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.

[Activation of cloned BKCa channels in Ang II-induced proliferation of HEK293 cells].

AIM: To investigate the mediating and regulating role of BK(Ca) channels in Ang II-induced cell proliferation. METHODS: Using Lipofectamine 2000, the pIRES-hSloalpha plasmid was transfected into HEK293 cells. The concentration-dependent cuve of Ang II-induced cell proliferation was tested by MTT colorimetry. The effect of IbTX, Ang II, Ang II + IbTX on the proliferating cell nuclear antigen (PCNA) expression and cell cycle of transfected HEK293-hSloalpha cells were detected by immunocytochemistry and flow cytometry, respectively. RESULTS: Ang II induced proliferation of transfected HEK293-hSloalpha cells in a concentration-dependent manner. PCNA expression of transfected HEK293-hSloalpha cells was enhanced by Ang II, and the percentage of S phase HEK293-hSloalpha cells was increased after Ang II treatment. However this effect was inhibited by IbTX, a selective BK(Ca) channel blocker. CONCLUSION: BK(Ca) channels play an potential role in mediating Ang II-induced proliferation of HEK293-hSloalpha cells.

Coexpression and characterization of the human large-conductance Ca(2+)-activated K (+) channel alpha + beta1 subunits in HEK293 cells.

Large-conductance Ca(2+)-activated K(+) channel is formed by a tetramer of the pore-forming alpha-subunit and distinct accessory beta-subunits (beta1-beta4) which contribute to BK(Ca) channel molecular diversity. Accumulative evidences indicate that not only alpha-subunit alone but also the alpha + beta subunit complex and/or beta-subunit might play an important role in modulating various physiological functions in most mammalian cells. To evaluate the detailed pharmacological and biophysical properties of alpha + beta1 subunit complex or beta1-subunit in BK(Ca) channel, we established an expression system that reliably coexpress hSloalpha + beta1 subunit complex in HEK293 cells. The coexpression of hSloalpha + beta1 subunit complex was evaluated by western blotting and immunolocalization, and then the single-channel kinetics and pharmacological properties of expressed hSloalpha + beta1 subunit complex were investigated by cell-attached and outside-out patches, respectively. The results in this study showed that the expressed hSloalpha + beta1 subunit complex demonstrated to be fully functional for its typical single-channel traces, Ca(2+)-sensitivity, voltage-dependency, high conductance (151 +/- 7 pS), and its pharmacological activation and inhibition.