Immuno-Blot PVDF membrane (Bio-Rad Hercules, CA). The membrane was blocked with Odyssey blocking buffer and incubated with the primary antibody at 4°C for 12 hr. The membrane was washed 4 times (each 5 min) with PBS containing 0.1% Tween 20 and followed by incubation with the secondary antibody for an additional 30 min. After washing, the membrane was scanned by Odyssey infrared imaging system (LI-COR, Lincoln, Nebraska) at wave-length of 680 or 800 nM. HO-1 and HO-2 antibodies were purchased from Santa Cruz (Santa Cruz, CA).
Patch-Clamp Experiments
Patch pipettes were drawn with PP81 Narishege puller and the electrode resistance was 3-5MΩ when filled with 138 mM KCl. Single BK channel activity in HUVEC was studied using the whole-cell recording technique. The advantage for using the whole cell recording was that the possibility of finding BK channels in endothelial cells increased. When the high resistance seal was formed, the cell membrane under the patch pipette was ruptured to form the whole cell patch configuration. If the seal resistance was not changed after forming whole-cell patch mode, we clamped the cell membrane potential from -120mV to +120mV in one second (ramp) to determine whether BK channel activity could be detected. If BK channel activity was detected, we clamped the cell membrane potential at +50 mV (depolarization) which activates BK channels and studied the channel activity at the same holding potential. An Axopatch200A amplifier (Axon Instruments, CA) was used for recording the BK channel activity. The signal was filtered at 1 kHz and digitized by an interface, digitada1200 (Axon Ins.). Data were collected at sample rate of 5 kHz and were analyzed with software, Axon patch 7.0. Channel activity was defined as NPo, that was calculated from data samples of 120 seconds duration in the steady state as follows:
NPo= S(t1 + 2t2 +...... iti)
where ti is the fractional open time spent at each of the observed current levels. The BK channel conductance was measured at several holding potentials.