Activation of cloned BK(Ca) channels in nitric oxide-induced apoptosis of HEK293 cells.
The large conductance Ca(2+)-activated K(+) (BK(Ca)) channels are highly expressed in vascular smooth muscle cells (VSMCs) and play an essential role in the regulation of various physiological functions. Besides its electrophysiological function in vascular relaxation, BK(Ca) has also been reported to be implicated in nitric oxide (NO)-induced apoptosis of VSMCs. However, the molecular mechanism is not clear and has not been determined on cloned channels. The present study was designed to clarify whether activation of cloned BK(Ca) channel was involved in NO-induced apoptosis in human embryonic kidney 293 (HEK293) cell. The cDNA encoding the alpha-subunit of BK(Ca) channel, hSloalpha, was transiently transfected into HEK293 cells. The apoptotic death in HEK-hSloalpha cells was detected using immunocytochemistry, analysis of fragmented DNA by agarose gel electrophoresis, MTT test, and flow cytometry assays. Whole-cell and single-channel characteristics of HEK-hSloalpha cells exhibited functional features similar to native BK(Ca) channel in VSMCs. Exposuring of HEK- hSloalpha cells to S-nitroso-N-acetyl-penicillamine increased the hSloalpha channel activities of whole-cell and single-channel, and then increased percentage of cells undergoing apoptosis. However, blocking hSloalpha channels with 1 mM tetraethylammonia or 100 nM iberiotoxin significantly decreased the NO-induced apoptosis, whereas 30 microM NS1619, the specific agonist of BK(Ca), independently increased hSloalpha currents and induced apoptosis. These results indicated that activation of cloned BK(Ca) channel was involved in NO-induced apoptosis of HEK293 cells.
Ma
Yu-Guang
YG
Department of Clinical Oncology, Xijing Hospital, Fourth Military Medical University, Xi'an, China.
Dong
Ling
L
Ye
Xiao-Long
XL
Deng
Chang-Lei
CL
Cheng
Jiu-Hua
JH
Liu
Wen-Chao
WC
Ma
Jin
J
Chang
Yao-Ming
YM
Xie
Man-Jiang
MJ
D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits / Q000378:metabolism*; D009569:Nitric Oxide / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000494:pharmacology; D026423:S-Nitroso-N-Acetylpenicillamine / Q000494:pharmacology; D019789:Tetraethylammonium / Q000494:pharmacology; D014162:Transfection
C494841:KCNMA1 protein, human; D051037:Large-Conductance Calcium-Activated Potassium Channel alpha Subunits; D010455:Peptides; D009569:Nitric Oxide; D019789:Tetraethylammonium; C064719:iberiotoxin; D026423:S-Nitroso-N-Acetylpenicillamine